1. Regulation of Gene Expression in Bacteria and Bacteriophages
Text authored by Dr. Peter J. Russell
Slides authored by Dr. James R. Jabbur
CHAPTER 17
Regulation of Gene Expression in Bacteria and Bacteriophages

2. Gene Regulation in Prokaryotes
Genes involved in cell growth and cell division are regulated. Their expression is controlled by the needs of the cell as it responds to its environment
Genes that are continuously expressed are constitutive genes (housekeeping genes). Examples include protein synthesis and glucose metabolism
All genes are regulated at some level, so that as resources dwindle the cell can respond with a different molecular strategy
Prokaryotic genes are often organized into operons that are cotranscribed. A regulatory protein binds an operator sequence in the DNA adjacent to the gene array and controls the production of polycistronic (polygenic) mRNA

3. lac operon of E. coli
As a paradigm, the lac operon is an inducible operon, normally kept off and turning “on” in the presence of an inducer substance, such as lactose
An inducer is a small molecule that joins with a regulatory protein to control transcription of the operon
The regulatory event
typically occurs at a
specific sequence of
DNA which is near
the protein-coding
sequence

4. Lactose as a Carbon Source for E. coli
E. coli expresses genes for glucose metabolism constitutively, but the genes for metabolizing other sugars are regulated in a “sugar specific” sort of way. Presence of the sugar stimulates the synthesis of the proteins needed
Lactose is a disaccharide (glucose + galactose). If lactose is E. coli’s sole carbon source, three genes are expressed
b-galactosidase (lacZ ) has two functions:
Breaking lactose into glucose and galactose. Galactose is converted to glucose, and glucose is metabolized by constitutively produced enzymes
Converting lactose to allolactose (an isomerization). Allolactose is involved in the regulation of the lac operon (it acts as an inducer)
Lactose permease (lac Y; M protein) is required for the transport of lactose across the cytoplasmic membrane
b-Galactoside transacetylase (lac A) transfers an acetyl group from acetyl-CoA to b-galactoside (for reasons that are not understood)

6. The lac operon shows coordinate induction:
In glucose medium, E. coli normally has very low levels of the lac gene products
When lactose is the sole carbon source, levels of the three enzymes increase simultaneously about a thousandfold (coordinate induction should indicate to you that these genes are under the same regulatory pathway – polycistronic in prokaryotes or several monocistronic in eukaryotes)
Allolactose is the inducer molecule
The mRNA for the enzymes has a short half-life. When lactose is gone, lac transcription stops, and enzyme levels drop rapidly

7. Animation: Regulation of Expression
Evidence for the Regulation of lac genes
Nonsense point mutations in the lac structural genes were used to map their locations (see figure b below for explanation)
The genes are tightly linked in the order: lacZ-lacY-lacA
The genes are transcribed on one polygenic RNA
Ribosomes translate the first gene in the mRNA and finish in proper position to initiate and translate the next gene…
Animation: Regulation of Expression
of lac Operon Genes

8. Mutations affecting the regulation of gene expression affect the normal control of the operon and were defined employing partial diploid, E. coli mutant strains
Operator mutations are cis-dominant (they control genes that located on the same DNA) – more on this later!
i.e. F+ lacO+ lacZ- lacY+ / lacOc lacZ+ lacY-
Inducer (lacI) gene regulatory mutations are trans-dominant (they control genes that are located on different DNA) – more later!
i.e. lacI+ lacO+ lacZ- lacY+ / lacI- lacO+ lacZ+ lacY-
Promoter mutations affect the expression of all three genes

9. Jacob and Monod’s Operon Model for the Regulation of lac Genes
The lac operon is a cluster of genes that are coregulated
The lacI gene has its own promoter and terminator, and it encodes the repressor protein that is always present in low concentration
The repressor functions as a tetramer
The repressor protein binds to the operator (lacO) and prevents RNA polymerase initiation to transcribe the operon genes (negative control)
The binding of the repressor to the operator is not absolute and so an occasional transcript is made, resulting in low levels of the structural proteins

10. Lactose absent

11. When E. coli are growing with lactose as the sole carbon source, b-galactosidase converts lactose into allolactose
The allolactose-bound repressor changes shape (allosteric shift) and dissociates from the lac operator, thereby inducing the expression of the lac operon ()
Mutations have different effects (next slides…)
Lactose present

12. lacI+ lacO+ lacZ- lacY+ / lacI+ lacOc lacZ+ lacY-
lacOc mutation results in constitutive gene expression, because the repressor cannot bind to the (lacOc) operator sequence
lacI+ lacO+ lacZ- lacY+ / lacI+ lacOc lacZ+ lacY-
Figure 19.8 Cis-dominant effect of lac0c mutation in a partial diploid strain of E. coli. (a) In the absence of the lac0c inducer, the operon is turned off, whereas the lac0c operon produces functional -galactosidase from the lacZ+ gene and nonfunctional permease molecules from the lacY- gene with a missense mutation. (b) In the presence of the inducer, the functional -galactosidase and defective permease are produced from the lac0c operon, whereas the operon produces nonfunctional -galactosidase from the gene (a missense mutation) and functional permease from the gene. Between the two operons in the cell, -galactosidase functional and permease are produced.
Lactose absent

13. Figure 19.3 Translation of the polycistronic mRNA encoded by lac utilization genes in (a) wild-type E. coli and (b) a mutant strain with a nonsense mutation in the -galactosidase (lacZ) gene.
Lactose present

14. lacI- lacO+ lacZ+ lacY+
lacI- mutation changes repressor protein conformation, preventing it from binding to the operator, resulting in constitutive expression
lacI- lacO+ lacZ+ lacY+
Figure 19.9 Effects of a lacl- mutation. (a) Effect on expression of lac operon in a haploid cell, where mutant, inactive Lac repressor molecules that cannot bind to the operator lac0+ are produced; the structural genes are transcribed constitutively. Effect in a partial diploid strain lacI+ lacO+ lacZ-lacY+ /lacI-lacO+ lacZ+ lacY in (b) the absence or (c) the presence of inducer. (The lacZ and lacY mutations are missense mutations.)

15. lacI+ lacO+ lacZ- lacY+ / lacI- lacO+ lacZ+ lacY-
Lactose absent

16. Lactose present

17. lacIs superrepressor mutation changes the repressor protein conformation, inhibiting binding to the inducer, causing constitutive binding to the operator, blocking expression
Lactose present

18. Positive Control of the lac operon
Positive control of this operon also occurs when lactose is E. coli’s sole carbon source (no glucose is present)
When glucose is scarce, the cyclic AMP concentration increases
Catabolite Activator Protein (CAP) binds to cyclic AMP (cAMP)
The CAP-cAMP complex is a positive regulator of the lac operon. It binds the CAP-site (a DNA sequence upstream of the operon’s promoter)
Binding recruits RNA polymerase to the promoter, initiating transcription
Animation: Positive Control of the lac Operon

19. When both glucose and lactose are in the medium, E
When both glucose and lactose are in the medium, E. coli preferentially uses glucose, due to catabolite repression
Glucose transport into the cell triggers the dephosphorylation of the enzyme IIIglc
IIIglc is responsible for activating the enzyme adenylate cyclase, which produces cAMP from ATP
With IIIglc inactivated, adenylate cyclase is turned off and no new cAMP is produced
This system (CAP-cAMP) is universal for catabolic genes of other sugars

20. Molecular Details of lac Operon

21. trp operon in E.coli
Unlike the inducible lac operon, the trp operon is repressible
Repressible operons normally remain ON, but turn off in the presence of the end product (in general, anabolic pathways are repressed when the end product is available)
If amino acids are available in the medium, E. coli will import them rather than make them, and the genes for amino acid biosynthesis are repressed. When amino acids are absent, the genes are expressed and biosynthesis occurs.

22. Organization of Tryptophan Biosynthetic Genes
Yanofsky and colleagues characterized the controlling sites and the genes of the trp operon
There are 5 structural genes, trpA through trpE
The promoter and operator are upstream of trp
The leader region is between the operator and trp
The attenuator region att is encoded within trpL
E. coli synthesizes tryptophan from a precursor molecule in a series of steps, with each reaction catalyzed by a specific enzyme

24. Regulation of the trp operon
Two mechanisms regulate the expression of the trp operon:
Repressor–operator interaction
(a similar mechanism to the inducible lac model, but the trp operon is repressed)
Transcription termination
A very funky model indeed!

25. Expression in the Presence of Tryptophan
The repressor for the trp operon is coded by the trpR gene, which is located some distance from the promoter (therefore, it is not seen in the figure)
trpR makes an aporepressor which is weak and cannot bind to the operator by itself
When concentrations of tryptophan are high in the cell, some tryptophan molecules bind as a corepressor to the aporepressor protein
This activates the aporepressor and it turns the operon off (it binds to the operator and stops transcription)
Repression reduces transcription of the trp operon about 70-fold

26. Expression in the Presence of low concentrations of Tryptophan
When tryptophan is limited, transcription is also controlled by attenuation
Attenuation produces short (140 bp) transcripts that do not encode structural proteins
Termination occurs at the attenuator site within the trpL region
The proportion of attenuated to full-length transcripts is directly related to the level of tryptophan
Attenuation can reduce trp operon transcription 8- to 10-fold. Together, repression and attenuation regulate trp gene expression over a 560- to 700-fold range (a fine tuning mechanism!)

27. The Molecular Model for Attenuation
Within the leader peptide mRNA are four regions that can form secondary structures by complementary base-pairing
Pairing of sequences 1 and 2 creates a transcription pause signal
Pairing of sequences 3 and 4 is a transcription termination signal (a rho-independent terminator)
Pairing of 2 and 3 is an anti-termination signal; so transcription will continue
Animation: Attenuation in the trp Operon of E. coli

28. The ribosome’s position is the key to attenuation!
Figure Four regions of the trp operon leader mRNA and the alternative secondary structures they can form by complementary base pairing.
The ribosome’s position is the key to attenuation!

29. When Tryptophan is scarce, Trp–tRNAs are unavailable and the ribosome stalls at the Trp codons in the leader sequence, covering attenuator region 1
When the ribosome is stalled in attenuator region 1, it cannot base-pair with region 2. Instead, region 2 pairs with region 3 when it is synthesized.
If region 3 is paired with region 2, it is unable to pair with region 4 when it is synthesized. Without the region 3–4 terminator, transcription continues through the structural genes
Figure Models for attenuation in the trp operon of E. coli. The light-blue structures are ribosomes that are translating the leader transcript. (a) Tryptophan-starved cells. (b) Non-tryptophan-starved cells.

30. When Tryptophan is abundant, the ribosome continues translating the leader peptide, ending in region 2. This prevents region 2 from pairing with region 3, leaving region 3 available to pair with region 4
Pairing of regions 3 and 4 creates a rho-independent terminator known as an attenuator. Transcription ends before the structural genes are reached.

31. Attenuation is involved in the negative regulation of many operons

32. Regulation of the ara operon
The prior examples have illustrated negative control. An example of positive control is exhibited in the ara operon (arabinose; sugar)

33. In the absence of arabinose and glucose, a dimer of AraC binds the inducer site and the operator (araI1, araO2). This binding prevents CAP-cAMP binding (to CAP site) and RNA polymerase binding (to PBAD)

34. Note: CAP-cAMP is necessary for RNA
If arabinose is present and glucose is absent, the ara operon is induced. Arabinose binds each AraC subunit, causing one subunit to release the araO2 site and bind to the araI2 site (the other subunit remains bound to araI1). This facilitates binding of CAP-cAMP to the CAP site and RNA polymerase binding to PBAD, resulting in transcription
Note: CAP-cAMP is necessary for RNA
polymerase to bind and cause transcription

35. Regulation of Gene Expression in Phage l
Phage use bacterial components for replication and control their use with phage gene products
Bacteriophage l has two possible pathways when it enters its E. coli host:
The lytic cycle, in which the phage takes over the cell and produces progeny phage
The lysogenic cycle, where the phage chromosome is inserted into the E. coli chromosome and replicates with the bacterial genome

36. Early Transcription Events
The l chromosome is linear, with “sticky” ends used to circularize it in the host cell
The regulatory system for choosing between the lytic and lysogenic pathways is contained in the l chromosome
Transcription begins at two promoters, PL and PR (promoters for leftward or rightward transcription, respectively)

37. The first gene transcribed from PR is cro (control of repressor and other). The Cro protein is involved in the genetic switch to the lytic pathway
The first gene transcribed from PL is N. The resultant N protein is a transcription anti-terminator, allowing RNA synthesis through termination regions into early genes
N stimulates cII protein expression, stimulating the expression of:
cI (l repressor)
O and P (DNA replication proteins)
Q (late gene activation – lysis and capsid proteins

38. The Lysogenic Pathway
The lysogenic pathway occurs when enough l repressor is made to turn off early promoters
cII is expressed, activating the PRE promoter, causing the transcription of cI (l repressor)
l repressor binds to 2 operators (OL and OR), blocking N and cro production
In addition, l repressor stimulates lysogenic gene expression and the integration of l DNA into the host chromosome
Lytic genes, including Q, are not expressed. Without the Q protein, phage coat and lysis proteins are not produced

39. Lytic pathway
An example of induction of the lytic pathway is exposure to UV light
UV causes bacterial RecA protein to stimulate l repressor proteins to autocleave and become inactivated
Absence of repressor at OR allows transcription of the cro gene
Cro protein decreases RNA synthesis from PL and PR, reducing the synthesis of cII, blocking synthesis of l repressor
Q protein levels are sufficient to allow transcription of late genes needed by the lytic pathway

40. Summary
Thus, l uses complex regulatory systems to control entry into the lytic or lysogenic pathway
The decision depends on competition between the repressor and the Cro protein
If the repressor dominates, lysogeny takes place
If the Cro protein dominates, the lytic pathway occurs