Ziegler Lynn M, Khaperskyy Denys A, Ammerman Michelle L, Ponticelli Alfred S
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214-3000, USA.
J Biol Chem. 2003 Dec 5;278(49):48950-6. doi: 10.1074/jbc.M309656200. Epub 2003 Sep 30.
Previous studies have shown that transcription factors IIB (TFIIB), IIF (TFIIF), and RNA polymerase II (RNAPII) play important roles in determining the position of mRNA 5'-ends in the yeast Saccharomyces cerevisiae. Yeast strains containing a deletion of the small, nonessential Rpb9 subunit of RNAPII exhibit an upstream shift in the positions of mRNA 5'-ends, whereas mutation of the large subunit of yeast TFIIF (Tfg1) can suppress downstream shifts that are conferred by mutations in TFIIB. In this study, we report an approach for the production of functional recombinant yeast holo-TFIIF (Tfg1-Tfg2 complex) and use of the recombinant protein in both reconstituted transcription assays and gel mobility shifts in order to investigate the biochemical alterations associated with the deltaRpb9 polymerase. The results demonstrated that upstream shifts in the positions of mRNA 5'-ends could be conferred by the deltaRpb9 RNAPII in transcription reactions reconstituted with highly purified yeast general transcription factors and, importantly, that these shifts are associated with an impaired interaction between the DeltaRpb9 polymerase and TFIIF. Potential mechanisms by which an altered interaction between the DeltaRpb9 RNAPII and TFIIF confers an upstream shift in the positions of mRNA 5'-ends are discussed.
先前的研究表明，转录因子IIB（TFIIB）、IIF（TFIIF）和RNA聚合酶II（RNAPII）在确定酿酒酵母中mRNA 5'端的位置方面发挥着重要作用。含有RNAPII小的、非必需Rpb9亚基缺失的酵母菌株在mRNA 5'端的位置上表现出上游移位，而酵母TFIIF大亚基（Tfg1）的突变可以抑制由TFIIB突变导致的下游移位。在本研究中，我们报道了一种生产功能性重组酵母全酶TFIIF（Tfg1 - Tfg2复合物）的方法，并将重组蛋白用于重组转录测定和凝胶迁移率变动分析，以研究与ΔRpb9聚合酶相关的生化改变。结果表明，在由高度纯化的酵母通用转录因子重构的转录反应中，ΔRpb9 RNAPII可导致mRNA 5'端位置的上游移位，重要的是，这些移位与ΔRpb9聚合酶和TFIIF之间相互作用受损有关。本文讨论了ΔRpb9 RNAPII与TFIIF之间相互作用改变导致mRNA 5'端位置上游移位的潜在机制。
