2. Regulating Gene Expression Microbes respond to changing environment – Alter growth rate – Alter proteins produced Must sense their environment – Receptors on cell surface Must transmit information to chromosome Alter gene expression – Change transcription rate – Change translation rate

3. Sensing the Environment Two-component phosopho-relay signal transduction – Receptor/Sensor Histidine-kinase protein in plasma membrane Binds to a signal cue Activates itself via phosphorylation – Cytoplasmic response regulator Takes phosphate from sensor Binds chromosome  Alters transcription rate of multiple genes

4. Operonic regulation Coding vs regulatory sequences Regulatory sequences: promoters, operator and activator sequences Regulatory proteins: repressors, activators – Repressors – Repressors bind operator sequences, block transcription Induction vs Derepression – Activator proteins – Activator proteins bind sequences near by promoters, facilitate RNA Pol binding, upregulate transcription

5. The E. coli lac Operon Lactose (milk sugar) is used for food – Cannot pass through plasma membrane Lactose permease allows entry PMF used to bring lactose inside cell – Must be converted to glucose to be digested  -galactosidase converts lactose to glucose and galactose  People also make  - galactosidase  If not, person is lactose- intolerant

6. The E. coli lac Operon The lacZ gene encodes  -galactosidase The lacY gene encodes lactose permease – Need both proteins to digest lactose Operon Operon – Multiple genes transcribed from one promoter

7. The E. coli lac Operon Repressor protein LacI blocks transcription – Repressor binds to operator – Blocks  factor from binding promoter Repressor responds to presence of lactose – Binds inducer (allolactose) or DNA, not both – Add lactose  repressor falls off operator Allolactose cause operon induction

8. Activation of the lac Operon by cAMP-CRP Maximum expression requires cAMP and cAMP receptor protein (CRP) - The cAMP-CRP complex binds to the promoter at -60 bp - Interacts with RNA pol, increase rate of transcription initiation CRP acts as activator only when bound to cAMP

9. Catabolite Repression Glucose is present, lac operon is OFF (no transcription) Two mechanisms involved 1.High glucose  low cAMP levels  CRP inactive – Can’t bind operon  low level of lac transcription PTS dependent glucose uptake P-transfer from IIA-P to IIB for Glucose uptake IIA becomes available IIA: inhibitor of AC cAMP level reduced

10. Catabolite Repression 2.Inducer exclusion: – High glucose  high IIA – IIA inhibits LacY permease – Reducing intracellular lactose Importance of catabolite repression – Sequential sugar catabolism – diauxic growth

11. Animation: The E. coli lac Operon

12. Arabinose operon Regulation by dual role regulatory protein AraC “AraC” acts as repressor to block transcription (no arabinose) Acts also as activator when bound to “arabinose” (the inducer) – Operators O1, O2 and araI control AraC and AraBAD proteins expression

13. Ara Operon Controls Senario I: No arabinose present – “AraC” forms long dimeric conformation, blocks transcription (binding O2, araI1) Senario II: arabinose added – changes AraC dimeric conformation acts as activator Stimulates binding of RNA polymerase + arabinose

14. Trp operon: Repression and Attenuation trp operon – Cell must make the amino acid tryptophan Trp operon codes and regulates biosythetic enzymes When tryptophan is plentiful, cell stops synthesis Regulation by two mechanisms 1.Repression: Trp repressor must bind tryptophan to bind DNA Opposite of lac repressor Repressor + Tryptophan Transcription repressed

15. 2. Attenuation: a regulatory mechanism in which translation of a leader peptide affects transcription of a downstream structural gene Transcriptional Attenuation of the trp Operon The attenuator region of the trp operon has 2 trp codons and is capable of forming stem-loop structures.

16. Transcriptional Attenuation Mechanism of the trp Operon High tryptophan Low tryptophan

17. Animation: Animation: Transcriptional Attenuation

18. Sigma Factor Regulation  factors regulate transcription of all genes Recognize promoter sequences differentially Specific to a subset of genes Direct RNA Pol to start transcription Alternative  factors used for global transcriptional regulation of related genes

19. How are sigma factors regulated? Temperature-sensitive mRNA 2°-structure – at 42°C  70 degraded rapidly – Allows translation of  32 (    only at high temperature Synthesis of proteins that inhibit  factors – Anti-  factors block  activity until needed – Anti-anti-  factors respond to environment

20. Small Regulatory RNAs found in bacterial intergenic regions Regulate transcription or interfere with translation antisense nature of sRNA allows its binding to mRNA - ex. pairing of RNAIII with 5’-end of mRNA prevents ribosome assembly, ie. halting translation – mRNA degradation by dsRNA specific enzyme RNaseIII – RNAIII a multi-locus global regulator of several vir. factors Protein A (spa) Coaggulase (Sa1000) Read more: RNAIII represses virulence factorsRNAIII represses virulence factors

21. CRISPR system: Clustered Regularly Interspersed Short Palindromic Repeats CRISPR arrays – Repeats: tandem array palindromes (4 to >100), 28-40 bases, form stem-loop in transcripts, 3’ terminus GAA(AC/G) – Spacers: 25-40 bp, identical to phage or plasmid and some chromosomal seq., mostly sense, some antisense Leader: ~550 bp, immediately 5’ to array, AT rich, promoter CAS: clustered gene families, only present in genomes containing CRISPRs, code for RNA or DNA active proteins, possibly involved in processing arrays and psiRNA 1. CRISPERS: Nature Reviews (Microbiology), 6:181, 2008 2. Evolution of CRISPRS CAS prteins, Nature Reviews(Microbiology), 9:467, 2011CRISPERS: Nature Reviews (Microbiology), 6:181, 2008Evolution of CRISPRS CAS prteins, Nature Reviews(Microbiology), 9:467, 2011

22. CRISPR: CRISPR: a global response to foreign DNA or RNA specificity sensitive to single nucleotide difference maintains memory a primitive nucleic acid based adaptive immunity?

23. Quorum Sensing Cells work together coordinately at high cell density – V. fischeri becomes bioluminescent – Many bacteria form biofilms Discovered in Vibrio fischeri, a bioluminescent bacterium that colonizes the light organ of the Hawaiian squid

24. Induction of a quorum-sensing genes need accumulation of a secreted small molecule called an autoinducer – Homoserine lactone for V. fishcheri At a certain extracellular concentration, the secreted autoinducer reenters cells - binds to a regulatory molecule, which in the case of Vibrio fischeri is LuxR - The LuxR-autoinducer complex then activates transcription of the luciferase target genes that confer bioluminescence Quorum Sensing: Bioluminescence induction

26. Animation: Animation: Quorum Sensing

27. Summary ● Regulatory proteins help the cell sense and react to changes in its internal environment. ● Two-component signal transduction systems help the cell sense its external environment. ● The lacZYA operon is regulated as follows: - Operon is off when LacI binds to the operator. - Operon is on when allolactose binds to LacI; cAMP- CRP are bound to the promoter (and there is no glucose around). ● The tryptophan operon is regulated by repression and attenuation (premature transcript termination).

28. Summary ● Sigma factors are controlled by alternate transcription and translation, proteolysis, and anti-sigma factors ● Small regulatory RNAs can bind to mRNA and inhibit translation and cause mRNA degrade. CRISPR is a global reaction against invading foreign RNA or DNA ● Bacterial genes are regulated by a hierarchy of regulators that form integrated gene circuits. - Example: Chemotaxis ● In quorum sensing, bacteria can communicate with each other at high cell densities via autoinducers