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1. Regulation of Gene Expression in
Prokaryotes
Surendra Marasini
M.Sc. Biochemistry
Third Year
16th Dec 2019

2. Overviews
• Introduction
• Principle of gene regulation
• Regulation of gene expression in prokaryotes
• Operon concept in reference to lac operon and trp operon

3. Introduction
• Gene expression: process in which information from a gene is
used in the synthesis of a functional gene product
• Out of around 4,000 genes in bacterial genome and around 29,000
genes in human genome only a fraction are expressed in a cell at
a time
• Most abundant proteins in bacterial cells – elongation factors
required for protein synthesis
• Most abundant enzyme in the biosphere – RuBisCO (Ribulose
1,5 bisphosphate carboxylase /oxygenase )

4. Continue..

5. Processes that may affect the steady state
concentration of protein

6. Trends in Understanding Gene Regulation
• Past focus has been on understanding transcription initiation.
• There is increasing elucidation of posttranscriptional and
translational regulation.
• Mechanisms can be elaborate and interdependent
• Regulation relies on precise protein-DNA and protein-protein
contacts.

7. The vocabulary of gene expression
• Housekeeping gene
– under constitutive expression
– constantly expressed in approximately all cells
– Genes for products that are required all the times
• Regulated gene
– Levels of the gene product rise and fall with the needs of the
organism.
– Such genes are inducible.eg: expression of DNA repair
enzymes
– Such genes are also repressible.eg: tryptophan represses

8. RNA polymerase binds to DNA at promoters
• Some E. coli genes are transcribed once per second, others less
than once per generation
• Much of this variation is due to variation in promoter
sequences
Fig: consensus sequence for many E.coli promoters

9. Activators Improve Contacts Between
RNA Polymerase and the Promoter
• Binding sites in DNA for activators are called enhancers.
• In bacteria, enhancers are usually adjacent to the promoter.
– often adjacent to promoters that are “weak” (bind RNA
polymerase weakly), so the activator is necessary
• In eukaryotes, enhancers may be very distant from the promoter.

10. So, what regulates the transcription
initiation??
• By the proteins that bind to or near promoters
• At least three types of proteins regulate transcription initiation
by RNA polymerase
I. Specificity factor – sigma subunit of RNAP
II. Repressors – impede the access of RNAP to the promoter
III. Activators – promotes the RNAP- promoter interaction
Fig: consensus sequence for promoters that regulate expression of E.coli heat shock genes

11. Negative Regulation
Fig: two types of negative regulation

12. Positive Regulation
Fig: two types of positive regulation

13. Many Bacterial Genes are clustered and
regulated through Operons
• An operon is a cluster of genes sharing a promoter and
regulatory sequences.
– Genes are transcribed together, so mRNAs are several genes
represented on one mRNA (polycistronic).
• First example: the lac operon,
• Operons containing 2-6 genes is common.
• Some operons contain 20 or more genes

14. The lac Operon Reveals Many Principles of
Gene Regulation
• Work of Francois Jacob and Jacques Monod − 1960
• Shows how three genes for metabolism of lactose are regulated
together as an operon:
– -galactosidase (lacZ)
• cleaves lactose to yield glucose and galactose
– lactose permease (galactoside permease; lacY)
• transports lactose into cell
– thiogalactoside transacetylase (lacA)- modify toxic galactosides
• They rely on negative regulation via a repressor.

15. Lactose Metabolism in E. Coli
• When glucose is abundant and lactose is lacking, cells make only
very low levels of enzymes for lactose metabolism.
– Transcription is repressed.
• If glucose is scarce and cells are fed lactose, the cells can use it as
their energy source.
• The cells suddenly express the genes for the enzymes for lactose
metabolism.
– Transcription is no longer repressed.

17. Inhibiting the Transcription of the lac Operon
via a Repressor Protein
• A gene called lacI encodes a repressor called the Lac repressor.
– It has its own promoter PI.
– The repressor can bind to three operator sites (O1–O3).
• The Lac repressor binds primarily to the operator O1.
– O1 is adjacent to the promoter.
– Binding of the repressor helps prevent RNA polymerase from
binding to the promoter.
• The repressor also binds to one of two secondary operators, with
the DNA looped between this secondary operator and O1

18. Structure of the lac operon

19. Regulatory proteins have discrete DNA
binding domains
• Generally bind to specific DNA sequences
• Affinity for these target sequences is 104 to 106 times higher
than that of other sequences
• Most regulatory proteins have discrete DNA binding domains
• Binding domains usually contain one or more of a relatively
small group of recognizable and characteristic structural
motifs
• Regulatory proteins must identify the surface feature on DNA to
bind specifically

20. Fig: Groups in DNA available for protein binding

21. Fig: specific amino acid residue – base interaction

22. Continue…
• Two DNA binding motifs that play prominent roles in binding of
DNA by regulatory proteins
 Helix- turn – helix
 Zn finger motifs
Fig: DNA binding motifs of lac repressor

23. Fig: Zinc fingers

24. How Lac Repressor Binds to DNA?
• Lac repressor is a tetramer.
– dimer of dimers
– Each dimer binds to the palindromic operator sequence.
– ~17−22 bp of contact
– Kd ~10−10 M
• The O1 sequence reflects the symmetry of the repressor.
• There are approximately 20 repressors per cell.

25. The lac Operon Is Governed by More Than
Repressor Binding
• The availability of glucose governs expression of lactose-
digesting genes via catabolite repression.
– When glucose is present, lactose genes are turned off.
– It is mediated by cAMP and cAMP receptor protein (CRP or
CAP -catabolite activator protein).
When Glucose Is Absent, lac Operon Transcription Is
Stimulated by CRP-cAMP
 cAMP binds near the promoter
 Stimulates transcription 50 folds
 Open complex doesn’t form without cAMP -CRP

26. When Glucose Is Absent, lac Operon
Transcription Is Stimulated by CRP-cAMP
• CRP-cAMP only has this effect
when the Lac repressor has
dissociated.
• cAMP is made when [glucose] is
low
Fig: CRP homodimer with bound CAMP

27. When Lactose Is Absent
Little to No Transcription Occurs

28. When Lactose Is Present, Transcription
Depends On Glucose Level

31. Amino Acid Biosynthesis Regulated by
Transcriptional Attenuation
• Bacterial operons are also found for biosynthetic pathways.
• The trp operon is regulated by transcription attenuation.
• Transcription begins but is then halted by a stop signal
(attenuator).
• The attenuator sequence is in the 5’-region of a leader sequence,
and it can make the ribosome stall.
Role of the Attenuator
– if transcription will be attenuated at the end of the leader
– or, if transcription will continue into the genes for Trp
synthesis

33. The Leader Region Can Form Different Stem-
Loop Structures
• The leader is 162 nucleotides long.
– includes segments 1−4
• If segments 3 and 4 base-pair, they form a hairpin structure
that is the attenuation signal.
• If segments 2 and 3 base-pair, transcription proceeds and the
trp synthetic enzymes are made.
– no attenuation

34. The Four Segments of the Trp Leader Region

35. Abundance of tRNATrp Leads to Formation of
the Attenuator
• Segment 1 is transcribed and immediately translated.
– The ribosome is close behind RNA Pol.
– Segment 1 contains important Trp codons.
• If tRNATrp is abundant, translation proceeds so that segment 2
is covered with the ribosome and can’t pair with segment 3.
– so segment 3 pairs with 4  attenuator
Low Availability of tRNATrp Signals Translation to
Continue
• If tRNATrp is scarce, the ribosome will stall at the Trp codons in
the mRNA.
 allows 2–3 pairs to form
Translation proceeds unhindered.

36. Trp Operon When Trp Synthesis Is Not
Needed (tRNATrp Is High)

37. Trp Operon When Trp Levels Are Low,
tRNATrp Not Abundant, and Trp
Synthesis Is Needed

38. The 3–4 Pair (Attenuator) and the 2–3 Pair

39. • The Trp operon also has a repressor that binds to DNA in the
presence of tryptophan.
• Trp repressor is a homodimer.
• When Trp is abundant, it binds to repressor, causes it to bind to
the operator, and slows expression of genes for Trp synthesis.
• It has helix-turn-helix motifs that interact with DNA via the
major groove.
A repressor protein also regulates Trp operon

40. Regulation of the SOS Response
• SOS Response = response to extensive DNA damage
– results in cell cycle arrest and activation of DNA repair
systems
• Normally, SOS genes are repressed by LexA repressor.
– LexA binds to operators at several genes.
• Damaged DNA produces a lot of single strands.
• ssDNA is bound by the protein RecA (or, in eukaryotes Rad51).
– activates RecA’s ability to interact with LexA repressor
• RecA binds to LexA repressor, causing it to self-cleave and
dissociate from DNA.
– RecA is called a co-protease.

41. Regulation of the SOS Response
in E. Coli

42. Link Between the SOS Response and Virus
Propagation
• Some repressors keep viruses in a dormant state within the
bacterial host.
• RecA (Rad51 in eukaryotes) can help cleave and inactivate
these other repressors.
– allows virus to replicate, lyse cell, and release new virus
particles

43. Translational Feedback Mechanism
• Each operon for an r-protein encodes a translational repressor.
– repressor binds to mRNA and blocks translation
• Repressor has greater affinity for rRNA than for mRNA.
– so translation is repressed only when synthesis of r-proteins
exceeds a level needed to make ribosomes

44. Fig: translational feedback in some ribosomal protein operons

45. rRNA Synthesis Is Also Regulated by Amino
Acid Availability
• The stringent response occurs when aa concentrations are low.
• Lack of aa produces uncharged tRNA.
• Uncharged tRNA binds to ribosomal A site.
• rRNA synthesis triggers a cascade that begins with binding
stringent factor protein (RelA) to ribosome.

46. Stringent Factor Catalyzes Formation of an
Unusual Guanosine-Based Messenger
• Stringent factor catalyzes formation of nucleotide guanosine
tetraphosphate (ppGpp).
– It is formed from adding diphosphate (pyrophosphate) to
the 3’-end of GTP.
– Then a phosphorylase cleaves a phosphate to yield ppGpp.
• Binding of ppGpp to RNA polymerase reduces rRNA
synthesis.

47. Fig: Stringent response in E.coli

48. Some RNAs Participate in Regulation
• “Cis” regulation: a molecule affects its own function
• “Trans” regualtion: a molecule is affected by another separate
molecule
– Example: mRNA of gene rpoS (RNA polymerase sigma
factor) that encodes S, a specificity factor used by E. coli
in stress conditions
• such as starvation when S needed to transcribe stress
response genes

49. Inhibition of Bacterial Translation by Small
RNA Molecules
• The ribosome-binding Shine−Dalgarno sequence is
sequestered into a stem-loop structure in the mRNA.
• In the presence of protein Hfq, small regulatory RNA OxyS
binds to the mRNA.
• The binding of OxyS blocks the ribosome binding site in
mRNA.
• OxyS RNA inhibits translation.

50. Cis Regulation by Riboswitches
• Riboswitch = domain of an
mRNA that can bind a small-
molecule ligand
• The binding of ligand affects
conformation of the mRNA
and its activity.
• Thus, riboswitches allow
mRNA to participate in their
own regulation and respond to
changing concentrations of the
ligand.

51. Riboswitches Are a Developing Area of
Research
• Riboswitches have been found to respond to many coenzymes,
metabolites, and so on.
• They are also found in eukaryotic introns and seem to regulate
splicing.
• Some riboswitches are unique to bacteria and are therefore a
target for antibiotics.

52. References
• Lehninger’s Biochemistry, sixth edition
• Lippincott's Biochemistry seventh Edition

53. Continued with regulation of gene expression
in eukaryotes

54. Features of Eukaryotic Gene Regulation
• Access of eukaryotic promoters to RNA polymerase is
hindered by chromatin structure.
– thus requires remodeling chromatin
• Positive regulation mechanisms predominate and are required
for even a basal level of gene expression.
• Eukaryotic gene expression requires a complicated set of
proteins.

55. Three Features of Transcriptionally Active
Chromatin
• Euchromatin = less-condensed chromatin, distinguished from
transcriptionally inactive heterochromatin
• Chromatin remodeling of transcriptionally active genes:
– nucleosomes repositioned
– histone variants
– covalent modifications to nucleosomes

56. Nucleosomes Can Be Restructured by Specific
Protein Complexes
• SWI/SNF (SWItch/Sucrose NonFermentable) complex
– remodels chromatin to irregularly space nucleosomes
– stimulates binding of transcription factors
– works with proteins of ISWI (imitation switch) family
– ATP-dependent alteration of spacing between nucleosomes,
and so on

57. Covalent Modification of Histones
• Methylation
• Phosphorylation
• Acetylation
• Ubiquitination
• Sumoylation
• Occur mostly in the N-terminal domain of the histones found
near the exterior of the nucleosome particle

58. Histone Modification Alters Transcription
• Covalent modification of histones allows recruitment of
enzymes and transcription factors.
• Methylation of Lys-4 and Lys-36 at histone3 (H3) and Arg of H3
and H4:
– results in transcriptional activation
– recruits histone acetyltransferases (HATs) that then acetylate
a particular Lys
– reversed by histone deacetylases (HDACs) that make
chromatin inactive
– Acetylation of Lys results in decreased affinity of histone
for DNA.

59. Positive regulation of Eukaryotic promoters
• Eukaryotic gene transcription is usually dependent on activator
proteins, not RNA Pol affinity.
• Most promoters are inaccessible, thus making repressors
redundant.
• Combinatorial control provides a more precise positive control
for gene regulation.
• Negative regulation exists but typically involves lncRNAs not
proteins.

60. RNAP II requires five types of different
promoters
• Transcription activators (enhancers)
– proteins that bind to upstream activator sequences (UASs)
• Architectural regulators to facilitate DNA looping
• Chromatin modification/remodeling proteins
• Coactivators
– act indirectly (with other proteins, not with DNA)
• Basal (general) transcription factors

61. Enhancer Proteins Are Diverse
• Can bind thousands of nucleotides away from the TATA box of
the promoter
• Can have DNA-binding, protein-binding, and/or signal
molecule-binding domains
– can bind with multiple proteins
• Some regulate a few genes; some regulate many hundreds of
genes

62. Architectural Regulators Regulate Looping

63. Continue…

64. Coactivators assist RNA polymerase
• Mediator complex binds to carboxyl-terminal domain(CTD) of
RNA Pol II
– required for both basal and regulated transcription at many
promoters
– later provides assembly surface for other complexes
• TATA-binding protein is first component of preinitiation
complex (PIC) at the typical TATA box of a promoter

65. Fig: the components of transcription activation

66. Genes of galactose metabolism in yeast

67. Features of Hormone mediated regulation
• Hormone-receptor complex binds to DNA regions called
hormone response elements (HREs).
• Hormone receptors have a DNA-binding domain with zinc
fingers.
• Hormone receptors also have a ligand-binding region at the C-
terminus that is highly variable between different receptors.

70. Fig: A typical steroid hormone receptor

71. Translational regulation of eukaryotic mRNA

72. Types of responses to a regulatory signal

74. References
• Molecular biology of the cell
• Lehninger’s Biochemistry
• Harper’s Biochemistry
• Lippincott’s Biochemistry