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Lecture 22 –RNA Processing I The CTD of large subunit of RNA Pol II is unique among the RNA Polymerases When stretched out, it forms a long scaffold CTD is made up of many repeats (heptapeptide repeats) oYSPTSPS in humans –occurs 52 times oYSPTSPS in yeast –occurs 26 times The repeat becomes phosphorylated on S5 and then S2 TFIIH and its associated protein kinase activity will phosphorylate Serine 5 of this heptapeptide oThis is associated with getting into an elongation phase Phosphorylation of the CTD of large subunit of RNA Pol II occurs Post-initiated RNA pol complex comes to the first nucleosome and stops, with all S5 phosphorylated by TFIIH It waits for S2 to be phosphorylated by P-TEFb S2 is recognized by splicing factors RNA Pol can elongate only after Serine 2 has been phosphorylated oTakes place when RNA Pol II gets held up in a nucleosome
Upon the initiation of transcription the 5’ end of the emerging pre-mRNA is modified Once initiated and start to form pre-mRNA, as it emerges from the transcription bubble, it needs to be capped It’s modified so that enzymes that degrade RNAs won’t chew it upCap is put on by an enzyme that recognizes the phosphorylation of S5 of CTD Maturation of nascent transcripts must occur to form functional mRNAs Capping enzyme will recognize 5’ end of pre-mRNA oPrecursor mRNAs (pre-mRNA) are modified at their 5’ endIt will remove a γ phosphate oA 7’ methylguanylate CAP is added to the 5’ terminal nucleotide through an unusual 5’-5’ linkageoRNAase can’t degrade pre-mRNA from 5’ end –it’s protectedCTD is critical for ensuring that the 5’ end is stabilized by adding the 7’ methylguanylate CAP, there is appropriate elongation that takes place, during elongation there are splicing factors required to make a mature mRNA in close proximity, and after elongation has been terminated, there is poly-adenylation. In animal cells and in higher plants, the 2’ hydroxyl of the ribose group of the first base is methylatedIn vertebrates the second base is also methylated Addition of the CAP protects the pre-mRNA from enzymatic degradation, while facilitating nuclear export and recognition by translation initiation factors From pre-mRNA to mRNA: splicing Unlike bacterial genes, eukaryotic genes can (and most of them do) have introns Exons are those regions of a transcript that are present in the mature mRNA, whereas introns get spliced out from the primary RNA transcript and are not part of the mature mRNA Introns are not junk –they can encode regulatory information Introns are more common in higher eukaryotes than in lower ones
Introns: Genes in Pieces Introns were discovered, because a discrepancy between mRNA size and gene size was observed for some genes They were even visualized by hybridization experiments, in which the mRNA of a gene was annealed to its corresponding coding DNA strand in its genomic context The mRNA of the adenovirus hexon gene hybridized to the DNA