[대학원 생명과학과 세미나 안내] 

연사 : Stephen Buratowski 교수(Harvard Medical School)

연제 : Dynamics of RNA Polymerase II Transcription

일시 : 2018년 4월 27일 (금) 오후 5시 

장소 : 하나과학관 A동 B131호

초청교수 : 김준 교수

Abstract

Many proteins associate with RNA polymerase II during gene expression.  Chromatin immunoprecipitation (ChIP) has been useful for constructing current models for how these factors exchange during the transcription cycle, but this technique has limited temporal and spatial resolution.  The location of crosslinking along the promoter-terminator axis is typically used as a proxy for time after initiation, but can also reflect positional cues such as the promoter or introns.  Factors crosslinking to the same position may be bound simultaneously, sequentially, or mutually exclusively.  
To better study dynamics of factor interactions, we used immobilized template reactions coupled with quantitative mass spectrometry. Yeast nuclear extracts were incubated with either naked or chromatinized DNA templates, and complexes were isolated at various time points before or after addition of NTPs. Proteomic analysis of pre-initiation, early elongation, and late elongation complexes shows our system reproduces many expected features of the transcription cycle. These include Rpb1 CTD phosphorylations, association of mRNA processing and elongation factors, and histone co-transcriptional histone modifications. Some elongation factors are only detected on chromatin templates, suggesting they interact with the nucleosomes during elongation rather than directly with the polymerase itself. A subset of factors depends on CTD phosphorylation. Interestingly, our results indicate the transcription cycle progresses as a function of time after initiation, rather than as a function of a distance from the promoter. In vivo ChIP results using polymerase mutants with altered elongation rates validate this finding.
Most surprisingly, following the first round of transcription we observed increased binding of TFIID TAF subunits to downstream promoter regions.  In vitro transcription and in vivo ChIP experiments suggest that TAF binding acts to promote subsequent re-initiation. Our results suggest a simple model to explain why some promoters show greater dependence on TAFs than others.