Molecular Analysis of RNA Polymerase II Elongation and Termination on Mammalian Genes

Download or read book Molecular Analysis of RNA Polymerase II Elongation and Termination on Mammalian Genes written by Kristopher Wade Brannan and published by . This book was released on 2014 with total page 154 pages. Available in PDF, EPUB and Kindle. Book excerpt: RNA polymerase II (pol II) pausing, elongation and termination are important mechanisms for controlling pol II distribution and transcriptional output during the transcription cycle on protein coding genes. This thesis focuses on mechanisms that influence pol II elongation through protein coding genes, and the state of elongating pol II during promoter proximal accumulation, elongation and termination. I address the following specific questions: 1) What factors are important for pol II termination? 2) What is the role of premature termination in limiting pol II elongation? 3) What is the role of CTD phosphorylation on pol II elongation and mRNA cleavage/polyadenylation? I report that decapping proteins and TTF2 interact with Xrn2 and that these factors localize by ChIP at 5' ends of genes. Knockdown of decapping and termination factors by shRNA caused a widespread re-positioning of pol II at 5' ends of genes away from start sites and toward distal positions both downstream and upstream. These results suggest that co-transcriptional decapping and premature termination by a torpedo mechanism is broadly employed to limit transcription of human genes. I also report that Fcp1 localizes at the 5' end of human genes and limits CTD phosphorylation at both Ser2P and Ser5P positions. Fcp1 knockdown caused a widespread redistribution of pol II at gene 5' ends away from transcription start sites (TSS) toward downstream positions, and localized increases in pol II Ser2P and Ser5P on highly expressed genes. Fcp1 knockdown also results in shifting in pA-site choice on ~1000 genes, primarily toward proximal positions. These results suggest that cotranscriptional dephosphorylation by Fcp1 is important for limiting both pol II elongation and usage of proximal alternative polyadenylation signals. Together these results have implications for new mechanisms regulating transcriptional control both at the elongation checkpoint and at the level of 3' end formation.