1. Lecture #13: Regulation of Gene Expression (Part 2)

2. Lecture #13 Reading Assignment Physiology & Biochemistry of Prokaryotes 3 rd Ed.: Ch. 10, pp. 291; Ch. 18, pp. 496-503, 554-561, 509-518 Brock Biology of Microorganisms 11 th Ed: Ch.8, pp. 221-227 Brock Biology of Microorganisms 12 th ed.: Ch.9, pp. 231-234, 244-248 Article(s) to be discussed  Majdalani Review (Bacterial Small RNA regulators) Small Things Considered Essay for Lecture #13

3. Topics:  Transcriptional control by attenuation  Two-component regulatory systems  Quorum sensing  Bacterial small RNA regulators

4. Transcriptional Regulation: Attenuation  A system for regulating gene expression by control of transcriptional termination  Does not involve DNA-binding proteins

5. Leader Peptide Sequence of the Trp Operon

6. Attenuation Control of the Trp Operon

7. Overall Trp Operon Control TrpR

8. Signal Transduction and Two-component Regulatory Systems  Sensor kinase has Histidine kinase activity  Response regulator has aspartate residue that becomes phosphorylated  In some systems, the de-phosphorylated regulator may be the active DNA-binding species.

9. Two-component Regulation: Phosphate Uptake System From: Tetsch and Jung. Molecular Microbiology (2009) 73(6), 982–991 phoA: alkaline phosphatasepstSCAB: phosphate-specific transporter phoE: outermembrane porinUgpBAEC: hexose-6-P and G-3-P uptake

10. Quorum Sensing: Control of the lux Operon Bobtail squid colonized with luminescent Vibrio harveyi (10 11 cells/ml) LuxR Vibrio Autoinducer (VAI)

11. ArcA/ArcB and LuxR-LuxI-mediated regulation of bioluminescence in V. fischeri 5 redundant small RNAs (qrr1…qrr5) function to destabilize luxR transcript when there’s low VAI levels From: Septer et al. 2012. Volume 7, Issue 11, e49590

12. Effect of Small RNA Regulators on Gene Expression Bacterial and plasmid small RNAs are indicated in red and Eukaryotic sRNAs are shown in blue Positive interactions are indicated by arrows Negative regulation is indicated by the bars From: Storz G., Altuvia, S., Wassarman K. 2005. An Abundance of RNA Regulators. Annu. Rev. Biochem. 74:199-217 cis acting: coded on the opposite strand of targeted mRNA trans acting: encoded at a location distant from the target Mechanisms: 1)Base pair with other nucleic acids 2)Bind to and modify proteins

13. Advantages of small RNAs over protein Regulators 1)Small RNAs require fewer resources to make than a regulatory protein. Many small RNAs are expressed when carbon and energy are limiting. 2)Small RNAs act at the post- transcriptional level allowing a fast regulatory response. 3) cis-encoded RNA regulators evolve with the target (Cheaper) (Faster) (Adapting)

14. General Mechanisms by which RNA Regulators Modulate Transcription Ex.: 6S RNA Ex.: SRA Ex.: RepE and RNAI From: Storz G., Opdyke, JA., Wassarman KM. 2006. Regulating Bacterial Transcription with Small RNAs. Cold Spring Harbor Symposia on Quantitative Biology vol LXXI. Pp 269-273.

15. Regulation of Transcription by the 6S sRNA 6S sRNA sequesters the polymerase complex during nutrient limitation, requiring the expression of genes controlled by an alternate sigma factor (sigma S) From: Pichon C., Felden, B. 2007. Proteins That Interact with Bacterial Small RNA Regulators. FEMS Microbiol. Rev. 31:614-625.

16. The Structure of the Small RNA Regulator DsrA DsrA functions to stimulate the translation of the rpoS mRNA DsrA functions to inhibit translation of hns mRNA

17. Stimulation of RpoS translation by DsrA DsrA requires RNA chaperone Hfq

18. Role of Hfq in Small Regulatory RNA Function From: Gottesman, S. et al. 2006. Small RNA Regulators and the Bacterial Response to Stress. Cold Spring Harbor Symposia on Quantitative Biology vol LXXI. Pp. 1-10. Possible Outcomes: 1)Promote base pairing between the small RNA and the target mRNA 2)Change the target mRNA’s accessibility to RNases. 3)Protect RNA from RNAase E digestion. Hfq mutants are viable but are slow growers.

19. From: Gottesman, S. et al. 2006. Small RNA Regulators and the Bacterial Response to Stress. Cold Spring Harbor Symposia on Quantitative Biology vol LXXI. Pp. 1-10.

20. Global Regulation by the Small RNA- binding Protein CsrA Liu, M. Y. et al. J. Biol. Chem. 1997;272:17502-17510 Transmission electron microscopy of negatively stained CsrA-CsrB complexes. A, native complex (micrograph); B, EDTA-treated preparation (digital image). CsrA (carbon storage regulator) is a 61 amino acid protein that binds RNA CsrB is a 250 nucleotide RNA that does not code for protein CsrA CsrB

21. Expression of genes affected by CsrA (E.coli) Pathways Neg. Regulated by CsrA:  Glycogen biosynthesis  Glycogen catabolism  Gluconeogenesis Pathways Pos. Regulated by CsrA:  Glycolysis  Glyoxylate shunt  Acetate metabolism  Flagella biosynthesis

22. CsrA regulates CCM

23. CsrB and CsrC RNA: Titrators of CsrA CsrB CsrC CsrB and CsrC levels increase 3-fold when E. coli cells enter stationary phase The BarA/UvrY 2- component regulatory system has been shown to regulate CsrB and CsrC expression

24. Regulation of CsrA

25. Proposed Model for Post-transcriptional Regulation of Gene Expression by CsrA and CsrB/CsrC RNA CsrA-CsrC complex From: Romeo, T. 1998. Global Regulation by the Small RNA-binding Protein CsrA and the Non- coding RNA molecule CsrB. Mol. Microbiol. 29:1321-1330. Encodes glycogen biosynthesis enzymes

26. Proposed Model for Post-transcriptional Regulation of Gene Expression by CsrA and CsrB/CsrC RNA Benefits of this Regulatory Strategy (1)There is a low energy investment and short synthesis time to produce an RNA regulator. (2) A single RNA can bind 18 regulator proteins, thereby amplifying its control (3) Post-transcriptional control results in almost immediate changes in gene expression.

27. CRISPR: adaptive RNA regulation From: Bhaya et al. 2011. CRISPR-Cas Systems in Bacteria and Archaea: versatile small RNSs for adaptive immunity. Annu. Rev Genet. 45:273-297. This system is found in bacteria and archaea. The spacers regions can be an indication of evolution.

28. CRISPR: self vs non-self From: Marraffini LA and Sontheimer EJ. CRISPR Interference: RNA-directed adaptive immunity in bacteria and archaea. Pairing with the repeat prevents cleavage of self-DNA.

29. CRISPR: a tool for genome editing Genes are targeted by creating a spacer with homology to the gene of interest. From: NEB.com