한빛사 논문
Yoo Jin Joo1, Scott B. Ficarro2,3, Yujin Chun1, Jarrod A. Marto2,3 and Stephen Buratowski1,*
1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA;
2 Department of Cancer Biology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA;
3 Blais Proteomics Center, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA
*Corresponding author
Abstract
RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4–Spt5, Paf1C, Spt6–Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.
Keywords : RNA polymerase II C-terminal domain (CTD), DSIF, Spt6, Paf1 complex, Cdk7, transcription elongation
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