1. Microbial Genetics MICB404, Spring 2008 Lecture #23 Global Regulation

2. Announcements –Guest lecture in the works Dr. Stacey Gilk will discuss work she’s done with protists. Genomic/genetic-directed therapeutic drug development. -Study guide posted -Supplemental reading material posted Storz and Haas (2007) A guide to small RNAs in microorganisms Today’s lecture –Global Regulation I

3. http://www.affymetrix.com/technology/index.affx http://www.nimblegen.com/technology/

4. 100 masks per chip (25-mers)

5. Mo Bio microbial gDNA isolation kit gDNA Fragmented DNA DNaseI treatment Terminal biotinylation Fragmented, Biotin-labeled DNA RMLchip Expression Array Target Hybridization Washing and Staining Scanning and Data Analysis Image adapted from www.affymetrix.com Comparative genome hybridization - CGH

7. Phi29 polymerase

8. Translational Regulation Differential translation -Downstream genes in a polycistronic mRNA are translated less frequently than genes closer to the 5’ end (genetic polarity) -lacZ Y A translated in a ratio of 10:5:2 -integrons, gene shuffling may determine expression level

9. Translational Regulation Genetic polarity - why? - Genes at the beginning of an operon are available for translation first, often before distal genes are even transcribed. - Rho termination factor

10. Posttranslational Regulation Protein degradation - cI in lambda - Sigma 38, ClpXP/RssB (P+, P-) - Sigma 32, DnaK

11. Posttranslational Regulation Protein degradation - N-end rule model: The a.a. at the N- terminus acts as a signal for proteases N-terminal amino acidHalf-life Met, Ser, Ala, Thr, Val, Gly>20 hours Ile, Glu30 min Try, Gln10 min Pro7 min Phe, leu, Asp, Lys3 min Arg2 min

12. Posttranslational Regulation Protein degradation - PEST model: Determined by regions rich in one of four amino acids. Proline Glutamic acid Serine Threonine Tend to be degraded in less than 2 hours

13. Posttranslational Regulation Protein structural change -Protein may be activated or deactivated by other factors -LacI/lactose -AraC/arabinose -TrpR/tryptophan

14. Posttranslational Regulation Feedback inhibition - The end product of a pathway inhibits the activity of the first enzyme in the pathway. - Valine sensitivity - Valine and isoleucine are synthesized by the same pathway - The first enzyme in the pathway, acetohydroxy acid synthase, is feedback inhibited by valine.

15. Regulatory RNA sRNA (trans-acting) - mechanisms - Basepairing with target mRNA -Antisense RNA - translation repression/activation - mRNA stability/degradation

16. Regulatory RNA sRNA (Trans-acting) - mechanisms - RNA-protein complexes - signal recognition, protein secretion Secretion recognition protein (SRP) 4.5S RNA + Ffh

17. Regulatory RNA sRNA (Trans-acting) - mechanisms - Structural mimics - 6S RNA binds to sigma 70 RNAP holoenzyme by resembling a promotor - doesn’t bind sigma 38 RNAP holoenzyme - stationary phase

18. Regulatory RNA sRNA (cis-acting)- riboswitches metabolite-binding mRNA -mRNA conformationalchanges modulate gene expression - aptamers

19. Global regulation Adaptation and response to changing environmental conditions Cellular mechanisms involving multiple genes & operons –Coordinated regulation Regulon – A set of operons that are all regulated by the same regulatory gene Stimulon – A collection of operons and regulons that respond to the same environmental conditions

20. Mechanisms –Transcriptional regulators DNA binding proteins –Ligands: inducers, co-repressors, etc. Activators Repressors –Sigma factors RNA polymerase subunits –alter promoter specificity of RNAP Global regulation

21. Mechanisms –Regulatory RNAs Transcriptional termination Protein binding Anti-sense RNAs –base-pairing to mRNA –affect translation –affect mRNA stability Global regulation

22. Stimulus  Physical  Chemical  Biological Response (Gene expression)  Induction  Repression

23. Mechanisms –Two-component systems Sensor Messenger Regulate a wide variety of cellular responses, including: –osmoregulation –chemotaxis –sporulation –antibiotic production –pathogenicity Global regulation

24. Mechanisms –Two-component systems Sensor –Histidine kinase Messenger –Response regulator Global regulation >50 in E. coli 10 sub-families, based on additional signal output domains that they employ

25. Two-component systems Sensor –Membrane-anchored –Senses environmental stimulus –ATP-dependent autophosphorylation of histidine residue phosphoryl group transferred to aspartic acid residue in response regulator –Activation of regulator results in change of protein function or gene expression

26. Two-component systems

28. Structural motifs within different types of transcription factors. DNA Binding Domains – Conserved Motifs Zinc Finger Motif Helix-turn-Helix Motif Helix-loop-Helix Motif Leucine Zipper Motif

29. DNA Binding Domains Leucine Zipper Motif Helix-turn-Helix Motif Zinc Finger Motif

30. Nitrogen Assimilation Cellular roles of nitrogen –amino acids –nucleotides –vitamins/cofactors Sources of N –NH 3, NO 3 -, organic nitrogenous compounds N 2 Utilized at NH 3 oxidation state

31. Nitrogen Assimilation glnA The amount of N available is sensed through the level of glutamine

32. P II –signal transduction protein Ntr Regulon

33. Glutamine synthetase requirement depends on N availability –glnA regulated according to [Gln] –Operon: glnA-ntrB-ntrC NtrB is sensor histidine kinase - autophophorylation NtrC is a transcriptional regulator Transcribed from σ 54 -dependent promoter –Nitrogen-responsive sigma factor –Promoter p 2 »2 other promoters, p 1 and p 3

34. σ 54 -dependent promoters –NtrC~P recruits σ 54 -RNAP to glnA operon Ntr Regulon

36. At low Glutamine synthetase conc.: NtrB autophosphorylates NtrB~P then transfers ~P to NtrC NtrC binds to glnA operon UAS to activate transcription

37. Ntr Regulon Low [Gln] P II protein modified by UMP NtrB free to autophosphorylate High [Gln] GlnD activated cleaves P II -UMP bond Free P II binds NtrB and inhibits autophosphorylation No phosphoryl transfer to NtrC response regulator glnA operon not induced

38. Ntr Regulon 3 promoters p 2 : NtrC~P and σ 54 -dependent, activated at low [Gln] p 1 : σ 70 -dependent, active at high [Gln]: expression of glnA p 3 : σ 70 -dependent, active at high [Gln]: expression of ntrBC glnA operon

39. Ntr Regulon NtrB –sensor histidine kinase P II P II -UMP low NH 3 high NH 3 P ATP binding

40. Ntr Regulon NtrC –response regulator –transcriptional activator NtrC

41. Ntr Regulon ~P  NtrB P II -UMP  P II Low NH 3 High NH 3 Low GlnA High GlnA

42. Wednesday’s lecture: –Global Regulation II –Reading Snyder and Champness, Chapter 13