Genomic location of the human RNA polymerase II general machinery: evidence for a role of TFIIF and Rpb7 at both early and late stages of transcription

被引:27
作者
Cojocaru, Marilena [1 ]
Jeronimo, Celia [1 ]
Forget, Diane [1 ]
Bouchard, Annie [1 ]
Bergeron, Dominique [1 ]
Cote, Pierre [1 ]
Poirier, Guy G. [2 ]
Greenblatt, Jack [3 ]
Coulombe, Benoit [1 ]
机构
[1] Inst Rech Clin Montreal, Lab Gene Transcript & Proteom, Montreal, PQ H2W 1R7, Canada
[2] Univ Laval, Ctr Hosp Univ QC, Quebec City, PQ, Canada
[3] Univ Toronto, Banting & Best Dept Med Res, Toronto, ON, Canada
基金
加拿大健康研究院;
关键词
chromatin immunoprecipitation; general transcription factor; genomic location; RNA polymerase II; tandem-affinity purification; transcription factor IIF (TFIIF);
D O I
10.1042/BJ20070751
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The functions ascribed to the mammalian GTFs (general transcription factors) during the various stages of the RNAPII (RNA polymerase II) transcription reaction are based largely on in vitro studies. To gain insight as to the functions of the GTFs in living cells, we have analysed the genomic location of several human GTF and RNAPII subunits carrying a TAP (tandem-affinity purification) tag. ChIP (chromatin immunoprecipitation) experiments using anti-tag beads (TAP-ChIP) allowed the systematic localization of the tagged factors. Enrichment of regions located close to the TIS (transcriptional initiation site) versus further downstream TRs (transcribed regions) of nine human genes, selected for the minimal divergence of their alternative TIS, were analysed by QPCR (quantitative PCR). We show that, in contrast with reports using the yeast system, human TFIIF (transcription factor IIF) associates both with regions proximal to the TIS and with further downstream TRs, indicating an in vivo function in elongation for this GTF. Unexpectedly, we found that the Rpb7 subunit of RNAPII, known to be required only for the initiation phase of transcription, remains associated with the polymerase during early elongation. Moreover, ChIP experiments conducted under stress conditions suggest that Rpb7 is involved in the stabilization of transcribing polymerase molecules, from initiation to late elongation stages. Together, our results provide for the first time a general picture of GTF function during the RNAPII transcription reaction in live mammalian cells and show that TFIIF and Rpb7 are involved in both early and late transcriptional stages.
引用
收藏
页码:139 / 147
页数:9
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