Introduction to Bacterial Gene Regulation 2024 PDF

Introduction to bacterial gene regulation Presented by Assoc/Prof Charlene Kahler Email: [email protected] Learning outcomes At the end of this lecture, you should be able to: – Understand the three different types of regulatory control: transcriptional, post-transcriptional and post-transl...

Introduction to bacterial gene regulation Presented by Assoc/Prof Charlene Kahler Email: [email protected] Learning outcomes At the end of this lecture, you should be able to: – Understand the three different types of regulatory control: transcriptional, post-transcriptional and post-translational – Describe sigma factors with respect to consensus sequence and their role in transcription – Describe the mechanism of action of riboswitches and which type of regulation they contribute to – Describe post-translational regulation of RpoH and RpoE and the controlling feedback loop Revision: The central dogma of molecular biology Transcription Translation Replication DNA RNA Protein Prescott, p297-301 3 Gene and protein naming convention A gene = lower case, italics eg. rpoH A protein = uppercase, no italics eg. RpoH Regulon = multiple genes in different locations controlled by the same type of promoter thus resulting co-ordinated expression Operon = multiple genes in the same location, controlled by a single promoter Revise your knowledge from Prescott. Features of genes Gene: Entire nucleic acid sequence necessary for expression of a gene product -C protein N- 5’ untranslated region (UTR) 5’ Transcription Start point +1 (tsp) 3’ mRNA Open reading frame 5’ -35 box 3’ untranslated region (UTR) 3’ Open reading frame -10 box Promoter = RNA polymerase binds to initiate transcription Start Codon ATG Shine Dalgarno sequence = Ribosomes bind to initiate translation of protein DNA Stop Codon TAA TGA TAG Transcription termination signal 5 Why do bacteria regulate gene expression? To express a subset of proteins to permit the bacterium to survive current conditions Examples of Global responses – SOS response (sudden global DNA damage) – Starvation response – Heat stress response Examples of Specific responses – lac operon to utilise lactose as an energy source – trp operon to synthesise tryptophan – Etc. https://www.youtube.com/watch?v=NYMTeqpr6JI Each response is different – lasting seconds to minutes to hours- so each mechanism must be triggered off and then on, and modulated in volume (weak, moderate or strong) Hierarchical control mechanisms for gene expression Mechanisms for controlling translation eg. Occlusion of the Shine Dalgarno sequence Mechanisms controlling transcription eg. Sigma factors, Transcriptional regulatory factors Mechanisms for controlling protein function eg. Sequestration, degradation Protein Translation Transcription Start point (tsp) -35 box -10 box mRNA Open reading frame How do bacteria regulate gene expression at the post-transcriptional and post-translational level? Post-transcriptional control mechanisms Inhibition of translation Secondary structure of mRNA that prevents binding of the ribosome (“cis” acting) (This lecture) Small interfering RNA (sRNA) that binds the mRNA and stops ribosomes binding (“trans” acting) (see Lecture: Attenuation and sRNA) – eg. RpoS in E. coli Attenuation (secondary structure in mRNA with co-translation) Post-translational control mechanisms (This lecture) – Sequestration and degradation of the regulator protein eg. RpoH sigma factor in E. coli eg. RpoE sigma factor in E. coli How do bacteria regulate gene expression at the transcriptional level? Mechanisms controlling gene expression at the transcriptional level – Different promoters bind different sigma factors of RNA polymerase (This lecture) – DNA binding proteins bind the promoter region acting as repressors or activators of transcription (See lecture Repressors/activators ) Transcription: RNA polymerase holoenzyme 5 Subunits: 2 large ,′ 2 copies of smaller  1 copy of 70 mRNA     70 -35 +1 -10 ' (upstream)  70 initiation factor interacts with promoter binds to specific sequences near –10, -35  controls frequency of initiation of transcription , ' polymerise NTPs, transcribe DNA Sigma factor binding to DNA FEMS Microbiol Rev, Volume 43, Issue 3, May 2019, Pages 304–339, https://doi.org/10.1093/femsre/fuz001 The content of this slide may be subject to copyright: please see the slide notes for details. Sigma Factors Sigma factors form a reversible complex with RNA polymerase and aids promoter selectivity Different classes – 70 family (RpoD) Group 1: Essential (RpoD) Group 2: Non-essential primary like sigma factors – Stationary phase  factors (38 or RpoS) Group 3: Alternative factors – Heat shock  factors (32 or RpoH) Group 4: RpoE subfamily – ECF  factors – 54 family (RpoN) Sigma factors: Structure of Sigma Factors Domain organization of different  70 – A.  70 (RpoD) – B.  32 (RpoH) – C.  E (RpoE) Domains – S1 = only in  70, auto-inhibition of DNA binding determinants – S2 = interacts with -10 of promoter – S3 = three helix domain – S4 = interacts with -35 of promoter Sigma factors: Sigma factor expression is dynamic Sigma 70 is the major sigma factor and essential Minor sigma factors are expressed in response to physiological signals such as starvation, temperature, growth phase Each sigma factor has a different affinity for RNA polymerase Each sigma factor recognizes different promoters and strength of binding is determined by the spacing of the -10 and -35 regions Increased sigma factor concentration results in increased amplitude of expression from those genes under this control http://meds.queensu.ca/~mbio318/EXTRA_MATERIAL.html Sigma factor pool changes with environment FEMS Microbiol Rev, Volume 43, Issue 3, May 2019, Pages 304–339, https://doi.org/10.1093/femsre/fuz001 The content of this slide may be subject to copyright: please see the slide notes for details. An example: Heat stress response is regulated by RpoH Heat stress proteins have RpoH promoter sequences Under normal conditions – RpoH expression is low – Therefore, the expression of RpoH controlled regulon is low Under heat stress – RpoH expression increases – Therefore the expression of RpoH controlled regulon increases – The cell has the right concentration of proteases and chaperones to remove mis-folded proteins so it can survive heat shock When the crisis is over (for example temperature decreases), the expression of RpoH declines to baseline levels How is RpoH regulated in E. coli? Post-translational level (sequestration and degradation) Post-transcriptional (mRNA structure prevents translation) Transcriptional level (promoter recognised by different sigma factors for induced expression) Post-translational control mechanisms: RpoH in E. coli Low levels of RpoH under normal growth conditions. After heat shock at 42oC for 5 min, RpoH becomes the dominant sigma factor. But mRNA transcription of rpoH (see below) only really increases at 20min HOW DOES THIS HAPPEN? There are mechanisms controlling the level of translation from mRNA but also a cycle of sequestration/degradation that controls RpoH levels in the cytoplasm and hence the amount associated with the RNA polymerase Post-translational control mechanisms: RpoH expression during normal growth Under normal growth conditions – Low levels of rpoH mRNA is transcribed from sigma 70 promoter – Low levels of RpoH protein is translated – low levels of activation of the regulon (sigma 32 dependent promoters) – Low levels of DnaJ/GrpE/DnaK are translated- these form a complex in the inner membrane- FtsH (a protease) associates with this complex and degrades proteins bound to DnaJ/DnaK/GrpE – Most RpoH is bound to DnaK/DnaJ/GrpE and FtsH degrades RpoH – Therefore, levels of RpoH are kept low How does the cell produce enough RpoH during heat shock? Wick & Egli. Adv. Biochem. Eng/Biotechnol. (2004) 89:145 GrpE DnaJ P70 DnaK RpoH FtsH rpoH RpoH P32 dnaK dnaJ, grpE, ftsH, clpB & ~30 genes Post-transcriptional/translational control mechanisms: RpoH expression during heat stress rpoH mRNA secondary structure acts as a thermostat and signal for degradation At normal temperature, the mRNA forms a tertiary structure (thermosensitive riboswitch) – occluding the Shine Dalgarno (SD) site to prevent translation – Triggers degradation, mRNA half-life of 40secs – So RpoH is low During heat shock, the structure of the mRNA unwinds – – – – allows ribosome access to the SD site Half-life of mRNA increases to 4 min Increased translation of RpoH Proportion of RNA pol association with RpoH increases, therefore induction of the genes encoding factors that protect from stress Post-transcriptional/translational regulation of RpoH Thermosensitive riboswitch – Improves mRNA stability and prevents access of the ribosome to the Shine-Dalgarno site thus preventing translation Post-translational control mechanisms: Relief of the RpoH degradation cycle During heat shock, the DnaK/DnaJ/GrpE chaperone HEAT GrpE DnaJ preferentially recognizes misfolded proteins in the cytoplasm associates with ClpB foldase that re-folds proteins to correct conformation RpoH is released from DnaK/DnaJ/GrpE chaperone DnaK ClpB FtsH RpoH associates with RNA polymerase increased transcription from RpoH (H)dependent promoters → high level transcription of the heat stress genes including DnaK/DnaJ/GrpE, GroES/EL, ClpP and FtsH Higher expression level of chaperones deals with mis-folded proteins to restore normal function RpoH P32 dnaK dnaJ, grpE, ftsH, clpB & ~30 genes Post-translational control mechanisms: Relief of the RpoH degradation cycle Once misfolded proteins are removed, RpoH binds to DnaK/DnaJ/GrpE RpoH levels decline in the cytoplasm and proportionally the amount of sigma factor binding RNA polymerase declines So decreased transcription from all RpoH-dependent promoters GrpE DnaJ Feedback loop RpoH DnaK RpoH P32 FtsH dnaK dnaJ, grpE, ftsH, clpB & ~30 genes Stress responses in the periplasm The thermosensory mRNA mechanism allows for rapid responses to short exposures to heat shock But RpoH maintains the protein folding system in the cytoplasm not the periplasm So how are stress responses dealt with over prolonged periods and in the periplasm? – rpoH is transcribed from more than two promoters σ70, normal low level constitutive expression σE, induced expression when RpoE is expressed – RpoE is responsible for the protein repair response in the periplasm Post-translational control mechanisms: RpoE in E. coli RpoE expression responds to misfolded proteins in the periplasm – Gram negative bacteria Must transmit response signal from the periplasm to cytoplasm compartments across the periplasmic membrane Under normal growth conditions – Sigma E is sequestered to the inner membrane via RseA and RseB This complex sits in the cytoplasmic membrane – The cytoplasmic domain binds to RpoE so only low levels of RpoE associate with RNA polymerase – DegS is a protease that remains inactive at normal temperature as it folds upon itself by the PDZ domain Transmission of environmental signals across bacterial compartments: RpoE in E. coli During stress – DegS unfolds, becomes active and degrades the mis-folded proteins – RpoE is released from RseA and RseB by YaeL protease RpoE associates with RNA polymerase and induces the stress response from RpoE dependent promoters including RpoH If the response is to heat, the increased mRNA from RpoH will unwind and will be translated – so both systems work together Feedback loop – Once all misfolded proteins are removed, RpoE re-associates with RseAB, shutting the response off Are Riboswitches commonly found in other bacterial sensory pathways? Riboswitch (or sensory RNA) – Folding of the 5’ UTR of the mRNA to influence Continuation of transcription (similar to attenuation, see lecture 22) mRNA half-life Translation – Alterations to folding dependent upon effectors Metabolites (e.g. rib operon in B. subtilis) tRNAs (e.g. T box system, expression of tyrosyl tRNA synthetase in Gram positives) Temperature Summary We have used sigma factors to explain – transcriptional control (promoters, specificity of sigma factors) – post-transcriptional control of translation (riboswitches, mRNA stability, occlusion of the Shine Dalgarno site) – Post-translational control (sequestration and proteolytic degradation of the sigma factors) These mechanisms of control are important to respond to short and long term responses and to moderate the amplitude of the response (a lot or a little) Although we have only used sigma factors as an exemplar, these control mechanisms are common for many transcriptional activator and repressor proteins- see next lecture! Reading Microbiology: an evolving science. 3rd Ed. Joan Slonczewski and J. W. Foster. Chapter 10. Prescott’s Microbiology. 11th Ed. Willey, Sherwood, Woolverton. Chapter 13.5-13.7 and Chapter 14 (all lectures in this module). Practice SAQs Describe the hierarchical mechanisms controlling gene expression. (slide 7) What is a sigma factor and how does it exert control of gene expression? (slide 10 ,11, 15) How does the structure of a sigma factor affect its affinity to its binding sites in promoters (slide 13 and 14) Using RpoH as an example, explain the hierarchical control mechanisms governing its role in normal conditions (slide 19) Using RpoH as an example, explain the hierarchical control mechanisms governing its role in heat stress and how does this restore homeostasis (slide 20-23) Using RpoE as an example, explain the hierarchical control mechanisms governing its role in normal conditions (slide 25) Using RpoE as an example, explain the hierarchical control mechanisms governing its role in heat stress and how does this restore homeostasis (slide 26) Recall- MCQs can be derived from any slide in the deck