5- Regulation of Gene Expression Lecture.ppt

Kuliah Regulasi Gen

1. Regulation
of Gene Expression

2. Cell metabolic activities have to be
precisely controlled, because:
•the need to coordinatetheir inter-
connecting relationship
•to prevent the wasteful synthesis
of unnecessary materials
•to prevent the accumulation of
toxic products

3. Two main levels of controlmechanisms:
•Regulation of enzyme production level
•Control of enzyme activity
Enzyme activity:
•Its affinityfor a specific substrate
•The ratesit carries out reactions
•It is genetically determined by its structure
•It is being influence by its surrounding
conditions

4. Factors that influenced overall
regulation of gene flow to protein
•the number of copies of the gene
•the efficiency of gene transcription
•the stability of the mRNA
•the efficiency of mRNA translationinto
protein
•the stability of the protein product
•post translationaleffects (protein folding,
covalent modification, phosphorylation)

6. Gene copy number
•Gene copy number is not an important
control on bacterial cellmetabolism
•Most gene in bacterial chromosomeare
presents as single copies
•Gene copy number become important if were
mediated by plasmid
•Some plasmids are present within the cell in
very high copy numbers, which is reflected in
the enhanced level of the carried gene
expression

7. Transcriptional control
•Promoters
•Alternative promoters
•Gene fusions and reporter genes
•Induction and repression: the lacoperon
–Regulatory mutants of the lacoperon
–Structure of repressors and operators
–Catabolite repression

8. Control mechanism
•Transcriptional control
Controlling gene expression by
regulating the amount of mRNA
produced
•Translational control
Controlling mRNA translation into
protein (after the production of
mRNA)

9. Transcriptional control
•Upstream activators sequences and
enhancers
•Terminators and anti-terminators
•Attenuation
–Regulation of the tryptophan operon
•Two-component regulatory systems
•Global control system

10. Translational control
•Ribosome binding
•Codon usage
•Stringent response
•Anti sense RNA

11. Promoters
•Promoters regulate the amount of
the RNA producedby the gene
•Promoter is the site, adjacent to the
5’end of the gene, to which RNA
polymerase will bindand initiate
transcription
•Each gene musthave a promoter
attached to it

12. Operon
•Operon is a set of genes as a single
part of transcriptional unit
•The whole operon is transcribe from a
single promoter, into one long mRNA
molecule, from which each proteins is
translated separately
•Therefore, regulation applies to the
whole operon, and the gene concerned
is called as coordinately regulated

13. Operon structure

15. Regulon
•Groups of genes at different sites on
the chromosome regulated in
concertedfashion
•Coordinate regulation is achieved by
the presence of related transcriptional
regulatory sequencesadjacent to
each gene

16. Regulon structure

17. The lacoperon in E. coli
Consist of:
•Structural gene of lacZ, coding for β-galactosidase
•Structural gene of lacY, coding for permease
(required for lactose uptake)
•Structural gene of lacA, coding for thiogalactoside
transacetylase
•To the 5’end of the lacoperon, it was a promoter
that overlap with operator
•The operator and the separate lacIgene (codes for
a repressor protein), are connected with the
inducibilityof the operon

20. Overall structure of the lacoperon

22. The promoter region in the lacoperon
Pribnow box

23. Structure of operator/promoter region of the lacoperon

27. Alternative promoters
•Specificity of promoters recognition is not
only a function of promoter sequence,
but also of the RNA polymerase
•RNA polymerase consists of α
2ββ’ and a
dissociable σfactor
•This σfactor determine promoter
specificity
•Replacement of σfactor with different
subunit will alter promoter recognition
of the RNA polymerase

29. Gene fusion and reporter gene
•lacZis oftenly utilized as a reporter gene
•DNA sequence containing putative
promoterand additional regulatory
sequenceis inserted adjacent to the
5’end of the coding lacZgene
•Expression of β-galactosidase is now
dependent on the test promoterand the
regulatory gene
•N-terminal portion of β-galactosidase is
not important for enzyme activity

30. Use of reporter genes to study gene regulation

31. Induction and repression: the lacoperon
•The binding of the repressor proteinon
the operator site prevents the RNA
polymerase from obtaining access to the
promoter, hence prevents the initiation
of transcription
•The repressor protein also has affinity to
allolactose (a derivative of lactose)
•In the absence of lactose, the repressor is
active, but in the presence of lactose, the
repressor is inactivated

35. Regulatory mutants of the lacoperon
•Constitutive mutants: the enzyme of the
lacoperon are produced at a maximum
level, even in the absence of any inducers
•Non-inducible mutants: gene
expressions are unaffected by the
presence or absence of the inducer, but
the level of enzymes is always low
•Super-repressor (lacI
q
) mutants:
characterized by an over production of the
repressor, probably due to a mutation in
the promoter of the lacIgene

36. Types of constitutive mutants
1.lacImutants (i
–
)
–defectivein the production of the
repressor protein
–the repressor protein is alteredso that
it cannot bind to the operator

37. 2. operator constitutive (O
c
) mutants
–The changes is in the operator site, preventing
its recognition by the repressor protein
–Partial diploid straincan be constructed
–In lacI
–
/ F lacI
+
strain the lacoperon is inducible,
the lacImutation is recessive
–ThelacIgene carried on the plasmidwill
produce an active repressor, capable of binding
to the chromosomal lacoperon
–The repressor is said to be trans-acting gene
–Operator constitutive mutants are described as
cis-dominant

38. Non-inducible mutants
•gene expressions are unaffected by the
presence or absence of the inducer
•the level of enzymes produced is always low
•Might be due to lacImutationthat abolished
the ability of the repressor protein to
recognize and respond to the inducer

39. Super-repressor mutants
•Over production of the repressor
•Probably due to the mutation in the promoter
of the lacIgene
•Could be learned by genetic manipulation
utilizing multi copy plasmidscarrying a β-
galactosidase gene
•the normal level of the lac repressor in the
cells is not sufficient to repressall plasmid-
borne genes
•Useful to provide an elevated levelof repressor

41. Structure of repressors and operators
The lac repressor:
•Is a multimeric protein
•Consisting of four identical sub-units
•Showing a secondary structure feature
characteristic of DNA binding protein
•Consisting of two α-helicesseparated by a
few amino acids, placed the two α-helices at a
defined angle to each other
•This conformation is known as helix-turn-helix
motif, enables to the protein to fit into the
major groove of the DNA, make specific
contact with DNA sequence

42. The lacoperator site:
•Exhibits dyad symmetry, part of the
sequence is repeated (imperfectly) in the
inverse orientation
•The dimeric or tetrameric structure of
the regulatory proteinenables each half
of it to recognize half of the binding site
•This structure increasing both specificity
and affinity of the operator

43. Structure of operator/promoter region of the lacoperon

45. Gene regulation
•Negative regulation: the genes
concerned are actively transcribed unless
are switched off by the action of repressor
(or by an attenuation mechanism)
•Positive regulation: the genes are not
usually transcribed unless an activator is
supplied

46. Catabolite repression
•The repression of a set of genes in the
presence of an easily metabolite substrate
•If E. coliis grown in a medium containing
glucose and lactose, it will preferentially
use the glucose, and the lactose will not be
metabolized until the glucose is used up
•The gene of the lacoperon are repressed
while glucose is present
•Similar effects are seen with other systems,
such as the arabinose, maltose, and
galactose operons

47. Catabolite repression of the lacoperon

49. Catabolite repression in the lacoperon
•In the presence of glucose:
–the level of ATPwithin the cell rises
–The level of cAMPdecreases
•The lacpromoter activity is dependent on the
stimulation by a combination of cAMP and CRP
•CRP (cAMP receptor protein) also known as
CAP (catabolite activator protein)
•If cAMP is low, the stimulation can not take
place, and lacoperon is not express

51. Different consequences of cAMP-CRP binding
•CRP, when bound to DNA, interacts directly with
the RNA polymerase to promote bindingto the
promoter
•Binding of cAMP-CRP complex preventsthe RNA
polymerase from binding to a site distinct from the
genuine promoter, that would otherwise abortive
•Binding of cAMP-CRP complex produces a
substantial bend in the DNA, which may facilitate
local opening of the helix

52. Upstream activator sequences (UAS)
•In eukaryotes, UAS are found at a variable
distance upstream from the promoter
•In some cases, they have a regulatory role
through the binding of regulatory proteinto
UAS
•In contrast to promoter, UAS function in
either orientation

53. Enhancer
•Found associated with many genesin
mammalian cells and their viruses
•In SV40, enhancer is a 72 bp repeated
sequence that can be located anywhere
within a wide range, about thousand bases
upstream or downstreamfrom the start
point of transcription, and could be in
either orientation
•Some enhancer may be involved in tissue
specific gene expression

54. •The mechanism by which enhancer and
UAS activate transcription is still unclear
•It is possible that UAS (but not enhancer)
act as binding sites for transcriptional
factorsthat then migrate along the DNA to
find the promoter
•It must be remembered that DNA in the
cell is not stretched out in straight line,
enabling protein to interact simultaneously
with two apparently remote sequences

55. Terminators
•Sequence that stop transcriptionat the
end of the gene or operon
•Characteristic feature: possessed a dyad
symmetry(an inverted repeat)
•The formed RNA can establish a stem and
loop structure
•In most terminator sequences, the stem-
loop structure is followed by a run of U
residues

58. Transcription termination
•RNA polymerase activity requires a short
length of DNA to unwound(about 17 bp),
which is unstable
•The synthesized mRNA is responsible to
keep the DNA bubble open
•Physiologically, RNA-DNA hybrid is
slightly more stable than the DNA-DNA
pairing

59. •The size of bubble is limited by topological
constraints
•Extension of unwound DNA increases
winding on either site, causes stress in the
molecule
•The larger the wound region, the greater
the stress
•Therefore, the remainder of RNA molecule
is dissociatedfrom the DNA template

60. •When RNA polymerase encounter termination
sequence, it will still form RNA
•Formation of stem-loop structureincludes a
portion of RNA that keep the DNA bubble open
•The bubble tend to close up, hindering the RNA
polymerase activity, causing pause
•The string U residues have weak hydrogen
bondingof A-U pairs
•Therefore, the RNA tends to dissociate from
DNA template, terminating RNA synthesis
•Some terminator is dependent on the activity of
rho factor

61. Attenuation within an operon
The presence of a transcriptional termination site
within an operonleads to reduced expression of the
distal gene. Genes a and b are required at higher
level than c and d.

62. The trp repressor:
•Known as TrpR protein
•Also consist of two or four identical
subunits
•The protein structure can be related to
the structure of DNA site to which this
protein binds

72. Attenuation and the synthesis of leucine, isoleucine, and valine

73. Two component regulation
•Bacteria have mechanisms for sensing
external conditionsvia signal transduction
pathways
•This involves trans-membrane protein
which sense external condition by
changing its conformation to act as
appropriate gene regulator(therefore it is
referred as a two component regulation)

74. Model of two-component regulatory system

75. Translational control
•Binding of ribosomes to mRNA requires
the presence of a specific purine-rich
sequence(known as Shine-Dalgarno
sequence), complementary to 3’ of 16s
ribosomal RNA
•This sequence is up to 7 bases upstream
from AUG start codon

76. Ribosome binding site (Shine-Dalgarnosequence)

77. •Polarity is an alternative expression
regulationto attenuation
•In polycistronic operons, the ribosome
may, after translating the first gene,
dissociate from the mRNA
•The next genemust have a site to which
ribosomes can attach for translation
•The efficiencies may vary, which may
results in less effective translation
Polarity of expression

78. Translation initiation in overlapping genes

79. Ribosomal frameshifting

80. Codon usage
•Every organism have codon usage preferences,
as most amino acids can be coded for more
than one codon
•In many cases, different tRNA is responsible for
recognition of different codon
•Some of these tRNA are known to be present in
the cell at low level
•Gene that requires this rare tRNA would be
expected to suffer delays in translation

81. Stringent response
•The cells need to coordinatethe
production of several proteins so that
equal amount are made and relate to the
amount of rRNA
•This is achieved by autogenous control of
translation
•Accumulation of ribosomal protein will
repress the translation of specific mRNA,
as this proteins may bind to the mRNA

82. •If rRNA is shortage, free ribosomal protein
will accumulate, bind to the relevant
mRNA, and prevent protein synthesis
•If the cells are starved, reduction on the
number of ribosome will occurred, which is
achieved by reducing rRNA production
•This is known as stringent response

83. Antisense RNA
•If region of a gene (the ribosome binding site
and translation initiation point) is transcribe in
both directions, a complementary RNA
molecule will be produced
•This molecule can hybridize to mRNAand
block ribosome bindingor prevent formation of
translation initiation complex
•Introduction of artificial antisense RNAinto cell
may reduce gene expression