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Transcription Part 1

Transcription I

How does RNA polymerase read the template strand? In the 3' to 5' direction and produces a transcription in the 5' to 3' direction identical in the the sequence to the bottom coding strand
What does unwinding DNA do? Create a transcription bubble (Chromatin has to undergo chromatin remodeling)
What does SSB do? Prevents reannealing
What needs to be recognized for transcription to occur and what needs to be present in the proceeding nt? Promoter has to be recognized and a 3' OH needs to be on the proceeding nt for continued polymerization
What happens to the incoming nucleoside triphosphate? At the alpha, the 3'OH undergoes a nucleophillic attack of incoming NTP.
After reaction occurs what is released? Pyrophosphate (PPi) which almost instantaneously cleaved into 2 inorganic phosphates (Pi) thereby driving the reaction irreversibly
What does the prokaryotic system transcription require? Requires assembly of a # of polypeptide chains
E. Coli RNA polymerase subunits alpha=holoenzyme assembly; initiation. beta=Nucleotide binding; chain initiation & elongation, 5'-3' RNA synthesis. b'=binding to DNA template(strong affinity for DNA, helps b latch on) sigma=recog & binding to promoters; open promoter complex
When can sigma be discarded? At point of initiation, only core needed for elongation & termination
RNA polymerase I 14 subunits, nucleolus, produces rRNA precursors, resistant to inhibition by alpha-amanitin.
RNA polymerase II 12 subunits, nucleus, precursor to mRNA, sensitive to alpha-amanitin
RNA polymerase III 17 subunits, nucleus, synthesizes 5SrRNA, tRNA & small RNAs, moderated sensitivity to alpha-amanitin
What do all 3 RNA polymerase have in common? All 3 have a common large subunit (lots of resemblance to B & B' of Pro)
Large subunit of RNA polymerase II At the C terminus domain, there is a repeat 7-amino acid peptide: CTD domain; 2 large subunits=claw like, small subunits cluster on the periphery of large subunits
Termination rho-independent rho-independent: does not require rho factor, intrinsic secondary structure (stem-loop) that is created by newly synthesized RNA. Induces RNA pol to pause. Stem loop is rich in GC residues followed by poly U tract.
Inhibitors of Transcription Actinomycin D: Both Pro & Euk, intercalator b/w G & C. Rifampicin: blocks B subunit in Pro system. Alpha-amantinin: inhibits RNA pol II in Euk
What does the 5' cap structure enable in Euk? Enables mRNA to be recognized by the initiation factor eIF-4F. Cap contributes to the integrity & stability of mRNAs by protecting the 5' ends from attack by phosphatases & %' exonucleases
Describe the process of polyadenylation Poly A tail is added post-transcriptionally to 3' end of Euk mRNA by poly A polymerase. Site of polyadenylation is determined by the hexanucleotide AAUAAA.
What does polyadenylation do? Enhances mRNA stability by protecting it against degradation mediated by 3' exonuclease. It has been postulated to aid in mRNA transport from nucleus to cytoplasm (histone mRNAs lack a poly A-tail)
RNA editing Introns are removed
How is rRNA transcript in Prokaryotes synthesized? As a longer precursor of 30S: contains rRNA-16S, 23S & 5S rRNA
How is rRNA transcribed in Eukaryotes? 35S & 47S (larger precursor)- 18S, 28S and 5.8S
How is tRNA spliced? BY RNase P which is an endonuclease that recognizes tertiary structures of primary transcript to effect change at 5' end of the tRNA precursor.
What does mature tRNA contain? CCA at the 3' end
What happens if CCA is in sequence and not at 3' end? RNase D is required to cleave til CCA is at the 3' end
What happens if CCA is not present? It may be generated post-transcriptionally by the enzyme tRNA nucleotidyl transferase in a reaction that uses 2 molecules of CTP & one molecule of ATP
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