transcription 1.pptx biochemistry of genetics

genetics

1. Sohaib F. Khawaja

2. RNA

3. RNA

4. DNA

5. Features

6. Termination

7. Termination

8. Reverse Transcription

9. Reverse Transcription

10. Replication

5: に
DNA

transcription

> N ATT u 11 y
RNA
J Translation

Transcription J Reverse

Protein

11. AURADURACURE *

™ .0060006000060.
Ve

RNA TAO CARE TAB

Vo

Protein Methionine (Met)-Threonine (Thr)-Aspartate (Asp)

12. DNA Transcription (RNA Synthesis)

DNA 3 5
AÏTIGIAIGITICICIAIAIGIT
Transcription

mRNA 5 000000000000 3

L__ | | | 1 Codons

13. Transcription

14. Polymerase
movement

RNA POLYMERASE

Unwinding
of DNA

ng
网 3 NINN,
N) | Nucleotide being added

to the 3' end of the RNA

RNA-DNA
hybrid region

15. FIGURE 10.49 53

DNA

Transcription |

RNA

| — A ューー、
| | à

Polypeptide

Berets eee oe

16. TRANSCRIPTION FACTOR

we
0 ANA
‚gun
svat » é fo
transcription “apres

actor. N)
Bag
버버 = gene

STRANDED DNA 、

tra, LA

RNA polymerase

17. LA
ppp

18. Definition

Cellular process in which RNA is
synthesized using DNA as a template
known as TRANSCRIPTION.

SN W/W

DNA RNA

19. Transcription

The synthesis of RNA molecules using
DNA strands as the templates so that
the genetic information can be
transferred from DNA to RNA.

20. RNA

» Polymer of ribonucleotide held together by 3 5’
phosphodiester bridge & are single stranded.

> Is the only molecule known to function both in the
storage & transmission of genetic information & in
catalysis.

» All RNAs except the RNA genomes of certain viruses
derived from information which is stored permanently
in DNA.

21. RNA

» Three major kinds of RNAs

» mRNA (5-10%)> transfer information of gene to
ribosome i.e. encodes the amino acids sequence .

» tRNA (10-20%) > reads codes on mRNA and
transfers appropriate AA to mRNA.

» TRNA (60-80%) > constituents of ribosome.

» Many additional specialized RNAs which has catalytic
activity or regulatory functions are present in the cell.

22. e Transmits information from DNA to make proteins.
has several types

Messenger RNA (mRNA) (5-10%) carries genetic
information from DNA to the ribosomes.

Transfer RNA (tRNA) (10-15%) brings amino acids to
the ribosome to make the protein.

Ribosomal RNA (rRNA) (75%) makes up 2/3 of
ribosomes where protein synthesis takes
place. In prokaryotic cell: 23S, 16S 5S

In eukaryotic cell: 28S, 18S 5.8S, 5S

23. rRNA mRNA tRNA miRNA

ribozyme

24. » In replication entire DNA molecule is normally copied.

> In transcription a particular gene or group of genes are
copied at any time, & some portions of DNA are never
transcribed.

! Gene is a segment of DNA that codes for a type of
Protein or for RNA & may present on any strand of
DNA (contain many genes.)

25. DNA

톡
Gene Gene

26. Differences between
replication and transcription

Replication Transcription
Template Both strand single strand
whole genome small portion of genome
Primer yes no
Enzyme DNA polymerase RNA polymerase
Product dsDNA ssRNA
Base pair A-T, G-C A-U, T-A, G-C

Proof reading yes no

27. Features of transcription
> 1) Itis highly selective.

» This selectivity is due to signals embedded in the nucleotide
sequence of DNA.

» Specific sequences mark the beginning and end of the DNA
segment which is to be transcribed.

» This signals instruct the enzyme
where to start & stop the transcription

when to start,
how often to start.

28. features

» 2) Many of the RNA transcripts are synthesized as
precursors that is known as primary transcripts.

» Which on modifications & trimming converted into
functional RNA .

> SITE:
> Transcription - Prokaryotes- cytoplasm(all RNAs).
Eukaryotes— Nucleus & mitochondria
a) Nucleolus - rRNA
b) Nucleoplasm ARNA & mRNA.

29. Features

) The basic biochemistry of RNA synthesis is similar in
prokaryotes & eukaryotes, but its regulation is more
complex in eukaryotes.

» RNA synthesis in prokaryotes is catalyzed by a large
enzyme called as

30. ) DNA dependent RNA polymerase
or RNA polymerase

A single enzyme, RNA polymerase, synthesizes all types
cellular RNAs in prokaryotes.

RNA polymerase use one of the DNA strand as template

on which complimentary ribonucleotides are incorporate to
synthesize RNA.

31. » The strand of DNA which is transcribed to RNA called as
template strand.

» Opposite strand is referred as coding strand.
coding strand (+sense)
> 5’—GTCAATCCGAACT——3'’

11111 1111 111 11111 1 11 |
> 3 —ELCAGTTAGBETTECA > 7

» Template (antisense) |

RNA 5~GUCAAUCCGAACU 3’

32. RNA POLYMERASES
> RNA polymerase synthesize RNA in the direction of
5’-3’ that means DNA template is read in 3’-5’
direction.

> Ribonucleotides required -- ATP, GTP, CTP & UTP.

> The prokaryotic RNA polymerase is a multimeric
enzyme consisting of six subunits, two identical a-
subunits, similar but not identical ß and $? and mw
sixth is o factor.
20,B,B’@ -- core enzyme
2a,B,B°o + 6 --- Holoenzyme

33. RNA Polymerase of prokaryotes

Subunit Function
0,0 Determine the DNA to be transcribed
B Catalyze polymerization
, Bind & open DNA

B a
template(unwinding)

0 Function is not known
Recognize the initiation sites called

O promoter

34. Functions of RNAP

» A single RNA polymerase performs multiple functions
in transcription process.

» 1- search & binds to promoter site

» 2- unwinds a short stretch of double helical DNA.

> 3- selects correct ribonucleotide & catalyze the
formation of phosphodiester bond (polymerization
according to base pair rule)

> (RNA)n+NTP === (RNA)n+l + PPi

> 4- detects termination signals

» 5- interacts with activator & repressor proteins that
regulate the rate of transcription.

35. Transcription in Prokaryotes

Three stages

> Initiation phase: RNA-polymerase recognizes the
promoter and starts the transcription.

> Elongation phase: the RNA strand is continuously
growing.

» Termination phase: the RNA-polymerase stops
synthesis and the nascent RNA is separated from the
DNA template.

36. Initiation

» Involves the interaction of RNAP with DNA at a specific
site or sequences of DNA.

» The sequence of DNA needed for RNA
polymerase to bind to the template and accomplish the
initiation reaction defines the promoter.

> Promoter are the characteristic sequences of DNA that
direct the RNA polymerases to initiate the transcription.

» usually located on coding strand.

» Simplest type of promoters found in prokaryotes.

37. ) Two general types of sequence elements are found.

» One sequence element is believed to promote initial binding of
the enzyme RNAP.

» Other element usually has high content of adenine & thymine.

» These sequences are 6 to 8 nt in length and located about -35
& -10 bp upstream of the start point of transcription.

» These are on coding strand indicates duplex DNA required for
transcription.

» Change in only one base pair in promoter region decrease
the rate of transcription

38. Coding strand Start point

upstream Down stream
-2,-1|+1,+2 ==>
HIIIIHITTIIIIIITIIIIIIIIITIITTIIITITITTITITT

5

y e 5
template Transcription unit
Enzyme move
TATA box/

Coding strand Pribnow box Start point

5 GACA TATAAT | (4-7) y
y AA A Aa! ||||||||||! y

x ACAACTGT 17 bp 000

template

39. Initiation process

Coding strand Start point Closed complex
5’
： 57
： | 9 10 +1
template Promoter |

open complex

40. Initiation

pppA + pppN 、 pppApN +ppi

I >

Core enzyme

41. Elongation

42. Transcription bubble

Transcription bubble

Nontemplate

RNA-DNA Active site
5° hybrid, 8 bp

_ 50

Direction of transcription

43. Termination

» The transcribed region of DNA template contain stop
signals.
» Prokaryotes have two classes of termination signals.
» 1, relies on protein factor called rho(p)
rho-dependent termination.
» 2 other is rho-independent termination.

44. Rho- dependent termination

» Signalled by a sequence in the template strand of the
DNA molecule

» Which are 40bp in length & are inverted repeat or
hyphenated. These signals recognized by a termination
protein, the rho (p) factor.

) Rho is an ATP-dependent RNA-stimulated helicase
which binds to the signals. Thus RNAP cannot move
further, so it dissociates from DNA that disrupts the
nascent RNA-DNA complex., release nascent RNA.

45. Termination

Rho factor identifies
Nascent RNA stop signal

4 Rho

46. Rho independent

Most -independent terminators have two
distinguishing features.
1. One is palindromic G-C rich region which is
followed by an A-T rich region.
Thus RNA transcript of this palindrome is self
complementary sequences, permitting the formation of
a hairpin structure.

2. The second feature is a highly conserved string of
A residues in the template strand that are transcribed
into U residues. The RNA transcript ends within or
just after them.

47. Diecionoltansrpion

Caving stand 5 GCGGGCT ——— pu
Teen

palindrome
1TTTTT
Coding strand a ~ nM
Template strand 3 ーーーーーーーーーーーーーーーーー AAAAAA 一 了
UUUUUUU3

osoooo”
DODDDoOcCe

RNA transcript

E

48. Transcription in Eukaryotes

» 1. Much more complicated.
» 2. Three different RNA polymerases.
» 3. Required many transcription factor protein.

> 4. Transcription initiation needs promoter and
upstream regulatory regions.

» 5. Enhancers /silencers are DNA sequences that
regulate the rate of initiation of transcription by
RNA polymerase II

49. RNA polymerases of eukaryotes

RNA-

polymerases 1 ut

IRNA mRNA 58 rRNA
products (288,188, (hn RNA) tRNA
5.88) snRNA
Sensitivity

to Amanitin No high moderate

Amanitin is a specific inhibitor of RNA polymerases .

50. Eukaryote Promoters
40 bp

Regulatory Enhancer/ CAAT TATA | | Transcription
element(HRE) Silencers BOX BOX unit

5 -70 & -80 er box
upstream
-25 to -30

CAAT TATA Regulatory Enhancer/
E | Box | [unit element(HRE) | | Silencers

메테

downstream

51. Post transcriptional modifications

» The nascent RNA, also known as primary transcript,
needs to be modified to become functional tRNAs,
tRNAs, and mRNA.

» Primary transcripts of mRNA are called as
heterogenous nuclear RNA (hnRNA).

) hnRNA are larger than matured mRNA ,

52. Post transcriptional modifications

! Splicing

» Addition of 5’ cap

» Creation of poly A tail
» RNA editing

53. Splicing

The structural genes are composed of coding and non-
coding regions that are alternatively separated.

< 7700 bp -|
L 1 2 3 4 5 6 7

A-G non-coding region 1-7 coding region

Noncoding sequences called intervening sequences
or Introns & coding sequences called Exons

54. Branch site

Exon 1 Intron y Exon 2
U A= AG
Precursor mRNA
ul
天
E Nucleophilic A residue
U1 U2 exposed

So

| 23 ususue ATP - dependent

complex Protein mediated

unwinding
-G 3

lariat

Complete spliceosome

55. Active
spliceosome

5 3
Us U2
lariat
formation
U5
ャ ーー 一 候
Ue U2

Intron release

5' 2. 12 극 ':

(b)

Production of auto antibodies
against small nuclear
rionucleoprotein (SnRNPs) cause
systemic lupus erytromatosis.

56. 5’capping

Most eukaryotic mRNAs have a 5’ cap, a residue of 7-
methyl guanosine linked to the 5’-terminal residue of the
mRNA through an unusual 5”,5”-triphosphate linkage.

Formation of cap require three steps.

1 5’ terminal phosphate group removes by phosphatase
enzyme leaving diphosphate.

2 GTP is added by releasing pyrophosphate.

3 7 N of guanine is methylated by methyl transferase
enzyme. Methyl group donor is S-adenosyl methionine.

57. Poly A tail

» At their 3’ end, most eukaryotic mRNAs have a
string of 40 to 200 adenine residues, making up
the poly(A) tail.

» Adenine nucleotides are added by enzyme
adenylate transferase.

58. RNA editing =

DD
1

mana WIN

Liver Translation

CAA
NH2 =, COOH
4536 1
Apo B 100 UAA

Intestine Translation 4

2152
ApoB48

59. Modification of tRNA

The primary transcripts of prokaryotic and eukaryotic
tRNAs are processed by the removal of sequences from
each end (cleavage) and in a few cases by the removal
of introns (splicing).

60. Primary transcript
y
OH
RNase D cut
RNase P cut
—
base modification
5' cleavage
3 cleavage

CCA addition

Intermediate

OH

splicing

Mature tRNA™

OH

61. Base modification
.

1. Methylation
(1) A-mA, G>mG

2. Reduction
U—DHU

3. Transversion
U>y

(3) 4, Deamination

62. Processing of eukaryotic rRNA 455

| 18 | 15.85 | | 288 |
| METHYLATION

a TER

Methyl groups | CLEAVAGE (NUCLEASES)

= En PA

63. Inhibitors

» Actinomycin D- it binds with DNA template & blocks
the movement of RNAP.

> Rifampicin- antibiotic used to treat tuberculosis. It
binds with B subunit of prokaryotic RNAP.

» A-Amanitin- Toxin produced by Amanita phalloides,
mushroom. It tightly binds with eukaryotic RNA
polymerase-Il. Thus it inhibits mRNA synthesis.

64. Reverse transcription

> Retrovirus possess RNA as genetic material
> Enzyme — RNA dependent DNA polymerase
» DNA complementary to viral RNA

) cDNA can be used as a probe to identify the sequence
of DNA in genes

65. RNA genome

Retrovirus N

Cytoplasm

| reverse transcription

Viral DNA

x integrati
Nucle
Chromosome

66. Reverse Transcription

» The first nucleotide (the start site) of a transcribed
DNA sequence is denoted as +1 and the second one
as +2: the

» nucleotide preceding the start site is denoted as -1.
These pes 00000 refer to the coding strand of
DNA. Recall that the

» sequence of the template strand of DNA is the
complement of that of the RNA transcript

In contrast,

67. Reverse Transcription

» the coding strand of DNA has the same sequence as
that of the RNA transcript except for thymine (T) in
place of uracil

» (U). The coding strand is also known as the sense (+)
KR and the template strand as the antisense (-)
strand,