Class 330 Notes Translation 2.10.12 Slide 2: Prokaryotic Transcription Slide 3 Prokaryotes Have Operons Genes with similar functions are usually grouped together within an operon. Usually a single promoter operates the transcription of this operon. Slide 4: Bacterial RNA Polymerase Comprised of four subunits. Two alpha form first as a dimer and then recruit beta and beta prime. Signma comes last. NTD (N-terminal domain) and CTD (C-termianl domain) Core enzyme is the minimal set that is required for activity. Slide 5 Enzyme is flipped on its back in the figure. Omega is believed to be involved in helping to form the core enzyme. Not absolutely required...Polymerase still works fine if you knock out omega gene. Needs sigma to sit down and bind to the DNA. Slide 4: Anatomy of a Promoter Specific sequences on the promoter region that the sigma subunits recognizes. Spacer of 17 bp +1 is the start of transcription. Hexamers. -35 and -10 region due to the six bps that make them up. Slide 7: Alignment of Different Bacterial Promoters Youc an see how these different promoters align compared to one anotherin. How this different alignment from consensus, is a way to control the strength of this promoter. Suboptimal spacer, RNA polymerase may not recognize this region and won't bind as strongly. Slide 8: Variations of the Basic Promoter. Some have UP elements Recognized by Alpha CTD. Have three contact sites. Improved binding Sigma binds to -10 and -35 "Extension of -10" Sigma recognizes this extended 10 region. Can compensate for the lack of -35 if that's that situation in the bacteria. Transcription factor binding sites.
If we throw TFBS we can further modulate. Slide 9: Template versus Strand. Non-Template Coding strand is the actually gene that we're transcribing. Template strand is the strand that serves as template to make that copy of RNA based off of coding strand. Slide 10: Different Sigma Factors Recognizes Different Promoters Can control gene regulation with different sigma factors. They can recognize different -35 and -10 regions. Slide 11: Sigma Factors Binds to -10 and -35 Sequences Region sigma 3 helps to bridge regions 2 and 4 Alpha subunits can reach well beyond this -35 and -10 regions. Every 10bps away from the region where the rest of RNA polymerase sits down. The periodicity of DNA. 1 full helical turn is approx. 10.5 bps. Slide 13: Steps of Transcription Slide 14: Transcription Initiation Slide 16: Abortive Initiation and Promoter Clearance RNA poly does not require a primer. Brings in ribonucleotide triphosphates (RNTP's) Not clear as to why it does this. An intrinsic property of enzyme, that entending these products of sufficient length...Flips the switch for enzyme to have to "go-ahead." Magic length of 10-12 nucleotides. Slide 17: Rifampin Binds near catalyitic site on Beta subunit of RNA polymerase. Does not inhibit binding of RNA polymerase....affects the initition and ability to clear the promoter. Slide 18: Mechanism and rifampcin is working by. Transcription bubble is formed. Rifampcin binds to active site and prevents the RNTP's from forming initiation chain required to continue synthesis. Slide 19; Elongation DNA is coming into enzyme. Rudder helps to disassociate the two strands but act as a road block to keep two separated strands apart and not rebinding. DNA leaves this exit channel.
Another channel where the DNA exits.