1. Chapter 16 Transcriptional Regulation in Prokaryotes

2. PRINCIPLES OF TRANSCRIPTIONAL REGULATION
Gene expression is controlled by regulatory proteins
Positive regulators or activators increase transcription Negative regulators or repressors decrease transcription.
Most activators and repressors act at the level of transcription initiation
Transcription initiation is the most energetically efficient step to regulate, ensuring that no energy is wasted for making an mRNA or a protein Regulation at this step is easier to do well. (a single promoter versus many mRNAs)
Advantages of regulating later steps -- It allows for more regulatory inputs. It reduces the response time.

3. Many promoters are regulated by activators that help RNA polymerase bind DNA and by repressors that block that binding
The basal level transcription without regulators

4. A repressor binding site: an operator
The repressor blocks polymerase binding to the promoter.
The activator recruits RNA polymerase to the promoter by cooperative binding.
-- recruitment

5. Some activators and repressor work by allostery and regulate steps in transcriptional initiation after RNA polymerase binding
In some cases, an activator stimulates the transition from a closed to open complex by inducing a conformational change. -- allostry

6. Action at a distance and DNA looping
Cooperative binding of proteins by DNA looping.
Architectural proteins facilitate protein-protein interactions by causing DNA bending.

7. Cooperative binding and allostery have many roles in gene regulation
Antitermination and beyond: not all of gene regulation targets transcription initiation

8. REGULATION OF TRANSCRIPTION INITIATION: EXAMPLES FROM BACTERIA
An activator and a repressor together control the lac genes
lac operon
lacZ, beta-galactosidase; lacY, lactose permease; lacA, thiogalactoside transacetylase
Lac repressor encoded by lacI binds the operator.
CAP (catabolite activator protein) or CRP (cAMP receptor protein) binds the CAP site.

10. CAP and lac repressor have opposing effects on RNA polymerase binding to the lac promoter
The lac operator, a 21 bp sequence, binds two subunits of lac repressor.

11. Since the lac operator overlaps the promoter, repressor binding prevents polymerase from binding to the promoter.
RNA polymerase binds the lac promoter poorly because the promoter sequence is not optimal.

12. CAP has separate activating and DNA-binding surfaces
The positive control (pc) mutants of CAP binds DNA but does not activate transcription. The amino acid substitutions in pc mutants identify the activating region of CAP, which interacts with aCTD.

14. CAP and Lac repressor bind DNA using a common structural motif
The protein binds as a homodimer to a site that is an inverted repeat.
The helix-turn-helix motif recognizes the half site.
The recognition helix fits into the major groove and interacts with the bases by H-bonds, indirect hydrogen bonds, or van der Waals forces. The second helix makes contact with the DNA backbone and ensures proper presentation of the recognition helix.

15. Lambda repressor

16. 1. Lac repressor binds as a tetramer.
2. In case of lambda repressor, regions outside the HTH motif also interacts with DNA.
3. Distortion of the DNA is induced in some cases. Ex) CAP

17. The activities of Lac repressor and CAP are controlled allosterically by their signals
beta-galactosidase and lactose permease
Expression of lac genes is leaky. Allolactose is the inducer.
Glucose lowers the concentration of cAMP.
CAP binds DNA only when it is complexed with cAMP.

18. Combinatorial control: CAP controls other genes as well
CAP acts at more than 100 genes in E. coli, working with an array of partners.
E. coli gal genes encode enzymes involved in galatose metabolism, and are regulated by CAP and galR.

19. Alternative σ factors direct RNA polymerase to alternative sets of promoters
The heat shock sigma factor σ32 is increased by stimulation of translation and by stabilizing the protein
σ54 is involved in nitrogen metabolism.
Bacterophage SPO1 infects B. subtilis.

20. NtrC and MerR: Transcriptional activators that work by allostery rather than by recruitment
NtrC triggers transition to the open complex.
MerR acts on DNA.

21. NtrC has ATPase activity and works from DNA sites far from the gene
At low nitrogen levels, NtrC is phosphorylated by NtrB and undergoes a conformational change that reveals the DNA binding domain.
NtrC directly contacts the σ54 at glutamine synthetase gene promoter.
Use ATP (ATPase activity) to induce the conformational change in RNAP leading to open complex formation.

22. MerR activates transcription by twisting promoter DNA
MerR binds between the -10 and -35 regions of MerT.
By binding mercury, MerR changes its conformation that causes DNA to twist. The distance between -10 and -35 elements and the alignment are changed by mercury binding.

24. Some repressors hold RNA polymerase at the promoter rather than excluding it
In the absence of galactose, Gal repressor keeps the target genes off in E. coli. Gal repressor inhibits transition from the closed to open complex.
P4 protein from bacteriophage phi29 (that grows on B. subtilis) binds to a weak promoter PA3 and activates transcription (by recruitment). It binds to a strong promoter PA2c and represses transcription (by inhibiting promoter escape).

25. AraC and Control of the araBAD Operon by Antiactivation
The araBAD operon functions in arabinose breakdown. It is activated in the presence of arabinose and in the absence of glucose. Two activators, araC and CAP, work together.
In the presence of arabinose, araC binds to araI1 and araI2.
In the absence of arabinose, araC binds to araI1 and araO2.

26. THE CASE OF PHAGE λ: LAYERS OF REGULATION
The lambda phage can propagate in two ways: lytically or lysogenically.
A lysogen is extremely stable, but the prophage can switch to lytic growth by lysogenic induction when exposed to agents that damage DNA.

27. Alternative patterns of gene expression control lytic and lysogenic growth
50kb genome with about 50 genes

28. PR and PL: strong constitutive promoters
PRM (repressor maintenance): a weak promoter, needs an activator

29. lytic growth: PR and PL on, PRM off
lysogenic growth: PRM on, PR and PL off

31. Regulatory Proteins and Their Binding Sites
Lambda repressor can both activate and repress transcription.
Cro (control of repressor and other things) only represses transcription.

32. operators OR and OL
affinity for lambda repressor: OR1> OR2=OR3
affinity for Cro: OR3> OR1=OR2

33. λ repressor binds to operator sites cooperatively
The lambda repressor binds OR1 and OR2 simultaneously because of interactions between dimers.

34. Repressor and Cro bind in different patterns to control lytic and lysogenic growth

35. Lysogenic induction requires proteolytic cleavage of λ repressor
E. coli senses and responds to DNA damage by activating RecA.
Activated RecA stimulates autocleavage of LexA and lambda repressor. LexA represses genes encoding DNA repair enzymes.
Autocleavage of lambda repressor triggers lysogenic induction by loss of dimerization and cooperativity of the repressor.
Efficient lysogenic induction requires tight control of the repressor level both by positive autoregulation and by negative autoregulation.

36. Negative autoregulation of repressor requires long-distance interactions and a large DNA loop
Repressor dimers at OR1 and OR2 interact with dimers at OL1 and OL2. This allows another two dimers to bind cooperatively at OR3 and OL3. At a lower concentration of the repressor OR3 and OL3 bind the repressor.

38. Another activator, λcII, controls the decision between lytic and lysogenic growth upon infection of a new host
CII is a transcription activator and helps polymerase bind to the weak PRE (repressor establishment).
Cro favors lytic growth by binding OR3 and blocking PRM .
CII favors lysogenic growth by directing transcription of repressor gene. N

40. The number of phage particles infecting a given cell affects whether the infection proceed lytically or lysogenically
As multiplicity of infection (moi) increases, the tendency toward lysogeny increases because more cII and cIII are made from more genomes in the infected host cell.
If there are few bacterial cells, the availability of host cells for the next round of infection will be limited.

41. Growth conditions of E. Coli control the stability of CII protein and thus the lytic/lysogenic choice
CII is very unstable and degraded by a specific protease called FtsH (HflB).
If growth is good, FtsH is very active, CII is degraded efficiently, repressor is not made, and phage grows lytically.
CIII stabilizes CII by functioning as a competing substrate for the protease.
CII-dependent promoters PI (for integration) and PAQ
PI directs transcription of the int gene that encodes the integrase enzyme that catalyzes site specific recombination of lambda DNA to the bacterial chromosome.
PAQ retards lytic development by producing RNA acting as an antisense RNA.

44. Transcriptional antitermination in λ development
N and Q proteins are antiterminators which regulate at stages after transcription initiation.

45. N protein regulates early gene expression.

46. Q protein regulates late gene expression from PR’.

47. The recognition sequences for antiterminators occur somewhere between the promoter and the terminator, not in the terminators where they act.

48. N binds nut sites in RNA and works with Nus proteins.
Q binds to QBE DNA sequence after RNA polymerase has left the promoter and transfers to the paused polymerase.

49. Sigma factor of polymerase is involved in Q function.
The nascent RNA-mediated destabilization of an interaction between sigma region 4 and the flap domain of the beta subunit is required for the bacteriophage lambda Q antiterminator protein to contact holoenzyme during early elongation.

50. Retroregulation: An interplay of controls on RNA synthesis and stability determines int gene expression
The int mRNA from PL under the influence of N is degraded by cellular nucleases, whereas int mRNA from PI activated by CII is stable and can be translated into integrase protein.

51. The sib site is a unique regulatory site and a target for RNase III
The sib site is a unique regulatory site and a target for RNase III. The expression of int from PL is inhibited by the sib. Because the sib site is downstream of the gene it affects, and because degradation proceeds backward, this process is called retroregulation.

52. Terminator formed
in RNA from PL
RNase III target

53. For lysogenic induction, retroregulation does not occur whether or not CII activity is high. Because the phage attachment site is between int and the sib site, int mRNA from PL is stable after integration.

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