Fermentation biotechnology for advanced biotechnology students

Fermentation biotechnology notes for the second semester second year

1. HABB_HBB 231: MOLECULAR
GENETICS
DR T. MUTEVERI

4. DNA STRUCTUREDNA STRUCTURE

5. NUCLEIC ACIDSNUCLEIC ACIDS

Nucleic acids are polymersNucleic acids are polymers

Monomer---nucleotidesMonomer---nucleotides

Nitrogenous basesNitrogenous bases

PurinesPurines

PyrimidinesPyrimidines

Sugar Sugar

RiboseRibose

DeoxyriboseDeoxyribose

PhosphatesPhosphates

+nucleoside=nucleotide+nucleoside=nucleotide
}
Nucleosides

6. The Sugars The Sugars

7. Bases of DNA (and RNABases of DNA (and RNA))
RNA only DNA only
Purines:
Pyrimidines:

8. Nucleic acids are composed of
repeating subunits called
nucleotides.
Each nucleotide is composed of
three units:
•phosphate,
•5-carbon sugar (pentose)
•A cyclic nitrogen-containing base

9. Nucleotides and NucleosidesNucleotides and Nucleosides

10. Chemical Structure of DNA and RNA
Figure 4.1
RNA DNA
Nucleotide
Nucleoside
1’
2’
4’
The C is
named 1’-5’
Resume

11. Nucleotides and NucleosidesNucleotides and Nucleosides
BASEBASE NUCLEOSIDENUCLEOSIDE DEOXYNUCLEOSIDEOXYNUCLEOSI
DEDE
AdenineAdenine AdenosineAdenosine 2-2-
deoxyadenosinedeoxyadenosine
GuanineGuanine GuanosineGuanosine 2-2-
deoxyguanosinedeoxyguanosine
CytosineCytosine CytodineCytodine 2-deoxycytodine2-deoxycytodine
UracilUracil UridineUridine Not usually Not usually
found found
ThymineThymine Not usually Not usually
foundfound
2-2-
deoxythymidinedeoxythymidine
Nucleotides = nucleosides + phosphate

12. NucleotidNucleotid
e Analogs e Analogs
as Drugsas Drugs

13. DNA Stabilization– DNA Stabilization–
Complementary Base PairingComplementary Base Pairing

14. Chemical Bonds Important in DNA
Structure

15. DNA Stabilization--H-bonding DNA Stabilization--H-bonding
between DNA base pair stacks between DNA base pair stacks

16. DNA structure:SummaryDNA structure:Summary

DNA is a double stranded molecule with 2 DNA is a double stranded molecule with 2
polynucleotide chains running in opposite polynucleotide chains running in opposite
directions.directions.

Both strands are complementary to each other.Both strands are complementary to each other.

The bases are on the inside of the molecules and The bases are on the inside of the molecules and
the 2 chains are joined together by double H-bond the 2 chains are joined together by double H-bond
between A and T and triple H-bond between C and G.between A and T and triple H-bond between C and G.

The base pairing is very specific which make the 2 The base pairing is very specific which make the 2
strands complementary to each other.strands complementary to each other.

So each strand contain all the required information So each strand contain all the required information
for synthesis (replication) of a new copy to its for synthesis (replication) of a new copy to its
complementary.complementary.

17. FORMS OF DNAFORMS OF DNA

18. A-form DNAA-form DNA::

Less common form of DNA , more common in RNALess common form of DNA , more common in RNA

Right handed helixRight handed helix

Each turn contain 11 b.p/turnEach turn contain 11 b.p/turn

Contain 2 different grooves:Contain 2 different grooves:

Major groove: very deep and narrowMajor groove: very deep and narrow

Minor groove: very shallow and wide (binding site for RNA)Minor groove: very shallow and wide (binding site for RNA)

19. Forms of DNA
B-form helixB-form helix::

It is the most common form of DNA in cells.It is the most common form of DNA in cells.

Right-handed helixRight-handed helix

Turn every 3.4 nm.Turn every 3.4 nm.

Each turn contain 10 base pairs (the distance Each turn contain 10 base pairs (the distance
between each 2 successive bases is 0.34 nm)between each 2 successive bases is 0.34 nm)

Contain 2 grooves;Contain 2 grooves;

Major groove (wide): provide easy access to basesMajor groove (wide): provide easy access to bases

Minor groove (narrow): provide poor access. Minor groove (narrow): provide poor access.

20. Z-form DNA:
Radical change of B-form
Left handed helix, very extended
It is GC rich DNA regions.
The sugar base backbone form Zig-Zag shape
The B to Z transition of DNA molecule may play
a role in gene regulation.

21. Denaturing and Annealing of DNA

The DNA double strands can denatured if The DNA double strands can denatured if
heated (95ºC) or treated with chemicals.heated (95ºC) or treated with chemicals.

AT regions denature first (2 H bonds)AT regions denature first (2 H bonds)

GC regions denature last (3 H bonds)GC regions denature last (3 H bonds)

DNA denaturation is a reversible process, as DNA denaturation is a reversible process, as
denatured strands can re-annealed again if denatured strands can re-annealed again if
cooled.cooled.

This process can be monitored using the This process can be monitored using the
hyperchromicity (melting profile).hyperchromicity (melting profile).

22. Hyperchromicity (melting profile)

It is used to monitor DNA denaturation and It is used to monitor DNA denaturation and
annealing.annealing.

It is based on the fact that single stranded (SS) It is based on the fact that single stranded (SS)
DNA gives higher absorbtion reading than DNA gives higher absorbtion reading than
double stranded (DS) at wavelength 260º.double stranded (DS) at wavelength 260º.

Using melting profile we can differentiate Using melting profile we can differentiate
between single stranded and double stranded between single stranded and double stranded
DNA. DNA.

23. Hyperchromicity (melting profile)
DS
SS
SS
Ab
260
Tm
Temperature
Tm (melting temp.): temp. at which 50% of DS DNA denatured to SS
•Heating of SS DNA: little rise of Ab reading
• Heating of DS DNA: high rise of Ab reading
Using melting profile we can differentiate between SS DNA and DS
DNA

24. Melting profile continue…..

Melting profile can be also used to give Melting profile can be also used to give
an idea about the type of base pair rich an idea about the type of base pair rich
areas using the fact that:areas using the fact that:

A═T rich regions: denatured first (low melting point)A═T rich regions: denatured first (low melting point)

G≡C rich regions: denatured last (higher melting G≡C rich regions: denatured last (higher melting
point)point)
DS
SS
GC rich DNA
AT rich DNA
GC/AT DNA
Tm1Tm2Tm3
Tm1: Small melting temp. of AT rich
DNA
Tm2: higher melting temp. of AT/GC
equal DNA
Tm3: Highest melting temp. of GC rich
DNA

25. Advantages to Double HelixAdvantages to Double Helix

Stability---protects bases from attack by Stability---protects bases from attack by
HH
22O soluble compounds and HO soluble compounds and H
22O itself.O itself.

Provides easy mechanism for Provides easy mechanism for
replicationreplication

26. Physical Structure (cont’d)Physical Structure (cont’d)

Chains are anti-parallel (i.e in opposite Chains are anti-parallel (i.e in opposite
directions)directions)

Diameter and periodicity are consistent Diameter and periodicity are consistent

2.0 nm2.0 nm

10 bases/ turn10 bases/ turn

3.4 nm/ turn3.4 nm/ turn

Width consistent because of Width consistent because of
pyrimidine/purine pairingpyrimidine/purine pairing

27. G-C ContentG-C Content

A=T, G=C, but ATA=T, G=C, but AT≠GC≠GC

Generally GC~50%, but extremely Generally GC~50%, but extremely
variablevariable

EX.EX.

Slime mold~22%Slime mold~22%

Mycobacterium~73%Mycobacterium~73%

Distribution of GC is not uniform in Distribution of GC is not uniform in
genomesgenomes

28. CONSEQUENCES OF GC CONTENTCONSEQUENCES OF GC CONTENT

GC slightly denser GC slightly denser

Higher GC DNA moves further in a Higher GC DNA moves further in a
gradientgradient

Higher # of base pairs=more stable Higher # of base pairs=more stable
DNA, i.e. the strands don’t separate as DNA, i.e. the strands don’t separate as
easily. easily.

29. Denaturation of DNADenaturation of DNA

Denaturation by Denaturation by
heating.heating.

How observed?How observed?
AA
260260

For dsDNA, For dsDNA,
AA
260260=1.0 for 50 =1.0 for 50 µg/mlµg/ml

For ssDNA and RNA For ssDNA and RNA
AA
260260=1.0 for 38 =1.0 for 38 µg/mlµg/ml

For ss oligosFor ss oligos
AA
260260=1.0 for 33 =1.0 for 33 µg/mlµg/ml

Hyperchromic shiftHyperchromic shift
The T at which ½ the DNA
sample is denatured is
called the melting
temperature (T
m)

30. Importance of TImportance of T
mm

Critical importance in any technique Critical importance in any technique
that relies on complementary base that relies on complementary base
pairing pairing

Designing PCR primersDesigning PCR primers

Southern blotsSouthern blots

Northern blotsNorthern blots

Colony hybridization Colony hybridization

31. Factors Affecting TFactors Affecting T
mm

G-C content of sampleG-C content of sample

Presence of intercalating agents Presence of intercalating agents
(anything that disrupts H-bonds or base (anything that disrupts H-bonds or base
stacking)stacking)

Salt concentrationSalt concentration

pH pH

LengthLength

32. RenaturationRenaturation

Strands can be induced to renature Strands can be induced to renature
(anneal) under proper conditions. (anneal) under proper conditions.
Factors to consider:Factors to consider:

TemperatureTemperature

Salt concentrationSalt concentration

DNA concentrationDNA concentration

TimeTime

33. RNA structure

It is formed of linear polynucleotideIt is formed of linear polynucleotide

It is generally single stranded It is generally single stranded

The pentose sugar is RiboseThe pentose sugar is Ribose

Uracile (U) replace Thymine (T) in the pyrimidine Uracile (U) replace Thymine (T) in the pyrimidine
bases.bases.
Although RNA is generally single stranded, Although RNA is generally single stranded,
intra-molecular H-bond base pairing occur intra-molecular H-bond base pairing occur
between complementary bases on the same between complementary bases on the same
molecule (secondary structure) molecule (secondary structure)

34. RNA structure

It can take 3 levels of structure;It can take 3 levels of structure;

Primary: sequence of nucleotidesPrimary: sequence of nucleotides

Secondary: hairpin loops (base pairing)Secondary: hairpin loops (base pairing)

Tertiary: motifs and 3D foldingsTertiary: motifs and 3D foldings

35. RNA structure
Transfer RNA (tRNA) structure

36. RNA RNA

TypesTypes

mRNAmRNA

tRNAtRNA

rRNArRNA

It’s still an RNA worldIt’s still an RNA world

snRNAsnRNA

siRNAsiRNA

RibozymesRibozymes

37. Types of RNA

Messenger RNA (mRNA)Messenger RNA (mRNA)::

Carries genetic information copied from DNA in the form of Carries genetic information copied from DNA in the form of
a series of 3-base code, each of which specifies a particular a series of 3-base code, each of which specifies a particular
amino acid.amino acid.

Transfer RNA (tRNA)Transfer RNA (tRNA)::

It is the key that read the code on the mRNA.It is the key that read the code on the mRNA.

Each amino acid has its own tRNA, which binds to it and Each amino acid has its own tRNA, which binds to it and
carries it to the growing end of a polypeptide chain.carries it to the growing end of a polypeptide chain.

Ribosomal RNA (rRNA)Ribosomal RNA (rRNA)::

Associated with a set of proteins to form the ribosomes.Associated with a set of proteins to form the ribosomes.

These complex structures, which physically move along the These complex structures, which physically move along the
mRNA molecule, catalyze the assembly of amino acids into mRNA molecule, catalyze the assembly of amino acids into
protein chain.protein chain.

They also bind tRNAs that have the specific amino acids They also bind tRNAs that have the specific amino acids
according to the code.according to the code.

38. Behavior in AcidsBehavior in Acids

Dilute or mild acidic conditionsDilute or mild acidic conditions

Intermediate conditions. EX. 1N HCl @ Intermediate conditions. EX. 1N HCl @
100100ºC for 15m : DepurinationºC for 15m : Depurination

Harsher treatment-EX. 2-6N HCl, higher Harsher treatment-EX. 2-6N HCl, higher
temps: Depyrimidination.temps: Depyrimidination.

NOTE: some phosphodiester bond NOTE: some phosphodiester bond
cleavage observed during depurination, cleavage observed during depurination,
much more during depyrimidinationmuch more during depyrimidination

39. Behavior in BasesBehavior in Bases

N-glycosidic bonds stable in mild N-glycosidic bonds stable in mild
alkaline conditionsalkaline conditions

DNA meltsDNA melts
Phosphodiester linkages in DNA and linkages in DNA and
RNA show very different behavior in RNA show very different behavior in
weak bases (EX 0.3 N KOH @37weak bases (EX 0.3 N KOH @37
ºº
C ~1 hr.)C ~1 hr.)

40. RNA Hydrolysis in Alkaline RNA Hydrolysis in Alkaline
SolutionsSolutions
2,3 cyclic
nucleotide

41. Hydrolysis by EnzymesHydrolysis by Enzymes

Nuclease—catalyzes hydrolysis of Nuclease—catalyzes hydrolysis of
phosphodiester backbone phosphodiester backbone

ExonucleasesExonucleases

EndonucleasesEndonucleases

General. Ex DNAse IGeneral. Ex DNAse I

Specific Ex. Restriction endonucleasesSpecific Ex. Restriction endonucleases

RibozymesRibozymes

42. DNA SEQUENCINGDNA SEQUENCING

Purpose—determine nucleotide Purpose—determine nucleotide
sequence of DNA sequence of DNA

Two main methodsTwo main methods
 Maxam & Gilbert, using chemical
sequencing
Sanger, using dideoxynucleotides

43. The Sanger TechniqueThe Sanger Technique

Uses Uses
dideoxynucleotides dideoxynucleotides
(dideoxyadenine, (dideoxyadenine,
dideoxyguanine, dideoxyguanine,
etc)etc)

These are These are
molecules that molecules that
resemble normal resemble normal
nucleotides but lack nucleotides but lack
the normal -OH the normal -OH
groupgroup..

44. 
Because they lack the -OH (which Because they lack the -OH (which
allows nucleotides to join a growing allows nucleotides to join a growing
DNA strand), replication stops.DNA strand), replication stops.
Normally, this would
be where another
phosphate
Is attached, but with no -
OH
group, a bond can not
form and replication
stops

45. The Sanger Method RequiresThe Sanger Method Requires

Multiple copies of single stranded Multiple copies of single stranded template template
DNADNA

A suitable A suitable primerprimer (a small piece of DNA that (a small piece of DNA that
can pair with the template DNA to act as a can pair with the template DNA to act as a
starting point for replication)starting point for replication)

DNA polymeraseDNA polymerase (an enzyme that copies DNA, (an enzyme that copies DNA,
adding new nucleotides to the 3’ end of the adding new nucleotides to the 3’ end of the
templatetemplate

A ‘pool’ of A ‘pool’ of normal nucleotidesnormal nucleotides

A small proportion of A small proportion of dideoxynucleotidesdideoxynucleotides
labeled in some way ( radioactively or with labeled in some way ( radioactively or with
fluorescent dyes) fluorescent dyes)

46. 
The template DNA pieces are replicated, The template DNA pieces are replicated,
incorporating normal nucleotides, but incorporating normal nucleotides, but
occasionally and at random occasionally and at random dideoxy (DD) dideoxy (DD)
nucleotides are taken up.nucleotides are taken up.

This stops replication on that piece of DNAThis stops replication on that piece of DNA

The result is a The result is a mix of DNA lengthsmix of DNA lengths, each , each
ending with a particular labeled ending with a particular labeled
DDnucleotide.DDnucleotide.

Because the different lengths ‘travel’ at Because the different lengths ‘travel’ at
different rates during electrophoresis, their different rates during electrophoresis, their
order can be determined.order can be determined.

47. Termination during
Replication

DNA
SEQUENCE
3’
G C A T T G G G A A C C
PRIMER
5’
C G T A
NO OF
BASES
1 2 3 4 5 6 7 8 9 10 11 12

G terminated C G T A A C C T T G
C G T A A C C T T G G

A terminated
C G T A A

Tterminated C G T A A C C T
C G T A A C C T T


C terminated C G T A A C
C G T A A C C
C G T A A C C C

49. DNA REPLICATIONDNA REPLICATION

50. 
DNA replication is an anabolic polymerization DNA replication is an anabolic polymerization
process, that allows a cell to pass copies of its process, that allows a cell to pass copies of its
genome to its descendants.genome to its descendants.

The key to DNA replication is the complementary The key to DNA replication is the complementary
structure of the two strands: structure of the two strands:

Adenine and guanine in one strand bond with Adenine and guanine in one strand bond with
thymine and cytosine, respectively, in the other. thymine and cytosine, respectively, in the other.

DNA replication is a simple concept - a cell separates DNA replication is a simple concept - a cell separates
the two original strands and uses each as a template the two original strands and uses each as a template
for the synthesis of a new complementary strand. for the synthesis of a new complementary strand.

Biologists say that DNA replication is Biologists say that DNA replication is semiconservative semiconservative
because each daughter DNA molecule is composed because each daughter DNA molecule is composed
of one original strand and one new strand. of one original strand and one new strand.

51. Processes in DNA ReplicationProcesses in DNA Replication

52. Initial Processes in DNA Initial Processes in DNA
ReplicationReplication

DNA replication begins at a specific sequence of nucleotides called an DNA replication begins at a specific sequence of nucleotides called an originorigin. .

First, a cell removes chromosomal proteins, exposing the DNA helix. First, a cell removes chromosomal proteins, exposing the DNA helix.

Next, an enzyme called Next, an enzyme called DNA helicase DNA helicase locally "unzips" the DNA molecule by locally "unzips" the DNA molecule by
breaking the hydrogen bonds between complementary nucleotide bases, which breaking the hydrogen bonds between complementary nucleotide bases, which
exposes the bases in a exposes the bases in a replication forkreplication fork. Other protein molecules stabilize the . Other protein molecules stabilize the
single strands so that they do not rejoin while replication proceedssingle strands so that they do not rejoin while replication proceeds

After helicase untwists and separates the strands, a molecule of an enzyme After helicase untwists and separates the strands, a molecule of an enzyme
called called DNA polymerase DNA polymerase III binds to each strand. III binds to each strand.

DNA polymerases replicate DNA in only one direction - 5' to 3' - like a jeweler DNA polymerases replicate DNA in only one direction - 5' to 3' - like a jeweler
stringing pearls to make a necklace, adding them one at a time, always moving stringing pearls to make a necklace, adding them one at a time, always moving
from one end of the string to the other. from one end of the string to the other.

Because the two original (template) strands are antiparallel cells synthesize new Because the two original (template) strands are antiparallel cells synthesize new
strands in two different ways. One new strand, called the leading strand, is strands in two different ways. One new strand, called the leading strand, is
synthesized continuously as a single long chain of nucleotides. synthesized continuously as a single long chain of nucleotides.

The other new strand, called the lagging strand, is synthesized in short The other new strand, called the lagging strand, is synthesized in short
segments that are later joined. segments that are later joined.

53. Synthesis of the Leading StrandSynthesis of the Leading Strand
A cell synthesizes a leading strand toward the replication fork in A cell synthesizes a leading strand toward the replication fork in
the following series of five steps the following series of five steps
1) An enzyme called 1) An enzyme called primase primase synthesizes a short RNA molecule that synthesizes a short RNA molecule that
is complementary to the template DNA strand. This is complementary to the template DNA strand. This RNA primer RNA primer
provides the 3' hydroxyl group required by DNA polymerase.provides the 3' hydroxyl group required by DNA polymerase.
2) Triphosphate deoxyribonucleotides form hydrogen bonds with 2) Triphosphate deoxyribonucleotides form hydrogen bonds with
their complements in the parental strand. Adenine nucleotides their complements in the parental strand. Adenine nucleotides
bind to thymine nucleotides, and guanine nucleotides bind to bind to thymine nucleotides, and guanine nucleotides bind to
cytosine nucleotides. cytosine nucleotides.
3) Using the energy in the high-energy bonds of the triphosphate 3) Using the energy in the high-energy bonds of the triphosphate
deoxyribonucleotides, DNA polymerase III covalently joins them deoxyribonucleotides, DNA polymerase III covalently joins them
one at a time by dehydration synthesis to the leading strand. one at a time by dehydration synthesis to the leading strand.

54. 4) DNA polymerase III also performs a proofreading function. About 4) DNA polymerase III also performs a proofreading function. About
one out of every 100,000 nucleotides is mismatched with its one out of every 100,000 nucleotides is mismatched with its
template; for instance, a guanine might become incorrectly paired template; for instance, a guanine might become incorrectly paired
with a thymine. with a thymine.
DNA polymerase III recognizes most such errors and removes DNA polymerase III recognizes most such errors and removes
the incorrect nucleotides before proceeding with synthesis. This the incorrect nucleotides before proceeding with synthesis. This
role, known as the role, known as the proofreading exonuclease proofreading exonuclease function, acts like the function, acts like the
delete key on a keyboard, removing the most recent error. delete key on a keyboard, removing the most recent error.
Because of this proofreading exonuclease function, only about Because of this proofreading exonuclease function, only about
one error remains for every ten billion (10one error remains for every ten billion (10
1010
) base pairs replicated. ) base pairs replicated.
5) Another DNA polymerase - DNA polymerase I -
­replaces the RNA
5) Another DNA polymerase - DNA polymerase I -
­replaces the RNA
primer with DNA. Note that researchers named DNA polymerase primer with DNA. Note that researchers named DNA polymerase
enzymes in the order of their discovery, not the order of their enzymes in the order of their discovery, not the order of their
actions. actions.

55. Synthesis of the Lagging StrandSynthesis of the Lagging Strand

The steps in the synthesis of a lagging strand are as follows The steps in the synthesis of a lagging strand are as follows

As with the leading strand, primase synthesizes RNA primers. As with the leading strand, primase synthesizes RNA primers.

Nucleotides pair up with their complements in the template-adenine Nucleotides pair up with their complements in the template-adenine
with thymine, and cytosine with guanine. with thymine, and cytosine with guanine.

DNA polymerase III joins neighboring nucleotides and proofreads. In DNA polymerase III joins neighboring nucleotides and proofreads. In
contrast to synthesis of the leading strand, however, the lagging contrast to synthesis of the leading strand, however, the lagging
strand is synthesized in discontinuous segments called strand is synthesized in discontinuous segments called Okazaki Okazaki
fragmentsfragments. Each Okazaki fragment requires a new RNA primer and . Each Okazaki fragment requires a new RNA primer and
consists of 1000 to 2000 nucleotides. consists of 1000 to 2000 nucleotides.

DNA polymerase I replaces the RNA primers of Okazaki fragments DNA polymerase I replaces the RNA primers of Okazaki fragments
with DNA and further proofreads the daughter strand. with DNA and further proofreads the daughter strand.

DNA ligase DNA ligase seals the gaps between adjacent Okazaki fragments to seals the gaps between adjacent Okazaki fragments to
form a continuous DNA strand. form a continuous DNA strand.

56. TRANSCRIPTIONTRANSCRIPTION

57. TRANSCRIPTIONTRANSCRIPTION

Cells transcribe four main types of RNA from DNACells transcribe four main types of RNA from DNA

RNA primer molecules for DNA polymerase to use during DNA RNA primer molecules for DNA polymerase to use during DNA
replication replication

messenger RNA (mRNA) molecules, which carry genetic messenger RNA (mRNA) molecules, which carry genetic
information from chromosomes to ribosomes information from chromosomes to ribosomes

ribosomal RNA (rRNA) molecules, which combine with ribosomal ribosomal RNA (rRNA) molecules, which combine with ribosomal
polypeptides to form ribosomes-the organelles that synthesize polypeptides to form ribosomes-the organelles that synthesize
polypeptides polypeptides

transfer RNA (tRNA) molecules, which deliver amino acids to the transfer RNA (tRNA) molecules, which deliver amino acids to the
ribosomes ribosomes

58. Initiation of TranscriptionInitiation of Transcription

RNA polymerases - the enzymes that RNA polymerases - the enzymes that
synthesize RNA
­bind to specific
synthesize RNA
­bind to specific
nucleotide sequences called promoters, nucleotide sequences called promoters,
each of which is located near the each of which is located near the
beginning of a gene and initiates beginning of a gene and initiates
transcription. transcription.

59. Initiation of TranscriptionInitiation of Transcription

In bacteria, a polypeptide subunit of RNA polymerase called the In bacteria, a polypeptide subunit of RNA polymerase called the
sigma factor sigma factor is necessary for recognition of a promoter. is necessary for recognition of a promoter.

Once it adheres to a promoter sequence, RNA polymerase unwinds Once it adheres to a promoter sequence, RNA polymerase unwinds
and unzips the DNA molecule in the promoter region and then and unzips the DNA molecule in the promoter region and then
travels along the DNA, unzipping the double helix as it moves. travels along the DNA, unzipping the double helix as it moves.

One type of RNA polymerase transcribes RNA primer, and a second One type of RNA polymerase transcribes RNA primer, and a second
type of RNA polymerase transcribes mRNA, rRNA, and tRNA. type of RNA polymerase transcribes mRNA, rRNA, and tRNA.

A cell uses different sigma factors and different promoter A cell uses different sigma factors and different promoter
sequences to provide some control over the relative amount of sequences to provide some control over the relative amount of
transcription. transcription.

RNA polymerases with different sigma factors do not adhere RNA polymerases with different sigma factors do not adhere
equally strongly to all promoters; there is about a 100-fold equally strongly to all promoters; there is about a 100-fold
difference between the strongest attraction and weakest one. The difference between the strongest attraction and weakest one. The
greater the attraction between a particular sigma factor and a greater the attraction between a particular sigma factor and a
promoter, the more likely that transcription will proceed. promoter, the more likely that transcription will proceed.
Ultimately, variations in sigma factors and promoters affect the Ultimately, variations in sigma factors and promoters affect the
amounts and kinds of polypeptides produced. amounts and kinds of polypeptides produced.

60. Elongation of the RNA TranscriptElongation of the RNA Transcript

61. Elongation of the RNA TranscriptElongation of the RNA Transcript

Like DNA polymerase, RNA polymerase links nucleotides in the 5' Like DNA polymerase, RNA polymerase links nucleotides in the 5'
to 3' direction only; however, RNA polymerase differs from DNA to 3' direction only; however, RNA polymerase differs from DNA
polymerase in the following ways: polymerase in the following ways:

RNA polymerase unwinds and opens DNA by itself; helicase is not RNA polymerase unwinds and opens DNA by itself; helicase is not
required. required.

RNA polymerase does not need a primer. RNA polymerase does not need a primer.

RNA polymerase is slower than DNA polymerase, proceeding at a RNA polymerase is slower than DNA polymerase, proceeding at a
rate of about 50 nucleotides per second. rate of about 50 nucleotides per second.

RNA polymerase incorporates ribonucleotides instead of RNA polymerase incorporates ribonucleotides instead of
deoxyribonucleotides. deoxyribonucleotides.

Uracil nucleotides are incorporated instead of thymine Uracil nucleotides are incorporated instead of thymine
nucleotides. nucleotides.

The proofreading function of RNA polymerase is less efficient, The proofreading function of RNA polymerase is less efficient,
leaving a base pair error about every 10,000 nucleotides. leaving a base pair error about every 10,000 nucleotides.

62. Termination of TranscriptionTermination of Transcription

63. Termination of TranscriptionTermination of Transcription::
Self-TerminationSelf-Termination

Self-termination occurs when RNA polymerase transcribes a terminator Self-termination occurs when RNA polymerase transcribes a terminator
sequence of DNA composed of two symmetrical series:sequence of DNA composed of two symmetrical series:

one that is very rich in guanine and cytosine bases, followed by a region rich in one that is very rich in guanine and cytosine bases, followed by a region rich in
adenine bases. adenine bases.

RNA polymerase slows down during transcription of the GC rich portion of the RNA polymerase slows down during transcription of the GC rich portion of the
terminator because the three hydrogen bonds between each guanine and terminator because the three hydrogen bonds between each guanine and
cytosine base pair make unwinding the DNA helix more difficult. cytosine base pair make unwinding the DNA helix more difficult.

This pause in transcription, which lasts about 60 seconds, provides enough time This pause in transcription, which lasts about 60 seconds, provides enough time
for the RNA molecule to form hydrogen bonds between its own symmetrical for the RNA molecule to form hydrogen bonds between its own symmetrical
sequences, forming a stem and loop structure that puts tension on the union of sequences, forming a stem and loop structure that puts tension on the union of
RNA polymerase and the DNA. RNA polymerase and the DNA.

When RNA polymerase transcribes the adenine-rich portion of the terminator, When RNA polymerase transcribes the adenine-rich portion of the terminator,
the relatively few hydrogen bonds between the adenine bases of DNA and the the relatively few hydrogen bonds between the adenine bases of DNA and the
uracil bases of RNA cannot withstand the tension, and the RNA transcript breaks uracil bases of RNA cannot withstand the tension, and the RNA transcript breaks
away from the DNA, releasing RNA polymerase.away from the DNA, releasing RNA polymerase.

64. Termination of TranscriptionTermination of Transcription::
Rho-Dependent TerminationRho-Dependent Termination

The second type of termination depends on a The second type of termination depends on a
termination protein, called Rho, that binds to termination protein, called Rho, that binds to
a specific RNA sequence near the end of an a specific RNA sequence near the end of an
RNA transcript. RNA transcript.

The protein moves toward the 3' end, pushing The protein moves toward the 3' end, pushing
between RNA polymerase and the DNA strand between RNA polymerase and the DNA strand
and forcing them apart; this releases RNA and forcing them apart; this releases RNA
polymerase and the RNA transcript. polymerase and the RNA transcript.

65. ANTIBOTICSANTIBOTICS

66. IntroductionIntroduction

Definition: Antibiotics are molecules that Definition: Antibiotics are molecules that
kill, or stop the growth of, kill, or stop the growth of,
microorganisms, including both bacteria microorganisms, including both bacteria
and fungi.and fungi.

Antibiotics that kill bacteria are called Antibiotics that kill bacteria are called
"bactericidal""bactericidal"

Antibiotics that stop the growth of Antibiotics that stop the growth of
bacteria are called "bacteriostatic" bacteria are called "bacteriostatic"

67. Introduction contdIntroduction contd

produced by or derived from produced by or derived from
microorganisms (i.e. bugs or germs microorganisms (i.e. bugs or germs
such as bacteria and fungi). such as bacteria and fungi).

The first antibiotic was discovered by The first antibiotic was discovered by
Alexander Fleming in 1928 in a Alexander Fleming in 1928 in a
significant breakthrough for medical significant breakthrough for medical
science. science.

68. Introduction contdIntroduction contd

Antibiotics are one class of Antibiotics are one class of
antimicrobials, a larger group which antimicrobials, a larger group which
also includes anti-viral, anti-fungal, and also includes anti-viral, anti-fungal, and
anti-parasitic drugs. Antibiotics are anti-parasitic drugs. Antibiotics are
chemicalschemicals

69. Antibiotics: Modes of ActionAntibiotics: Modes of Action

• • Inhibitors of DNA synthesisInhibitors of DNA synthesis

• • Inhibitors of bacterial protein Inhibitors of bacterial protein
synthesissynthesis

• • Inhibitors of bacterial cell wall Inhibitors of bacterial cell wall
synthesissynthesis

70. Antibiotic Inhibitors of TranscriptionAntibiotic Inhibitors of Transcription

Rifampicin and actinomycin are two Rifampicin and actinomycin are two
antibiotics that inhibit transcription, antibiotics that inhibit transcription,
although in quite different ways.although in quite different ways.

71. RifampicinRifampicin

specifically inhibits the initiation of RNA specifically inhibits the initiation of RNA
synthesissynthesis. .

does not block the binding of RNA does not block the binding of RNA
polymerase to the DNA template; polymerase to the DNA template;

interferes with the formation of the first interferes with the formation of the first
few phosphodiester bonds in the RNA few phosphodiester bonds in the RNA
chain.chain.

blocks the channel into which the RNA-blocks the channel into which the RNA-
DNA hybrid generated by the DNA hybrid generated by the
Polymerase must passPolymerase must pass

72. Rifampicin contdRifampicin contd

Rifampicin does not hinder chain Rifampicin does not hinder chain
elongation once initiated, because the elongation once initiated, because the
RNA-DNA hybrid present in the enzyme RNA-DNA hybrid present in the enzyme
prevents the antibiotic from binding.prevents the antibiotic from binding.

73. ActinomycinActinomycin

inhibits transcription by an entirely inhibits transcription by an entirely
different mechanism.different mechanism.

Actinomycin D binds tightly and Actinomycin D binds tightly and
specifically to double-helical DNA and specifically to double-helical DNA and
thereby prevents it from being an thereby prevents it from being an
effective template for RNA synthesiseffective template for RNA synthesis

It does not bind to single-stranded DNA It does not bind to single-stranded DNA
or RNA, double-stranded RNA, or RNA-or RNA, double-stranded RNA, or RNA-
DNA hybridsDNA hybrids

74. Actinomycin contdActinomycin contd

the phenoxazone ring of actinomycin the phenoxazone ring of actinomycin
slips in between neighboring base pairs slips in between neighboring base pairs
in DNA. This mode of binding is called in DNA. This mode of binding is called
intercalationintercalation..

75. Eukaryotic TranscriptionEukaryotic Transcription

76. Main difference between Main difference between
Eukaryotic and prokaryotic Eukaryotic and prokaryotic
transcriptiontranscription

Five different enzymes catalyze Five different enzymes catalyze
transcription in eukaryotes, and transcription in eukaryotes, and

the resulting RNA transcripts undergo the resulting RNA transcripts undergo
three important modifications,three important modifications,

Transcription and translation are Transcription and translation are
separted in time and spaceseparted in time and space

77. Five different enzymes

78. Eukaryotic Transcription and Eukaryotic Transcription and
Translation Are Separated in Space Translation Are Separated in Space
and Timeand Time

transcription in eukaryotes, a much transcription in eukaryotes, a much
more complex process than in more complex process than in
prokaryotesprokaryotes

transcription and translation take place transcription and translation take place
in different cellular compartments: in different cellular compartments:
transcription takes place in the transcription takes place in the
nucleus, whereas translation takes place nucleus, whereas translation takes place
in the cytoplasmin the cytoplasm..

79. Eukaryotic Transcription and Eukaryotic Transcription and
Translation Are Separated in Space Translation Are Separated in Space
and Timeand Time

The spatial and temporal separation of The spatial and temporal separation of
transcription and translation enables transcription and translation enables
eukaryotes to regulate gene expression eukaryotes to regulate gene expression
in much more intricate ways, in much more intricate ways,
contributing to the richness of contributing to the richness of
eukaryotic form and function.eukaryotic form and function.

80. Post transcription processing Post transcription processing
in eukaryotesin eukaryotes

A second major difference between A second major difference between
prokaryotes and eukaryotes is the prokaryotes and eukaryotes is the
extent of RNA processing. extent of RNA processing.

Although both prokaryotes and Although both prokaryotes and
eukaryotes modify tRNA and rRNA, eukaryotes modify tRNA and rRNA,
eukaryotes very extensively process eukaryotes very extensively process
nascent RNA destined to become mRNAnascent RNA destined to become mRNA. .

81. Post transcription processing Post transcription processing
in eukaryotesin eukaryotes

Primary transcripts (pre-mRNA molecules), Primary transcripts (pre-mRNA molecules),
the products of RNA polymerase action, the products of RNA polymerase action,
(1)acquire a cap at their 5’ ends and a (1)acquire a cap at their 5’ ends and a
(2) poly(A) tail at their 3’ ends. (2) poly(A) tail at their 3’ ends.
(3) Most importantly, (3) Most importantly, nearly all mRNA precursors nearly all mRNA precursors
in higher eukaryotes are splicedin higher eukaryotes are spliced

Introns are excised from primary Introns are excised from primary
transcripts, and exons are joined to form transcripts, and exons are joined to form
mature mRNAs with continuous messages. mature mRNAs with continuous messages.

82. Post transcription processing Post transcription processing
in eukaryotesin eukaryotes

Some mRNAs are only a tenth the size Some mRNAs are only a tenth the size
of their precursors, which can be as of their precursors, which can be as
large as 30 kb or more.large as 30 kb or more.

83. 5’ capping5’ capping
7-Methyl guanosine caps are added to
the 5 ends of the primary transcripts

The 5’ cap on most eukaryotic mRNAs is The 5’ cap on most eukaryotic mRNAs is
a 7-methyl guanosine residue joined to a 7-methyl guanosine residue joined to
thethe
initial nucleoside of the transcript by a initial nucleoside of the transcript by a
5-5 phosphate linkage.5-5 phosphate linkage.

84. TRANSLATIONTRANSLATION

85. TRANSLATION
Translation is the process whereby ribosomes
use the genetic information of nucleotide
sequences to synthesize polypeptides
composed of specific amino acid sequences.
process by which the genetic information stored in the sequence of
nucleotides in an mRNA is translated, according to the
specifications of the genetic code, into the sequence of amino acids
in the polypeptide gene product

86. How do ribosomes interpret the nucleotide
sequence of mRNA to determine the
correct order in which to assemble amino
acids?

87. The Genetic Code
Genes are composed of sequences of
three nucleotides that specify amino acids.
For example, the DNA nucleotide sequence
TTT specifies the amino acid lysine, and
TTA codes for asparagine.

89. 5’ end 5’ end Middle base Middle base 3’ end3’ end
UUCCAAGG
UU phephesersertyrtyrcyscys UU
phephesersertyrtyrcyscys CC
leuleuserserendendendend AA
leuleuserserendendtrptrp GG
CC leuleuproprohishisargarg UU
leuleuproprohishisargarg CC
leuleuproproglnglnargarg AA
leuleuproproglnglnargarg GG

90. Code QuizCode Quiz
Click for the answer.Click for the answer.
1. CCU codes for: ?1. CCU codes for: ?
2. CGA codes for: ?2. CGA codes for: ?
3. UCA codes for: ?3. UCA codes for: ?
1. pro
2. arg
3. ser
5’5’UUCCAAGG3’3’
UU
PhePheSerSerTyrTyrCysCys
UU
PhePheSerSerTyrTyrCysCys
CC
LeuLeuSerSerEndEndEndEnd
AA
LeuLeuSerSerEndEndTrpTrp
GG
CC
LeuLeuProProHisHisArgArg
UU
LeuLeuProProHisHisArgArg
CC
LeuLeuProProGlnGlnArgArg
AA
LeuLeuProProGlnGlnArgArg
GG
AA
IleIleThrThrAsnAsnSerSer
UU

91. Characteristics of the Genetic Characteristics of the Genetic
CodeCode
1. 1. is composed of nucleotide tripletsis composed of nucleotide triplets. Three . Three
nucleotides in mRNA specify one amino nucleotides in mRNA specify one amino
acid in the polypeptide product; thus, each acid in the polypeptide product; thus, each
codon contains three nucleotides.codon contains three nucleotides.
2. 2. is nonoverlappingis nonoverlapping. Each nucleotide in . Each nucleotide in
mRNA belongs to just one codon except in mRNA belongs to just one codon except in
rare cases where genes overlap and a rare cases where genes overlap and a
nucleotide sequence is read in two nucleotide sequence is read in two
different reading frames.different reading frames.

92. The Genetic Code contdThe Genetic Code contd

3. 3. is comma-freeis comma-free. There are no commas . There are no commas
or other forms of punctuation within or other forms of punctuation within
the coding regions of mRNA molecules. the coding regions of mRNA molecules.
During translation, the codons are read During translation, the codons are read
consecutively.consecutively.

4. 4. is degenerateis degenerate. All but two of the amino . All but two of the amino
acids are specified by more than one acids are specified by more than one
codon.codon.

93. The Genetic Code contdThe Genetic Code contd

5. 5. is orderedis ordered. Multiple codons for a given . Multiple codons for a given
amino acid and codons for amino acids with amino acid and codons for amino acids with
similar chemical properties are closely related, similar chemical properties are closely related,
usually differing by a single nucleotide.usually differing by a single nucleotide.

6. 6. contains start and stop codonscontains start and stop codons. Specific . Specific
codons are used to initiate and terminate codons are used to initiate and terminate
polypeptide chains.polypeptide chains.

7. 7. is nearly universalis nearly universal. With minor exceptions, . With minor exceptions,
the codons have the same meaning in all the codons have the same meaning in all
living organisms, from viruses to humans.living organisms, from viruses to humans.

94. Participants in Translation:
Messenger RNA
Messenger RNA carries genetic information (in the form of RNA nucleotide
sequences) from a chromosome to ribosomes.
In prokaryotes a basic mRNA molecule contains sequences of nucleotides
that are recognized by ribosomes:
an AUG start codon, sequential codons for other amino acids in the
polypeptide, and at least one of the three stop codons. A single molecule of
prokaryotic mRNA often contains a start codon and instructions for more
than one polypeptide arranged in series.
Because both transcription and the subsequent events of translation occur
in the cytosol of prokaryotes, prokaryotic ribosomes can begin translation
before transcription is finished.

96. Participants in Translation:
Transfer RNA
tRNA molecule is a
sequence of about 75
ribonucleotides that curves
back on itself to form three
main hairpin loops (a)
For simplicity, tRNA will be
represented in subsequent
figures by an icon shaped
like the illustration (b)

97. Transfer RNA contd
A molecule of tRNA transfers the correct amino acid to a
ribosome during polypeptide synthesis. To this end, tRNA has
an acceptor stem for a specific amino acid at its 3' end, and
an anticodon triplet in its bottom loop.
The existence of only one specific charging enzyme for each
amino acid ensures that every tRNA molecule carries only
one specific amino acid.
Anticodons are complementary to mRNA codons, and each
acceptor stem is designed to carry one particular amino acid,
which varies with the tRNA.

98. Transfer RNA contd
amino acids are attached to the tRNAs by
high-energy bonds between the carboxyl
groups of the amino acids and the 3-
hydroxyl termini of the tRNAs.
The tRNAs are activated or charged with
amino acids in a two-step process,
catalyzed by the same enzyme, aminoacyl-
tRNA synthetase.

99. Transfer RNA contd
tRNAs are transcribed from genes.
tRNAs are transcribed in the form of larger
precursor molecules that undergo
posttranscriptional processing (cleavage,
trimming, methylation, and so forth).
mature tRNA molecules contain several
nucleosides that are not present in the
primary tRNA gene transcripts.

100. Participants in Translation:
Ribosomes
about half protein and half RNA
composed of two subunits, one large and
one small,

dissociate when the translation of an mRNA
molecule is completed
Re-associate during the initiation of translation.

101. Ribosomes contd
Each subunit contains a large, folded RNA
molecule on which the ribosomal proteins
assemble.
Ribosome sizes are most frequently
expressed in terms of their rates of
sedimentation during centrifugation, in
Svedberg (S) units.

102. Ribosomes contd
The ribosomal RNA molecules, like mRNA
molecules, are transcribed from a DNA
template.
In eukaryotes, rRNA synthesis occurs in the
nucleolus and is catalyzed by RNA
polymerase I.

103. Ribosomes contd
The transcription of the rRNA genes
produces RNA precursors that are
much larger than the RNA molecules
found in ribosomes.
These rRNA precursors undergo
posttranscriptional processing to
produce the mature rRNA molecules.

104. Ribosomes contd
In E. coli, the rRNA gene transcript is a 30S
precursor, which undergoes endonucleolytic
cleavages to produce the 5S, 16S, and 23S
rRNAs plus one 4S transfer RNA molecule
In mammals, the 5.8S, 18S, and 28S rRNAs
are cleaved from a 45S precursor whereas
the 5S rRNA is produced by
posttranscriptional processing of a separate
gene transcript.

105. Ribosomes and ribosomal RNA

106. Prokaryotic ribosomes, also called 70S
ribosomes based on their sedimentation rate in
an ultracentrifuge, are extremely complex
associations of ribosomal RNAs and
polypeptides. Each ribosome is composed of
two subunits: 50S and 30S.
The 50S subunit is in turn composed of two
rRNA molecules (23S and 5S) and about 34
different polypeptides, whereas the 30S subunit
consists of one molecule of 165 rRNA and 21
ribosomal polypeptides. The ribosomes of
mitochondria and chloroplasts are also 70S
ribosomes composed of the similar subunits and
polypeptides.

107. Eukaryotic ribosomes
In contrast, both the cytosol and the rough endoplasmic
reticulum (RER) of eukaryotic cells have 80S ribosomes
composed of 60S and 40S subunits.
These subunits contain larger molecules of rRNA and
more polypeptides than the corresponding prokaryotic
subunits, though researchers do not agree on the exact
number of polypeptides.
The term eukaryotic ribosome is understood to mean only the 80S
ribosomes of the cytosol and RER. Since the ribosomes of
mitochondria and chloroplasts are 70S, they are called prokaryotic
ribosomes even though they are in eukaryotic cells.

108. rRNA continued
Each ribosome also has three tRNA binding
sites that are named for their function:
1) The A site accommodates a tRNA
delivering an amino acid.
2) The P site holds a tRNA and the
growing
polypeptide.
3) Discharged tRNAs exit from the E site.

109. Stages of Translation
Initiation: the events
1) The smaller ribosomal
subunit attaches to
mRNA at a ribosome
binding site (also
known as a Shine-
Dalganno sequence
after its discoverers),
with a start codon at
its P site.

110. Initiation contd
2) tRNA £Met (whose
anticodon is
complementary to
the start codon)
attaches at the
ribosome's P site;
GTP supplies the
energy required for
binding.

111. Initiation contd
3) The larger
ribosomal subunit
attaches to form
a complete
initiation complex.

112. Stages of Translation:
Elongation
1) The transfer RNA whose
anticodon matches the next
codon - in this case,
phenylalanine (Phe) - delivers
its amino acid to the A site.
Another protein called
elongation factor escorts the
tRNA along with a molecule of
GTP. Energy from GTP is
used to stabilize each tRNA
as it is added to the A site.

113. 2) A ribozyme in the larger
ribosomal subunit forms a
peptide bond by dehydration
synthesis between the terminal
amino acid of the growing
polypeptide chain (in this case,
N-formylmethionine) and the
newly intro
­duced amino acid.
The polypeptide is now
attached to the tRNA
occupying the A site.
3) Using energy supplied by more
GTP, the ribosome moves one
codon down the mRNA. This
transfers each tRNA to the
adjacent binding site; that is,
the first tRNA moves from the
P site to the E site, and the sec
­
ond tRNA (with the attached
polypeptide) moves to vacated
P site.

114. 4) The ribosome releases
the “empty" tRNA from
the E site. In the cytosol,
the appropriate enzyme
recharges it with
another molecule of its
specific amino acid.
5) The cycle repeats, each
time adding another
amino acid (in this case,
threonine, then alanine,
and then glutamine).

115. Stages of Translation:
Termination
Termination does not involve tRNA; instead, proteins called
release factors halt elongation.
It appears that release factors somehow recognize stop
codons and modify the larger ribosomal subunit in such a
way as to activate another of its ribozymes, which severs the
polypeptide from the final tRNA (resident at the P site). The
ribosome then dissociates into its subunits.
Termination of translation should not be confused with
termination of transcription. The polypeptides released at
termination may function alone as proteins, or they may
function together in quarternary protein structures.

116. Eukaryotic and Prokaryotic
Protein Synthesis Differs
1. Ribosomes.
Eukaryotic ribosomes are larger - consist of a 60S
large subunit and a 40S small subunit, which form
an 80S particle having a mass of 4200 kd, compared
with 2700 kd for the prokaryotic 70S ribosome.
2. Initiator tRNA.
In eukaryotes, the initiating amino acid is methionine
rather than N-formylmethionine. in vitro).

117. 3.
Initiation

Eukaryotes, in contrast with prokaryotes, do not
use a specific purine-rich sequence on the 5'
side to distinguish initiator AUGs from internal
ones.
Instead, the AUG nearest the 5' end of mRNA is
usually selected as the start site. A 40S ribosome
attaches to the cap at the 5 end of eukaryotic
mRNA and searches for an AUG codon by
moving step-by-step in the 3 direction .

118. Initiation
In almost all cases, eukaryotic mRNA
has only one start site and hence is
the template for a single protein. In
contrast, a prokaryotic mRNA can
have multiple Shine-Dalgarno
sequences and, hence, start sites,
and it can serve as a template for the
synthesis of several proteins.

119. Elongation and termination

4. Elongation and termination.

Eukaryotic elongation factors EF1a and EF1b g are
the counterparts of prokaryotic EFTu and EF-Ts.
The GTP form of EF1a delivers aminoacyl-
tRNA to the A site of the ribosome, and
EF1b g catalyzes the exchange of GTP for
bound GDP. Eukaryotic EF2 mediates GTP-
driven translocation in much the same way
as does prokaryotic EF-G.

120. Elongation and termination.
Termination in eukaryotes is carried out by
a single release factor, eRF1, compared
with two in prokaryotes.
Finally, eIF3, like its prokaryotic
counterpart IF3, prevents the re-
association of ribosomal subunits in the
absence of an initiation complex.

121. Antibiotics THAT
INHIBIT TRANSLATION

122. Antibiotics that inhibit
translation
Puromycin
 inhibits protein synthesis by causing nascent
prokaryotic polypeptide chains to be released
before their synthesis is completed.

It is an analog of the terminal
aminoacyladenosine part of aminoacyl-tRNA
It binds to the A site on the ribosome and
inhibits the entry of aminoacyl-tRNA.

123. Antibiotics that inhibit
translation
Streptomycin and other aminoglycoside
antibiotics
 interferes with the binding of formylmethionyl-
tRNA to ribosomes thereby preventing the
correct initiation of protein synthesis.
neomycin, kanamycin, and gentamycin interfere
with the decoding site located near nucleotide
1492 in 16S rRNA of the 30S subunit

124. Antibiotics that inhibit
translation
Chloramphenicol acts by inhibiting peptidyl
transferase activity. Erythromycin binds to
the 50S subunit and blocks translocation.

126. Regulation of Genetic Regulation of Genetic
ExpressionExpression
About 75% of genes are expressed at all timesAbout 75% of genes are expressed at all times; that is, they are ; that is, they are
constantly transcribed and translated and play a persistent role in constantly transcribed and translated and play a persistent role in
the phenotype. the phenotype.
These genes code for RNAs and polypeptides that are needed in These genes code for RNAs and polypeptides that are needed in
large amounts by the cell
­ for example, integral proteins of the
large amounts by the cell
­ for example, integral proteins of the
cytoplasmic membrane, structural proteins of ribosomes, and cytoplasmic membrane, structural proteins of ribosomes, and
enzymes of glycolysis. enzymes of glycolysis.
Other genes are regulatedOther genes are regulated so that the polypeptides they encode are so that the polypeptides they encode are
synthesized only when a cell has need of them. Protein synthesis synthesized only when a cell has need of them. Protein synthesis
requires a large amount of energy, which can be conserved if a cell requires a large amount of energy, which can be conserved if a cell
forgoes production of unneeded polypeptides. forgoes production of unneeded polypeptides.
Cells regulate protein synthesis in many waysCells regulate protein synthesis in many ways. They may stop . They may stop
translation directly or stop polypeptide synthesis by stopping mRNA translation directly or stop polypeptide synthesis by stopping mRNA
transcription. transcription.

127. Control of TranslationControl of Translation
Some regulation of genetic expression is at the level of Some regulation of genetic expression is at the level of
translationtranslation; that is, cells control which mRNA molecules ; that is, cells control which mRNA molecules
are translated into polypeptides. are translated into polypeptides.
One way a cell establishes control involves so-called One way a cell establishes control involves so-called
riboswitchesriboswitches. .
–A riboswitch is a molecule of mRNA that changes its shape in A riboswitch is a molecule of mRNA that changes its shape in
response to an alteration in temperature or a shift in the response to an alteration in temperature or a shift in the
concentration of a nutrient, such as a vitamin, nucleotide base, concentration of a nutrient, such as a vitamin, nucleotide base,
or amino acid. or amino acid.
–Riboswitches fold in such a way as to block ribosomes and Riboswitches fold in such a way as to block ribosomes and
translation of the polypeptide they encode when that polypeptide translation of the polypeptide they encode when that polypeptide
is not needed by the cell. is not needed by the cell.

128. Control of TranslationControl of Translation
Another method of translational control Another method of translational control
involves short interference RNA involves short interference RNA (siRNA), (siRNA),
which is an RNA molecule complementary which is an RNA molecule complementary
to a portion of mRNA, tRNA, or a gene. to a portion of mRNA, tRNA, or a gene.
–Such RNA molecules are also called Such RNA molecules are also called
antisense RNA. antisense RNA. siRNA binds to its siRNA binds to its
complementary nucleic acid, rendering its complementary nucleic acid, rendering its
target inactive. target inactive.

129. OPERONOPERON
An operon consists of a promoter and a series of genes, which code for An operon consists of a promoter and a series of genes, which code for
enzymes and structures such as channel proteinsenzymes and structures such as channel proteins..
Some operons are controlled by an adjacent regulatory element called an Some operons are controlled by an adjacent regulatory element called an
operator where a operator where a repressor protein repressor protein binds to stop transcriptionbinds to stop transcription. .
Such operons are either repressed (turned off) or induced (turned on) by Such operons are either repressed (turned off) or induced (turned on) by
proteins coded by a regulatory gene (located elsewhere). proteins coded by a regulatory gene (located elsewhere).
Inducible operons are not usually transcribed and must be activated by Inducible operons are not usually transcribed and must be activated by
inducers. inducers.
Repressible operons operate in reverse fashion-they are transcribed Repressible operons operate in reverse fashion-they are transcribed
continually until deactivated by continually until deactivated by repressors.repressors.

130. The Lactose Operon, an Inducible OperonThe Lactose Operon, an Inducible Operon
The lactose (lac) operon is inducible operon. It includes a The lactose (lac) operon is inducible operon. It includes a
promoter, an operator, and three genes that encode for promoter, an operator, and three genes that encode for
protein involved in the catabolism of lactose. protein involved in the catabolism of lactose.
The operon is controlled by a regulatory gene that is The operon is controlled by a regulatory gene that is
constantly transcribed and translated to produce a constantly transcribed and translated to produce a
repressor protein that attaches to DNA at the repressor protein that attaches to DNA at the lac lac operator. operator.
This repressor prevents RNA polymerase from moving be
­
This repressor prevents RNA polymerase from moving be
­
yond the promoter, stopping synthesis of mRNA. Thus, the yond the promoter, stopping synthesis of mRNA. Thus, the
lac lac operon is usually inactive. operon is usually inactive.

131. Whenever lactose becomes available, the cell takes in lactose and converts Whenever lactose becomes available, the cell takes in lactose and converts
it to allolactose - an inducer that changes the quaternary structure of the it to allolactose - an inducer that changes the quaternary structure of the
repressor so that it is inactivated and can no longer attach to DNArepressor so that it is inactivated and can no longer attach to DNA. .
This absence of binding al
­lows transcription of the three structural genes to
This absence of binding al
­lows transcription of the three structural genes to
proceed
­the operon has been induced and has become active. Ribosomes
proceed
­the operon has been induced and has become active. Ribosomes
translate the newly synthesized mRNA to produce enzymes that catabolize translate the newly synthesized mRNA to produce enzymes that catabolize
lactose. lactose.
Once the lactose supply has been depleted, there is no more inducer, and Once the lactose supply has been depleted, there is no more inducer, and
the repressor once again becomes active, suppressing transcrip
­tion and
the repressor once again becomes active, suppressing transcrip
­tion and
translation of the translation of the lac lac operon. In this manner, its conserve energy by operon. In this manner, its conserve energy by
synthesizing enzymes for the ca
­tabolism of lactose only when lactose is
synthesizing enzymes for the ca
­tabolism of lactose only when lactose is
available to them. available to them.
Such inducible operons are often involved in controlling catabolic pathways Such inducible operons are often involved in controlling catabolic pathways
whose polypeptides are not needed whose polypeptides are not needed

132. The Tryptophan Operon, a The Tryptophan Operon, a
Repressible Operon Repressible Operon

133. The The tryptophan (trp) operon,tryptophan (trp) operon, which consists of a promoter, an which consists of a promoter, an
operator, and five genes that code for the enzymes involved in the operator, and five genes that code for the enzymes involved in the
synthesis of tryptophan, is an example of such a repressible operon.synthesis of tryptophan, is an example of such a repressible operon.

Just as with the Just as with the laclac operon a regulatory gene codes for a repressor operon a regulatory gene codes for a repressor
molecule that is constantly synthesized. In contrast to inducible molecule that is constantly synthesized. In contrast to inducible
operons however, the repressor of repressible operons is normally operons however, the repressor of repressible operons is normally
inactive.inactive.
In the case of the repressible In the case of the repressible trptrp operon whenever tryptophan is not operon whenever tryptophan is not
present in the environment, the present in the environment, the trptrp operon inactive: operon inactive:
The appropriate mRNA is transcribed the enzymes for tryptophan The appropriate mRNA is transcribed the enzymes for tryptophan
synthesis are translated, and tryptophan is producedsynthesis are translated, and tryptophan is produced (Figure a). (Figure a).
When tryptophan is available, it activates the repressor by binding to When tryptophan is available, it activates the repressor by binding to
it. The activated repressor then binds to the operator, halting the it. The activated repressor then binds to the operator, halting the
movement of RNA polymerase and halting transcriptionmovement of RNA polymerase and halting transcription (Figure b). (Figure b).
In other words, tryptophan acts as a In other words, tryptophan acts as a corepressorcorepressor of its own of its own
synthesis.synthesis.

134. The roles of operons in the The roles of operons in the
regulation of transcriptionregulation of transcription
Type of Type of
regulationregulation
Type of metabolic Type of metabolic
pathway regulatedpathway regulated
Regulating Regulating
conditioncondition
Inducible Inducible
operonsoperons
Catabolic pathwaysCatabolic pathwaysPresence of Presence of
substrate of substrate of
pathwaypathway
Repressible Repressible
operonsoperons
Anabolic pathwaysAnabolic pathwaysPresence of Presence of
product of product of
pathwaypathway

135. MUTATIONSMUTATIONS

136. What is a mutation?What is a mutation?

The term The term mutationmutation refers to both: refers to both:

the change in the genetic material and the change in the genetic material and

the process by which the change occurs. the process by which the change occurs.

An organism that exhibits a novel An organism that exhibits a novel
phenotype resulting from a mutation is phenotype resulting from a mutation is
called a called a mutantmutant

137. Chromosomal vs point Chromosomal vs point
mutationsmutations

Mutational changes in the Mutational changes in the genotypegenotype of an of an
organism include changes in chromosome organism include changes in chromosome
number and structure as well as changes in number and structure as well as changes in
the structures of individual genes.the structures of individual genes.

Mutations that involve changes at specific sites Mutations that involve changes at specific sites
in a gene are referred to as in a gene are referred to as point mutationspoint mutations..

They include the substitution of one base They include the substitution of one base
pair for another or the insertion or deletion pair for another or the insertion or deletion
of one or a few nucleotide pairs at a specific of one or a few nucleotide pairs at a specific
site in a gene. site in a gene.

138. Point mutationsPoint mutations

the term mutation sometimes is used in the term mutation sometimes is used in
a narrow sense to refer only to a narrow sense to refer only to changes changes
in the structures of individual genesin the structures of individual genes..

we explore the process of mutation as we explore the process of mutation as
defined in the narrow sense.defined in the narrow sense.

139. Point mutationsPoint mutations

Mutation is the Mutation is the ultimate source of all genetic ultimate source of all genetic
variationvariation;;

provides new genetic variability and allow provides new genetic variability and allow
organisms to adapt to new environmentsorganisms to adapt to new environments

provides the raw material for micro-evolution. provides the raw material for micro-evolution.

140. Spontaneous MutationsSpontaneous Mutations
structures of the bases in DNA are not static
H atoms can move from one position in a purine or
pyrimidine to another position - e.g from an amino group to
a ring nitrogen
Such chemical fluctuations are called tautomeric shifts.
tautomeric shifts are rare but important because some alter
the pairing potential of the bases.

The more stable The more stable keto forms keto forms of thymine and guanine and the of thymine and guanine and the
amino forms amino forms of adenine and cytosine may undergo tautomeric of adenine and cytosine may undergo tautomeric
shifts to less stable shifts to less stable enol and imino formsenol and imino forms, respectively, respectively


141. Tautomeric forms of the four common
bases in DNA. The shifts of hydrogen
atoms between the number 3 and
number 4 positions of the pyrimidines
and between the number 1 and number
6 positions of the purines change their
base-pairing potential.

142. Spontaneous MutationsSpontaneous Mutations

When the bases are present in their rare enol or When the bases are present in their rare enol or
imino states, they can form imino states, they can form adenine-cytosineadenine-cytosine and and
guanine-thymineguanine-thymine base pairs base pairs

143. A guanine (1) undergoes a tautomeric shift to its rare enol form (G*) at
the time of replication (2). In its enol form, guanine pairs with thymine
(2). During the subsequent replication (3 to 4), the guanine shifts back to
its more stable keto form. The thymine incorporated opposite the enol
form of guanine (2) directs the incorporation of adenine during the next
replication (3 to 4). The net result is a G:C to A:T base-pair substitution.

144. ::
Types of Point Mutation Types of Point Mutation
Mutations range from large changes in an organism's
genome, such as the loss or gain of an entire
chromosome, to the most common type of mutation
- point mutations - in which just one nucleotide base
pair is affected.
Mutations include base pair insertions, deletions, and
substitutions.
Substitution of a nucleotide of similar shape - a
purine for a purine or pyrimidine for a pyrimidine-is
called a transition.
Substitution of a purine for a pyrimidine or vice versa
is called a transversion.

145. The following analogy illustrates some types of mutations. The following analogy illustrates some types of mutations.

Suppose that the DNA code was represented by the letters Suppose that the DNA code was represented by the letters
THECATATEELKTHECATATEELK. .

Grouping the letters into triplets (like codons) yields Grouping the letters into triplets (like codons) yields
THE CAT ATE ELK.THE CAT ATE ELK.

The The substitution of a single lettersubstitution of a single letter could either change the meaning of the sentence, could either change the meaning of the sentence,
as in as in THE RAT ATE ELKTHE RAT ATE ELK, or result in a meaningless phrase, such as , or result in a meaningless phrase, such as THE CAT RTE ELKTHE CAT RTE ELK. .

Insertion or deletion of a letter produces more serious changes, such as Insertion or deletion of a letter produces more serious changes, such as
TRH ECA TAT EEL KTRH ECA TAT EEL K or or TEC ATA TEE LKTEC ATA TEE LK. .

Insertions and deletions are also called Insertions and deletions are also called frame shift mutationsframe shift mutations because nucleotide because nucleotide
triplets subsequent to the mutation are displaced, creating new sequences of codons triplets subsequent to the mutation are displaced, creating new sequences of codons
that result in vastly altered polypeptide sequences. that result in vastly altered polypeptide sequences.

Frameshift mutations affect proteins much more seriously than mere substitutions Frameshift mutations affect proteins much more seriously than mere substitutions
because a frame shift affects all co dons subsequent to the mutation. because a frame shift affects all co dons subsequent to the mutation.

Mutations can also involve inversion Mutations can also involve inversion (THE ACT ATE KLE),(THE ACT ATE KLE),
duplication duplication (THE CAT CAT ATE ELK ELK),(THE CAT CAT ATE ELK ELK), or or
transposition transposition (THE ELK ATE CAT).(THE ELK ATE CAT).

Such mutations and even larger deletions and insertions are Such mutations and even larger deletions and insertions are gross mutationsgross mutations..

146. Effects of MutationsEffects of Mutations

Some base-pair substitutions Some base-pair substitutions
produce produce silent mutations silent mutations
because the substitution does because the substitution does
not change the amino acid not change the amino acid
sequence because of the sequence because of the
redundancy of the genetic redundancy of the genetic
code.code.

For example, when the DNA For example, when the DNA
triplet AAA " is changed to triplet AAA " is changed to
AAG, the mRNA codon will be AAG, the mRNA codon will be
changed from UUU to UUC; changed from UUU to UUC;
however, because both codons however, because both codons
specify the amino acid specify the amino acid
phenylalanine, there is no phenylalanine, there is no
change in the phenotype - the change in the phenotype - the
mutation is silent because it mutation is silent because it
affects the genotype only. affects the genotype only.

147. Effects of MutationsEffects of Mutations CONTD CONTD

Of greater concern are substitutions that Of greater concern are substitutions that
change a codon for one amino acid into a change a codon for one amino acid into a
codon for a different amino acid. codon for a different amino acid.

A change in a nucleotide sequence A change in a nucleotide sequence
resulting in a codon that specifies a resulting in a codon that specifies a
different amino acid is called a different amino acid is called a missense
mutation; what gets transcribed and ; what gets transcribed and
translated makes sense, but not the right translated makes sense, but not the right
sense. sense.

The effect of missense mutations depends The effect of missense mutations depends
on where in the protein the different on where in the protein the different
amino acid occurs. amino acid occurs.

When the different amino is in a critical When the different amino is in a critical
region of a protein, the protein becomes region of a protein, the protein becomes
nonfunctional; however, when the nonfunctional; however, when the
different amino acid is in a less important different amino acid is in a less important
region, the mutation has no adverse region, the mutation has no adverse
effect. effect.

148. Effects of MutationsEffects of Mutations
A third type of A third type of
mutation occurs when mutation occurs when
a base-pair a base-pair
substitution changes substitution changes
an amino acid codon an amino acid codon
into a stop codon. into a stop codon.
This is called a This is called a
nonsense mutation. .
Nearly all nonsense Nearly all nonsense
mutations result in mutations result in
nonfunctional proteins. nonfunctional proteins.

149. Frameshift
mutations (that is, (that is,
insertions or insertions or
deletions) typically deletions) typically
result in drastic result in drastic
missense and missense and
nonsense mutations, nonsense mutations,
except when the except when the
insertion or deletion insertion or deletion
is very close to the is very close to the
end of a gene end of a gene

150. MutagensMutagens

Mutations occur naturally during the life of an Mutations occur naturally during the life of an
organism. Such organism. Such spontaneous mutations spontaneous mutations result from result from
errors in replication and repair as well as from errors in replication and repair as well as from
recombination recombination in which relatively long stretches of in which relatively long stretches of
DNA move among chromosomes, plasmids, and DNA move among chromosomes, plasmids, and
viruses, introducing frame shift mutations. viruses, introducing frame shift mutations.

Further, though cells have repair mechanisms to Further, though cells have repair mechanisms to
reduce the effect of mutations, the repair process reduce the effect of mutations, the repair process
itself can introduce additional errors. itself can introduce additional errors.

Physical or chemical agents called Physical or chemical agents called mutagens, which , which
include radiation and several types of DNA-altering include radiation and several types of DNA-altering
chemicals, induce mutations. chemicals, induce mutations.

151. Radiation Radiation

Ionizing radiationIonizing radiation, such as , such as X raysX rays and and gamma raysgamma rays, can cause , can cause
some of the molecules within cells to lose electrons, becoming some of the molecules within cells to lose electrons, becoming
highly reactive ions and free radicals. highly reactive ions and free radicals.

Some of these reactive ions and free radicals can combine with Some of these reactive ions and free radicals can combine with
bases on DNA, resulting in errors in DNA replication and therefore bases on DNA, resulting in errors in DNA replication and therefore
mutations. Even more seriously, these groups can react with the mutations. Even more seriously, these groups can react with the
sugar-phosphate backbone of DNA, causing breaks in sugar-phosphate backbone of DNA, causing breaks in
chromosomes.chromosomes.

Nonionizing radiation in the form of Nonionizing radiation in the form of UV light UV light is also mutagenic is also mutagenic
because it can cause adjacent thymine bases to covalently bind to because it can cause adjacent thymine bases to covalently bind to
another, producing thymine dimmers. another, producing thymine dimmers.

Such dimmers can cause serious harm or death to a cell if they Such dimmers can cause serious harm or death to a cell if they
are not repaired, since these dimmers prevent the cell from are not repaired, since these dimmers prevent the cell from
properly transcribing or replicating such DNA.properly transcribing or replicating such DNA.

152. Chemical Mutagens : :
Nucleotide Analogs Nucleotide Analogs

nucleotide analogs are compounds that nucleotide analogs are compounds that
are structurally similar to normal are structurally similar to normal
nitrogenous bases, but with different nitrogenous bases, but with different
base-pairing properties. base-pairing properties.

These compounds can become These compounds can become
incorporated into growing DNA during incorporated into growing DNA during
replication, replacing their related base. replication, replacing their related base.

Once incorporated, the nucleoside Once incorporated, the nucleoside
analog can inhibit further replication, or analog can inhibit further replication, or
cause mismatching in a future round of cause mismatching in a future round of
replication. replication.

For example, 5-bromouracil is a For example, 5-bromouracil is a
nucleoside analog of thymine, but it nucleoside analog of thymine, but it
often pairs with guanine rather than often pairs with guanine rather than
adenine. Incorporation of 5-bromouracil adenine. Incorporation of 5-bromouracil
can therefore lead to a base substitution can therefore lead to a base substitution
mutation of a guanine for an adenine.mutation of a guanine for an adenine.

153. Chemical Mutagens :
Nucleotide-Altering Chemicals Nucleotide-Altering Chemicals

some chemical mutagens directly alter the some chemical mutagens directly alter the
structures of the nitrogenous bases of structures of the nitrogenous bases of
DNA. DNA.

For example, nitrous acid can chemically For example, nitrous acid can chemically
alter adenine bases so that they base pair alter adenine bases so that they base pair
with cytosine, rather than thymine. with cytosine, rather than thymine.

During replication, this change causes base During replication, this change causes base
substitution mutations in the daughter substitution mutations in the daughter
DNA.DNA.

155. Chemical Mutagens : Chemical Mutagens :
Frameshift Mutagens Frameshift Mutagens

some chemical some chemical
mutagens cause small mutagens cause small
insertions or insertions or
deletions of deletions of
nucleotide base pairs, nucleotide base pairs,
which can lead to which can lead to
frameshift mutations. frameshift mutations.

Examples of such Examples of such
frameshift mutagens frameshift mutagens
include acridine, a include acridine, a
dye commonly used dye commonly used
as a mutagen in as a mutagen in
genetic researchgenetic research

156. Frequency of MutationFrequency of Mutation

Mutations are rare events. Mutations are rare events.

Organisms could not live or effectively reproduce themselves. Organisms could not live or effectively reproduce themselves.
About one of every ten million (10About one of every ten million (10
77
) genes contains an error. ) genes contains an error.

Mutagens typically increase the mutation rate by a factor of 10-Mutagens typically increase the mutation rate by a factor of 10-
1000 times; mutagens induce an error in one of every 101000 times; mutagens induce an error in one of every 10
44
to 10 to 10
66

genesgenes. .

Most mutations are deleterious because they code for Most mutations are deleterious because they code for
nonfunctional proteins or stop transcription entirely. Cells, nonfunctional proteins or stop transcription entirely. Cells,
without functional proteins cannot metabolize; any deleterious without functional proteins cannot metabolize; any deleterious
mutations are removed from the population when the cells die. mutations are removed from the population when the cells die.

Rarely, a cell acquires a beneficial mutation that allows it to Rarely, a cell acquires a beneficial mutation that allows it to
survive, reproduce, and pass the mutation to its descendants. survive, reproduce, and pass the mutation to its descendants.

For example, a bacterium night randomly acquire a mutation that For example, a bacterium night randomly acquire a mutation that
confers resistance to an antibiotic. confers resistance to an antibiotic.

157. DNA Repair DNA Repair

Although a mutation might rarely Although a mutation might rarely
convey an advantage, most mutations convey an advantage, most mutations
are deleterious. are deleterious.

Methods for repairing damaged DNA, Methods for repairing damaged DNA,
including light and dark repair of including light and dark repair of
pyrimidine dimers, base-excision repair, pyrimidine dimers, base-excision repair,
mis
­match repair, and an S0S response.
mis
­match repair, and an S0S response.

158. Repair of Pyrimidine Dimers

Many cells contain Many cells contain DNA photolyase, DNA photolyase, an enzyme that is activated an enzyme that is activated
by visible light to break the bonds between adjoining pyrimidine by visible light to break the bonds between adjoining pyrimidine
nucleotides, reversing the mutation and restoring the original nucleotides, reversing the mutation and restoring the original
DNA sequence. DNA sequence.

Light repair mechanism is advantageous for the prokaryote, but Light repair mechanism is advantageous for the prokaryote, but
it presents a difficulty to scientists studying UV-induced it presents a difficulty to scientists studying UV-induced
mutations-
­they must keep such strains in the dark, or the
mutations-
­they must keep such strains in the dark, or the
mutants revert to their previous form. mutants revert to their previous form.

Dark repair involves a different repair enzyme-one that doesn't Dark repair involves a different repair enzyme-one that doesn't
require light. Dark repair enzymes cut the damaged section of require light. Dark repair enzymes cut the damaged section of
DNA from the molecule, creating a gap that is repaired by DNA DNA from the molecule, creating a gap that is repaired by DNA
polymerase I and DNA ligase. Dark repair operates either in polymerase I and DNA ligase. Dark repair operates either in
light or in the dark. light or in the dark.

159. Base-Excision Repair
Sometimes DNA polymerase III incorporates Sometimes DNA polymerase III incorporates
an incorrect nucleotide during DNA replication. an incorrect nucleotide during DNA replication.
If the proofreading function of DNA If the proofreading function of DNA
polymerase III does not repair the error, cells polymerase III does not repair the error, cells
may use another enzyme system in a process may use another enzyme system in a process
called base-excision repair. called base-excision repair.
This enzyme system excises the erroneous This enzyme system excises the erroneous
base, and then DNA polymerase I fills in the base, and then DNA polymerase I fills in the
gap gap

160. Mismatch Repair

A similar repair mechanism is called mismatch repair. A similar repair mechanism is called mismatch repair.

Mismatch repair enzymes scan newly synthesized DNA looking Mismatch repair enzymes scan newly synthesized DNA looking
for mismatched bases, which they remove and replace. for mismatched bases, which they remove and replace.

How does the mismatch repair system determine which strand How does the mismatch repair system determine which strand
to repair? If it chose randomly, 50% of the time it would choose to repair? If it chose randomly, 50% of the time it would choose
the wrong strand and introduce mutations. the wrong strand and introduce mutations.

Mismatch repair enzymes, however, do not choose randomly. Mismatch repair enzymes, however, do not choose randomly.

They distinguish between a new DNA strand and an old strand They distinguish between a new DNA strand and an old strand
because old strands are methylated. because old strands are methylated.

Recognition of an error as far as 1000 base pairs away from an Recognition of an error as far as 1000 base pairs away from an
unmethylated portion of DNA triggers the mismatch repair unmethylated portion of DNA triggers the mismatch repair
enzymes. Once a new DNA strand is methylated, mismatch enzymes. Once a new DNA strand is methylated, mismatch
repair enzymes cannot correct any errors that remain. repair enzymes cannot correct any errors that remain.

161. SOS Response SOS Response
Sometimes damage to DNA is so extreme that Sometimes damage to DNA is so extreme that
regular repair mechanisms cannot cope with the regular repair mechanisms cannot cope with the
damage. damage.
In such cases, bacteria resort to what geneticists call In such cases, bacteria resort to what geneticists call
an S0S response involving a variety of processes, an S0S response involving a variety of processes,
such as the production of novel DNA polymerases (IV such as the production of novel DNA polymerases (IV
and V) capable of copying less-than-perfect DNA. and V) capable of copying less-than-perfect DNA.
These polymerases replicate DNA with little regard to These polymerases replicate DNA with little regard to
the base sequence of the template strand. the base sequence of the template strand.
Of course, this introduces many new and potentially Of course, this introduces many new and potentially
fatal mutations, but presumably SOS repair allows a fatal mutations, but presumably SOS repair allows a
few offspring of these bacteria to survive. few offspring of these bacteria to survive.

163. CANCER

164. •The top six cancers in males were
kaposi's sarcoma (15.9 %)
•liver (13.3 %)
•prostate (10.7 %)
•esophagus (6.0 %)
•non-Hodgkin's lymphoma (5.8 %)
•stomach (4.5%).
In females, the leading cancers
were:
•cervix (25.4 %)
•breast (17.4%)
•Kaposi's sarcoma (6.2 %)
•liver (5.5%)
•stomach (3.8 %)
•non-Hodgkin's lymphoma (3.8 %).
Major Cancer Types in Sub-Saharan Africa, Both Sexes, All
Ages
 (
Source: Ferlay et al. 2004)

165. INTRODUCTION
Cancer – caused by mutations in genes that normally
play a role in the regulation of cell cycle, leading to
uncontrolled cell growth.
Cells acquire mutations in these genes as a result of
spontaneous or environmentally-induced DNA
damage.
Mutant cells have growth and survival advantage
over normal cells leading to the evolution of a tumor.

166. Cancer & the Cell Cycle
In tumour cells, checkpoints in the cell cycle are typically
deregulated
This deregulation is caused by genetic defects in the machinery
that alternately raises and lowers the abundance of the
cyclin/CDK complexes
e.g mutation can occur in genes encoding cyclins
or the CDKs, OR in the genes encoding the proteins
that respond to specific cyclin/CDK complexes
Cells in which the START checkpoint is dysfunctional are
especially prone to become cancerous
These cells move into S phase without repairing their
damaged DNA
Over a series of cell cycles, mutations resulting from
replication of unrepaired DNA may accumulate & cause
further deregulation of cell cycle

167. Cancer –causing mutations occur in the
following genes
•Genes normally involved in the cell cycle:
–1. Proto-oncogenes (oncogenes)
–2. Tumor suppressor genes
–3. Genes controlling apoptosis
–4. Genes regulating DNA repair
•Other genes involved:
–Genes regulating angiogenesis
–Genes enhancing invasion and metastasis

168. CLASSES OF GENES INVOLVED IN ONSET OF
CANCER

169. Properties of Cancer Cells
•What does a cell need to be “cancerous”?
–Independent growth (growth autonomy)
–Insensitive to inhibition of growth
–Resistant to apoptosis
–No aging (continuous dividing)
–Sustained angiogenesis
–Ability to invade and metastsize

170. The 3 phases in the development of
cancer cells
•Initiation – a single cell
undergoes a mutation that
causes it to divide
repeatedly
•Promotion – a tumor
develops and cells within
the tumor mutate
•Progression – a cell mutates
in such a way that allows it
to invade surrounding
tissue

171. What are angiogenesis and metastasis?
•Angiogenesis: formation of new blood
vessels to supply nutrients and oxygen to
the tumor
•Metastasis: cells move into the
bloodstream or lymphatic vessels to make
new tumors at distant sites from the
primary tumor

172. The genetic basis for cancer
•Proto-oncogenes – products promote the cell
cycle and prevent cell death (apoptosis)
•Tumor-suppressor genes – products inhibit the
cell cycle and promote apoptosis
•Mutations in the genes above can cause
cancer, in fact proto-oncogenes that have
mutated are cancer-causing genes called
oncogenes

173. Comparing these genes in normal and
cancer cells

174. Types of cancer
•Oncology – study of cancer
•Carcinomas: cancers of the epithelial tissue
•Adenocarcinomas: cancers of glandular
epithelial cells
•Sarcomas: cancers of muscle and connective
tissues
•Leukemias: cancers of the blood
•Lymphoma: cancers of lymphatic tissues

175. Causes of cancer
•Genetics
•Environmental carcinogens
–Radiation
–Environmental carcinogens (tobacco smoke
and pollutants)
–Viruses

176. Genetic causes of cancer
•Examples of genes associated with cancer:
–BRCA1 and BRCA2 – tumor-suppressor genes that
are associated with breast cancer
–RB – a tumor-suppressor gene that is associated
with an eye tumor
–RET – proto-oncogene that is associated with thyroid
cancer
•Mutations of these genes predispose individuals
to certain cancers but it takes at least one more
acquired mutation during their lifetime to
develop cancer

177. Environmental causes of cancer
•Radiation:
–Environmental factors such as UV light (in sunlight or tanning
lights) and x-rays can cause mutation in DNA
•Organic chemicals:
–Tobacco smoke: increases cancer of lungs, mouth, larynx and
others
–Pollutants: substances such as metals, dust, chemicals and
pesticides increase the risk of cancer
•Viruses:
–Hepatitis B & C: virus that can cause liver cancer
–Epstein-Barr virus: can cause Burkitt’s lymphoma
–Human papillomavirus: can cause cervical cancer

178. Seven warning signs of cancer
•Change in bowel or bladder habits
•A sore that does not heal
•Unusual bleeding or discharge
•Thickening or lump in breast or elsewhere
•Indigestion or difficulty in swallowing
•Obvious change in wart or mole
•Nagging cough or hoarseness

179. Some routine screening tests for
cancer
•Self-examination – monthly
exams of breasts and
testicles starting at age 20
•Colonoscopy – every 5 years
starting at age 50
•Mammogram – yearly after
age 40
•Pap smear – should begin
these 3 years after vaginal
intercourse or no later than
age 21

180. Health Focus: Self exams

181. Detecting skin cancer
•A – asymmetry
•B – border is
irregular
•C – color varies from
one area to another
•D – diameter is
larger than 6mm

182. Other ways to detect cancer
•Tumor marker tests – blood tests for tumor
antigens/antibodies
–CEA (carcinoembryonic antigen) antigen can be detected in
someone with colon cancer
–PSA (prostate-specific antigen) test for prostate cancer
•Genetic tests – tests for mutations in proto-oncogenes
and tumor-suppressor genes
–RET gene (thyroid cancer)
–P16 gene (associated with melanoma)
–BRCA1 (breast cancer)
•A diagnosis of cancer can be confirmed by performing a
biopsy

183. Standard cancer treatments
•Surgery – removal of small cancers
•Radiation therapy – localized therapy that causes
chromosomal breakage and disrupts the cell cycle
•Chemotherapy – drugs that treat the whole body that
kills cells by damaging their DNA or interfering with
DNA synthesis
•Bone marrow transplants – transplant bone marrow
from one individual to another

184. Newer cancer therapies
•Immunotherapy – inject immune cells that are genetically
engineered to bear the tumor’s antigens
•Passive immunotherapy – antibodies that are linked to
radioactive isotopes or chemotherapeutic drugs are
injected into the body
•p53 gene therapy – a retrovirus in clinical trial that is
injected into the body where it will infect and kill only
tumor cells (cells that lack p53 = tumor cells)
•Angiogenesis inhibition - Angiostatin and endostatin are
drugs in clinical trials that appear to inhibit angiogenesis

185. Immunotherapy

186. Bioethical focus: Control of tobacco
•Food for thought:
•Smoking diminishes the health of the smoker and damages
nearly every major organ
•Within minutes of smoking, a smoker’s body begins to heal
•Smoking low-tar or low-nicotine is no different than smoking any
other cigarette
•The tobacco industry targets young people (9 out of 10 smokers
start before age 18)
•It is the single most preventable cause of death and disease in
the US
•Give your thoughts:
•Who should pay for the medical bills associated with smoking?
•Should the government prevent the sale of tobacco or leave it up
to the individual?

187. TUMOR SUPPRESSOR GENES
Tumor suppressor genes can be defined as genes which
encode proteins that normally inhibit the formation of
tumors.
Their normal function is to inhibit cell proliferation, or act
as the “brakes” for the cell cycle.
Mutations in tumor suppressor genes contribute to the
development of cancer by inactivating that inhibitory
function.
Mutations of this type are termed loss-of-function
mutations. As long as the cell contains one functional copy
of a given tumor suppressor gene (expressing enough
protein to control cell proliferation), that gene can inhibit
the formation of tumors.

188. Inactivation of both copies of a tumor suppressor
gene is required before their function can be
eliminated. Therefore, mutations in tumor
suppressor genes are recessive at the level of an
individual cell. As we will see, the inactivation of
tumor suppressor genes plays a major role in cancer.

189. Examples of tumor suppressor genes

190. p53: a key tumor suppressor
p53:
a key tumor suppressorp53,
Gene located on chromosome 17p13.1,
is the single most common target for genetic alteration in human tumors. In
fact, more than 50% of human tumors contain mutations in this gene! Thus it is
among the most important “brakes” on tumor formation.
Homozygous loss of the p53 gene is found in virtually every type of cancer,
including carcinomas of the breast, colon, and lung – the three leading causes
of cancer deaths.
In most cases, the inactivating mutations affecting both p53 alleles are
acquired in somatic cells. In some cases, although it is rare, individuals inherit a
mutant p53 allele.
As with RB1, inheritance of one mutant allele predisposes these individuals to
develop malignant tumors because only one additional “hit” is needed to
inactivate the second, normal, allele. Inactivation of the second p53 allele
leads to increased cell proliferation, decreased apoptosis, and tumor
development.

191. p53: a key tumor suppressor
•p53 restrains tumor formation by two different
mechanisms (Figure 4).
•p53 activates the p21 Cdk inhibitor gene in response to DNA
damage and stress. Loss of p53 in cells prevents the p21 gene
from being transcribed, leading to the increased activity of the
multiple Cdks normally turned off by p21 and resulting in
increased cell proliferation.
•p53 restrains tumor formation is by inducing apoptosis
(programmed cell death)

192. ONCOGENES
Normal genes that actively promote division of cells are
called proto-oncogenes, while their mutated, cancer-
causing forms are called oncogenes.
In contrast to tumor suppressor genes, which put the
brakes on cell proliferation, proto-oncogenes actively
promote proliferation
Mutations that convert proto-oncogenes to oncogenes
typically increase the activity of the encoded protein or
increase the expression of the normal gene.
Such mutations are dominant or gain-of-function
mutations. Therefore, only one copy of the gene needs to
be mutated in order to promote cancer.

193. VIRUSES AND CANCER
Viruses are infectious agents that must replicate inside a
host cell.
Viruses contain their own genome (either DNA or RNA)
protected by a coat made up of protein or protein plus
lipid.
viruses use the host cell’s translation apparatus (e.g.
ribosomes) of the host cells to translate their mRNAs.
Some viruses promote cancer in humans (e.g. HIV; Epstein-
Barr Virus, EBV; and human papilloma viruses ,HPVs).

194. VIRUSES AND CANCER
Human papilloma viruses (HPVs) - transmitted through sexual
contact & plays a pathogenic role in most cases of cervical cancer
at least 77 subtypes of HPV that are distinguished by variations in
their DNA sequences (HPV-16 or HPV-18 DNA found in 70% of
cervical tumors)
An additional 20% of tumors contain HPV DNA corresponding to
one of 20 other cancer-associated subtypes.
HPVs have a double-stranded DNA genome.
There are 7 viral genes that are expressed early during infection
(E1-E7) and 2 genes that are expressed late (L1-L2).
Two of the early genes, E6 and E7, promote tumor formation. The
E6 protein associates with a cellular protein called E6AP (E6-
Associated Protein). The E6-E6AP protein complex catalyzes
covalent attachment of a small protein called ubiquitin to p53.
The attachment of a polyubiquitin chain to a protein targets it to
the proteasome for degradation. Thus, E6 inactivates p53 by
causing the degradation of the protein via the cell’s normal
protein degradation machinery.

195. PROPERTIES OF CANCER CELLS
Self-Sufficiency in Growth Signals
Normal cells cannot proliferate in the absence of stimulatory signals, but
cancer cells can. Many oncogenes act by mimicking normal growth
signaling through one of several mechanisms.
Insensitivity to antigrowth signals
Antigrowth signals can block proliferation by two mechanisms: 1) forcing
cells out of the active proliferative cycle into the quiescent (G
0
)
state, until appropriate growth signals put them back into the cell
cycle; or 2) inducing differentiation, which permanently removes their
proliferative potential. Cancer cells evade these anti-proliferative
signals.
Evading Apoptosis
As discussed earlier, the ability of tumor cell populations to expand in
number is determined not only by the rate of cell proliferation but
also by the rate of cell attrition. Programed cell death, or apoptosis,
represents a major source of this attrition. Resistance to apoptosis as
can be acquired by cancer cells through a variety of strategies.

196. PROPERTIES OF CANCER CELLS CONTD
Limitless Replicative Potential
The first three acquired capabilities – lack of requirement for growth
signals, insensitivity to antigrowth signals, and resistance to apoptosis
– all lead to an uncoupling of the cell’s growth program from signals in
its environment. However, it has been shown that this uncoupling alone
does not ensure expansive tumor growth. Most types of mammalian
cells carry an intrinsic mechanism that limits their proliferation by
keeping track of the number of cell generations. This mechanism uses
the number of telomere repeats at the ends of chromosomes, which
erode through successive cycles of