Lecture 5 (slides) - Transcription and RNA Processing

Transcription and RNA Processing Chapters 13  Section Outline From Chapter 13… 1.Basic features of RNA 2.The Process of Gene Expression 3.Transcription in Prokaryotes 4.Transcription in Eukaryotes From Chapter 14… 5.RNA molecules and RNA processing  4. Transcription in Eukaryo...

Transcription and RNA Processing Chapters 13  Section Outline From Chapter 13… 1.Basic features of RNA 2.The Process of Gene Expression 3.Transcription in Prokaryotes 4.Transcription in Eukaryotes From Chapter 14… 5.RNA molecules and RNA processing  4. Transcription in Eukaryotes Most Eukaryotes Have At Least Three RNA Polymerases  3. Transcription in Prokaryotes Recall Sigma factor () recognizes and binds to the -35 and -10 consensus sequences in the promoter region, properly positioning the RNA polymerase to begin transcription. The -10 consensus sequence is prone to unwinding due to is AT rich content.  4. Transcription in Eukaryotes There are specific promoter sequences for genes transcribed by RNA polymerases I, II or III. In eukaryotes, accessory proteins recognize each of these specific types of promoters (through interaction with their DNA sequences) and bind/recruit the appropriate polymerase to begin transcription (i.e., Pol. I, Pol. II or Pol. III). Promoter-specific accessory proteins Promoter DNA sequences 5’ 3’ Pol. I, Pol. II and Pol. III are only recruited to Pol. I Pol. II Pol. III their promoter specific accessory proteins.  4. Transcription in Eukaryotes Eukaryotic promoters are more complex than those in prokaryotes An example below is an RNA polymerase II promoter. It consists of a core promoter and a regulatory promoter that aid in positioning transcription proteins and RNA polymerase II to begin transcription Abbreviations: Y = pyrimidine, either C or T R= purine, either A or G N= any nucleotide, A,G,C orT  4. Transcription in Eukaryotes Initiation Initiation involves step-wise assembly of general Transcription Factors of Pol. II (TFII A, B, D, E, F and H). (replaces the sigma factor in prokaryotes) TFIID complex contains the TATA Binding Protein (TBP) and is first to assemble at the TATA box followed by the remaining general transcription factors (TFs) and Pol. II. Forms the “preinitiation complex” or PIC: that is sufficient to initiate basal (low levels) transcription. More complex transcriptional regulation involves a multi-subunit complex called a “Mediator” that permits interactions with other activator/repressor proteins bound to upstream/downstream regulatory regions or enhancer sequences.  4. Transcription in Eukaryotes Initiation Regulatory promoter Core promoter Each gene has a unique regulatory promoter, having distinct regulatory elements and unique cofactors to influence transcription. Assembly of TF’s and Pol II causes 11-15 bp of surrounding DNA to unwind  4. Transcription in Eukaryotes Puffs (Balbiani rings) Puffs in Drosophila polytene salivary chromosomes are sites of localized unwinding due to gene transcription  4. Transcription in Eukaryotes Elongation Polymerase moves along template strand leaving transcription factors intact at the promoter for reinitiation of transcription with new polymerase. RNA pol. maintains an 11-15 rNTPs transcription bubble during elongation. DNA-RNA hybrid bends at a right angle. Positions the –OH group at the active site where new nucleotides are added to the 3’ end of the growing chain. Newly synthesized RNA is separated from DNA and exits through another cleft. 4. Transcription in Eukaryotes Termination RNA Pol I: requires a termination factor similar to rho factor in prokaryotes. RNA Pol III: ends after transcribing a terminator sequence that produces a string of U’s that is downstream from a hairpin. Similar to rho- independent termination. 4. Transcription in Eukaryotes Termination RNA Pol II: transcription continues past termination sequence. - RNA is cleaved at a consensus sequence in the RNA. 4. Transcription in Eukaryotes Termination Cleaved RNA results in 2 RNAs: one that will encode a protein and the other with its 5’ trailing out of the RNA polymerase. Rat1 (5’→ 3’ exonuclease) attaches to the 5’ end and degrades the remaining RNA strand Transcription is terminated when Rat1 reaches the transcription machinery (recall how rho binds a consensus sequence and moves up the RNA, but does not degrade it ) Note that Rat1 does not bind the 5’ end of the protein coding mRNA. This is protected with a 5’ cap. 4. Transcription in Eukaryotes A transcription unit includes all of the following EXCEPT a(n) a. enhancer. b. promoter. c. coding sequence. d. terminator. e. All of the above are included. What would the result be if a specific sigma subunit of bacterial RNA polymerase were mutated? a. Nothing would result; sigma is not essential. b. RNA polymerase would still bind DNA at specific sites but will fall off after joining together only a few RNA nucleotides. c. RNA polymerase would initiate transcription at random on the DNA. d. The core enzyme would not be as stable as usual. e. None of the above. What is the function of the DNA Regulatory promoter? a) It contains the Operator site to control expression. b) Binds transcription factors that can positively or negatively influence transcription. c) Involved in the formation of the preinitiation complex (PIC) to initiate transcription d) Contains the TATA box e) Recruits the ribosomal complex for translation Which eukaryotic RNA polymerase transcribes mRNA? a) RNA polymerase III b) RNA polymerase I c) RNA polymerase IV d) RNA polymerase II Which subunit of prokaryotic RNA polymerase directs it to the target gene? a) Alpha b) Beta c) Delta d) Sigma e) Omega A rho-independent transcription termination includes a. inverted repeats that when transcribed fold into a hairpin. b. a string of 7–9 U’s in the DNA template. c. a region in the RNA molecule that causes RNA polymerase to pause. d. All of the above. e. Both a and c.