Biochem IIA 2024 Lecture 8: Transcription in Prokaryotes PDF

Biochemistry IIA Lecture 8: Transcription in Prokaryotes Dr Adrian Hunter [email protected]  Visualising Transcription Two genes being transcribed (rRNA genes), visualised by electron microscopy Multiple transcripts from each gene transcribed concurrently Direction of...

Biochemistry IIA Lecture 8: Transcription in Prokaryotes Dr Adrian Hunter [email protected]  Visualising Transcription Two genes being transcribed (rRNA genes), visualised by electron microscopy Multiple transcripts from each gene transcribed concurrently Direction of transcription? IT’S THAT WAY  Picture credits (from last lecture) E. coli by Dr Gary E. Kaiser, cropped, via LibreTexts Biology, https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/ Microbiology_Labs_II/06%3A_Gram_Stain_and_Capsule_Stain/6.02%3A_Gram_Stainin g_Procedure, CC-BY-3.0 S. cerevisiae by Mogana Das Murtey and Patchamuthu Ramasamy via Wikimedia Commons, https://commons.wikimedia.org/wiki/File:Saccharomyces_cerevisiae_SEM.jpg, CC-BY- 3.0 C. elegans by Bob Goldstein via Wikimedia Commons, https://commons.wikimedia.org/wiki/File:CrawlingCelegans.gif, CC-BY-SA-3.0  Transcription Three Stages: Initiation Elongation Termination  E. coli Promoter Promoters required to recruit & position RNA polymerase Bacterial promoters usually doesn’t know where to start Sigma factor required Sigma factor binds RNA polymerase and provides sequence-specific DNA binding RNA polymerase unwinds DNA, starts copying template strand Sigma factor dissociates  Sigma Factor Binding DNA By Mark S. Paget - https://www.mdpi.com/2218-273X/5/3/1245, CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=77565861  Bacterial Transcription Cycle DNA helix unwound by RNA RNA polymerase Termination Polymerase RNA Polymerase bonds nucleotides base-paired to template DNA Release of s Factor Elongation Initiation  Transcription Elongation (Bubble) Start with bacteria … Papachristodoulou et al., Biochem & Mol Biol 5th Ed., Fig 24.6 RNA Polymerase  Termination of Transcription in E. coli Two mechanisms of termination: 1. “Rho independent” Termination preceded by GC-rich sequence G-C base pair to form stable stem-loop structure Followed by U-rich sequence (weak RNA-DNA bonding – why?) mRNA detaches  Termination of Transcription in E. coli Two mechanisms of termination 2. “Rho Dependent” Rho attaches to newly transcribed RNA during transcription Moves behind RNA polymerase Rho is a helicase At termination site, RNA polymerase pauses (G-C rich sequence), Rho catches up, unwinds DNA-RNA complex to release RNA  Control of Transcription In prokaryotes and eukaryotes, gene expression regulated mostly at level of transcription Most transcriptional regulation at level of initiation (but can be pausing, elongation, termination) Many copies of mRNA transcribed, with generally short half lives  Question When a protein coding gene is expressed, thousands of copies of a short lived RNA molecules are made. What’s the advantage to having many copies of short lived species, rather than few copies of long lived species (the latter would be much more energy efficient) ?  Question RNA polymerase does not have any mechanism to repair errors (eg. mismatch repair). Why is this not required?  Control of Transcription - Prokaryotes Cells need control of transcription – both on and off, plus strength of transcription Need rapid response to changing environment How? Control recruitment and activity of RNA polymerase Achieved by 1. Strength of promoters -10 and -35 sequences (close to consensus = strong transcription) different sigma factors (varying affinities for different promoters) 2. Activators and repressors Influence recruitment of sigma factors, polymerases  Lac Operon Classic example of transcriptional control in E. coli. Purpose is to hydrolyse lactose so the glucose can be used for energy: b-galactosidase Papachristodoulou et al., Biochem & Mol Biol 5th Ed., Fig 26.1 Multiple genes regulated together. No point expressing these genes if lactose is absent. No point expressing these genes if glucose is present.  Lac Operon - Overview permease monosaccharides lactose (most, yummy) lac repressor + CAP β-galactosidase + allolactose (some) cAMP RNA pol PI lacI CBS P O lacZ lacY lacWTF??  Lac Operon – transcriptional regulators The lac repressor is active in its native state (binds DNA and represses transcription) Allosteric binding of allolactose prevents DNA binding allolactose The CAP protein is inactive in its native state (cannot bind DNA). Allosteric binding of cAMP is required for DNA binding cAMP  Lac Operon by condition No lactose, no glucose With lactose, no glucose + + cAMP RNA cAMP RNA pol pol CBS P O CBS P O Operon repressed Operon activated No lactose, with glucose With lactose, with glucose RNA RNA pol pol CBS P O CBS P O Operon repressed Operon weakly active  Lac Operon – Key Concepts Binding of CAP and the lac repressor is Sequence-specific Non-covalent Reversible Even when the operon is repressed, a little bit of transcription still occurs.  Lac Operon – Key Concepts Activators recruiting RNA polymerase/sigma factor a common control mechanism in bacteria, eg CAP in the lac operon Cooperative binding a key concept in biochemistry - two hands are better than one (increased affinity) - binding affinity of 2 > sum of each individual Release - rebind or diffuse away Release, - rebind  Question What would be the consequence for an E. coli cell that lacked a CAP site adjacent to the lac promoter?