Scotty Merrell Department of Microbiology and Immunology B4140 dmerrell@usuhs.mil Regulation of Gene Expression II

1. Scotty Merrell Department of Microbiology and Immunology B4140 dmerrell@usuhs.mil Regulation of Gene Expression II

2. Regulation of Gene Expression

3. Translational Control of gene expression: The Ribosome binds the Shine-Dalgarno sequence upstream of the AUG start codon http://www.mag.keio.ac.jp/~rsaito/Research/Survey/trans_pro.html

4. Translational control can occur at the level of RNA-RNA interaction (Antisense RNA) 1. Negative control: translation initiation can be inhibited by antisense RNA which pairs with 5’ mRNA and blocks SD sequence. 2. Positive control: antisense RNA binds with 5’ untranslated region preventing formation of a secondary structure that blocks the Shine-Dalgarno sequence.

5. Where does antisense RNA come from? A. Antisense RNA can be transcribed from the opposite strand of the same DNA fragment of the gene, thus the antisense RNA complements the sense RNA completely. B. Antisense RNA can be transcribed from a different genetic location from the sense RNA and usually is not completely complimentary.

6. Inhibition of translation by antisense RNA: (a) No antisense RNA SD ompF mRNA Translation (b) Antisense RNA present SD SD is blocked ompF mRNA No translation Antisense RNA Example: ompF in E. coli P micF P/O ompF OmpF is one of the two major outer membrane Activator Activator porin proteins, that permit RNAP RNAP the passive diffusion of small, hydrophilic Inhibition molecules into the Antisense periplasm of E . coli . RNA Why would OmpF expression be modulated by environmental signals? OmpF

7. Activation of translation by antisense RNA: a-Toxin expression in S. aureus Why is -toxin expression modulated by the environment?

8. Take home message: Production of a protein can be regulated at the level of translation. This can be positive or negative regulation and occurs independent of transcription.

9. Post-Translational Control -- Proteolysis Degradation of a regulatory protein leads to changes in gene expression Example: E. coli SOS repair pathway-- LexA -- transcriptional repressor, control the expression of numerous genes involved in DNA repair, such as recA, uvrA, uvrB, uvrC, etc.

10. We see low level expression of all genes due to repression

11. DNA damage Repression is relieved---high level expression

12. Proteolysis is important for many diverse functions Swarmer Cell Stalked Cell Predivisional Cell Schematic diagram of the C.crescentus cell cycle. SW, swarmer cell; ST, stalked cell; PD, predivisional cell. The eukaryotic nomenclature has been adapted for the stages of the cell cycle. G1 corresponds to the SW cell where initiation of DNA replication is inhibited by the CtrA protein (Quon et al., 1998). The S phase includes the ST cell, where DNA replication is initiated, and the early PD cell. G2 corresponds to the late PD cell, where the newly replicated chromosomes are segregated to the cell poles and cell division gives rise to two daughter cells.

13. CtrA is a major regulator in Caulobacter. ClpP mutant CtrA ClpX mutant

14. CtrA is a major regulator in Caulobacter. CtrA degradation is irregular in ClpP and ClpX mutants ClpP mutant ClpX mutant

15. The cell-cycle is altered as a result WT Clp mutant Mixed Population G1 G2 Rifampicin treated

16. Does anyone remember what the target or mechanism of action is of rifampicin (rifampin)?

17. Does anyone remember what the target or mechanism of action is of rifampicin (rifampin)? Targets the DNA-dependent RNA polymerase-- Inhibits RNA synthesis.

18. ClpP and ClpX are constitutively expressed What could this mean in terms of the CtrA regulation we observed?

19. Model for C.crescentus cell-cycle control by the ClpXP protease. ClpXP is required for degradation of CtrA during the G1-to-S transition and presumably also for removal of CtrA from the ST compartment of the late PD cell (G2). The phosphorylated form of CtrA blocks replication in the SW cell and in the SW compartment of the late PD cell (OFF) (Quon et al., 1998). Degradation and dephosphorylation of CtrA result in the onset of DNA replication in the ST cell and in the ST compartment of the late PD cell (ON) (Domian et al., 1997). Since CtrA alone cannot account for the essential nature of ClpXP we postulate one or several additional substrates (marked with '?') for the protease that have to be degraded to allow cells to proceed through the cell cycle. The observed cell-cycle arrest of the clp mutants suggests that stabilization of the additional substrate(s) result in a replication block similar to the one in C.crescentus cells expressing a stable and constitutive CtrA protein. The additional ClpXP substrates could fall into two groups: (i) proteins that do not affect activity of CtrA and upon stabilization lead to a CtrA-independent cell-cycle arrest; and (ii) proteins that affect CtrA activity [i.e. components of the phosphorelay that results in phosphorylation of the CtrA protein (Wu et al., 1998)] and upon stabilization would lead to increased concentrations of phosphorylated CtrA protein. Increased levels of phosphorylated CtrA combined with an increased stability of CtrA would then account for the G1 cell-cycle arrest observed in clp mutants.

20. Post-Translational Control -- Conformation Changes in the conformation of a regulatory protein lead to changes in gene expression Example: E. coli iron response-- Fur -- ferric uptake regulator a transcriptional repressor that controls the expression of numerous genes involved in iron uptake and storage.

21. Here iron is acting as ____________?

22. Take home message: Gene expression can be controlled post-translationally by proteolysis and changes in conformation of regulatory proteins

23. In Summary

24. Other things we should think about in terms of regulation Sigma factors: How do they work and how are they regulated

25. Sigma factors: How do they work? Most bacteria encode multiple sigma factors (some of which are specialized and only used at certain times). Each binds a different promoter consensus sequence

26. Alignment showing sigma 70 promoter conservation

27. The 4 major sigma factors of E. coli : Sigma70 Primary sigma factor, or housekeeping sigma factor. Encoded by rpoD . When bound to RNAP Core allows recognition of -35 and -10 promoters. No other factors required for RNAP binding and transcription initiation. Sigma54 Alternative sigma involved in transcribing nitrogen-regulated genes (among others). Encoded by rpoN (ntrA). When bound to RNAP Core allows recognition of different -26 -12 promoters. Requires an additional activator to allow open complex formation for transcription. Sigma32 Heat shock factor involved in activation of genes after heat shock. Encoded by rpoH (htpR). Turned on by heat shock (either at the transcription or protein level). Activates multiple genes involved in the heat shock response. SigmaS (sigma38) Stationary phase sigma factor. Encoded by rpoS . Turned on in stationary phase. Activates genes involved in long term survival, eg. peroxidase.

28. Some sigma factors are always expressed but inactive without a coactivator Some are only expressed when the genes they regulate are required Some are expressed but degraded Some are expressed but inactive due to anti-sigma factors

29. Anti-sigma Factors  Anti- Anti-sigma factors tightly (yet reversibly) bind sigma factors and prevent their activity.

31. How might anti-sigma factors regulated?

32. Anti-sigma factors and flagellar gene expression in Salmonella Flagellar genes are expressed in 3 major classes. Class 1 genes= regulatory proteins Class 2 genes= basal body and hook components Class 3 genes= chemotaxis genes and flagellin

33. fliA encodes 28 flgM So, how are Class 3 genes ever expressed since FlgM is expressed as a class 2 gene and acts as an anti-sigma factor?

34. fliA encodes 28 So, how are Class 3 genes ever expressed since FlgM is acting as an anti-sigma factor?

36. Figure 5 Model for the regulation of the stress response alternative sigma factor, σB, in Bacillus subtilus. (A) The σB structural gene, sigB, is transcribed in an eight-gene operon from a general "housekeeping" σA-dependent promoter. It is also autoregulated and will transcribe the rsbV—rsbW—sigB—rsbX four-gene operon. RsbW is an anti-σB factor and its activity is modulated by the other members of the operon in response to either energy stress (measured as a drop in ATP levels) or to environmental stress. (B) Under stress-free conditions, RsbW binds σB to prevent σB-dependent expression of stress-response genes. RsbW is also a kinase and under stress-free conditions will phosphorylate and inactivate its antagonist, RsbV. Under poor growth conditions, energy stress occurs, ATP levels drop, and unphosphorylated RsbV accumulates and inhibits RsbW. σB is free to transcribe stress-response genes. In the absence of environmental stress, RsbT is free to interact with RsbU phosphorylase to prevent it from dephosphorylating RsbV-phosphate; under these conditions, RsbW is free to inhibit σB-dependent expression of stress-response genes. RsbT is also a kinase that will phosphorylate and inactivate its antagonist, RsbS. In the presence of environmental stress, RsbX, a RsbS-PO4 phosphatase, is activated to dephosphorylate RsbS-PO4. In addition, stress also induces dephosphorylation of RsbR. Unphosphorylated RsbS is free to interact with RsbT and/or RsbR, disrupting the RsbT-RsbU complex. The net effect of this interaction is to titrate RsbR, RsbS, and RsbT. This frees RsbU to dephosphorylate RsbV-PO4. Unphosphorylated RsbV is free to interact with RsbW, disrupting the RsbW-σB complex. σB is free to transcribe stress-response genes.

37. So anti-sigma factors are also regulated: 1). Secretion from the cell 2). Sequestration by an anti-anti-sigma factor 3). Interaction with effectors Assignment: Think about how you might identify regulators of an anti-sigma factor

38. Quorum sensing and population density as a means of regulation What is quorum sensing?

39. Quorum sensing--the ability of bacteria to communicate and coordinate behavior via signaling molecules. Autoinducers--the signaling molecules produced and used for quorum sensing. Intra-speciescommunication--communication among the same species Inter-species communication--communication between different species

40. Bacterial Communication controls gene expression Quorum sensing allows bacteria to monitor population density. Why is this important? Vs Vs Gene expression program A Gene expression program B Gene expression program C

41. Quorum sensing regulates virulence gene expression in many bacterial pathogens Vibrio cholerae cholera Streptococcus pneumoniae otitis media and pneumonia Pseudomonas aeruginosa cystic fibrosis complications Staphylococcus aureus abscesses and endocarditis Escherichia coli diarrheal disease

42. How does Quorum sensing work? Autoinducer Bacterial Cell The local concentration of signal is low due to diffusion

43. At high cell densities the local concentration of Inducer becomes high Bacteria sense this and alter gene expression

44. Gram negative and Gram positive bacteria use different signaling molecules (Acyl Homoserine lactones vs peptides)

45. Quorum sensing in Gram negative bacteria: The Vibrio fischeri paradigm This marine bacterium colonizes the light organ of the Hawaiian squid, Eupryumna scolopes in a symbiotic relationship. The bacteria acquire nutrients and the light they produce helps protect the squid from predation. Additionally, factors produced by the bacteria are required for normal tissue development in the squid. Koropatnick TA, Engle JT, Apicella MA, Stabb EV, Goldman WE, McFall-Ngai MJ. Microbial factor-mediated development in a host-bacterial mutualism. Science. 2004 Nov 12;306(5699):1186-8.

46. How do these bacteria produce light? The lux genes encode components of Luciferase. Quorum sensing comes into play in terms of regulation of expression of those genes by LuxR and LuxI.

47. LuxI is the autoinducer (AI) synthase, which produces AHL. AHL is freely diffusible across the cell membrane. LuxR is the cytoplasmic AI receptor/DNA binding transcriptional activator. It is active only when bound to AI. Thus, as the amount of AI increases (due to increased population density) more AI enters the cell, affects LuxR and activates its regulatory ability.

48. This type of signaling is Intra-species communication--you are monitoring your own kind. How is this possible since many different bacteria produce AIs? (Formally known as AI-1). Each species produces a unique AI-1 molecule.

49. Unlike the Gram negative AHL, the AI-1 peptides produced by Gram-positive bacteria are not freely diffusible across the membrane. Transporters are required to move the peptides. Staphylococcus aureus Agr system. agrD encodes the AI-1 peptide. AgrB transports and processes the peptide. AgrC and AgrA act as a two-component-like system that senses AI accumulation and then regulates downstream gene expression.

50. This type of signaling is Intra-species communication--you are monitoring your own kind. How is this possible since many different bacteria produce AIs? Each species produces a unique AI-1 molecule.