The carboxyl-terminal domain of RNA polymerase U contains a tandemly repeated heptapeptide sequence. Previous work has show that this sequence is phosphorylated at multiple sites by a template-associated protein kinase, in a reaction that is closely associated with the Initiation of RNA synthesis. We have purified this kinase to apparent homogeneity from human (HeLa) cells. (4,5). The CTD is composed of repeats of the heptapeptide (SerPro-Thr-Ser-Pro-Ser-Tyr) (6, 7). Phosphorylation occurs cooperatively at multiple serine and threonine residues and induces a conformational change, as evidenced by altered immunoreactivity and sedimentation rate (8,9). The CTD is essential in yeast, Drosophila, and cultured murine cells (10-13). Mutations in the CTD impair the response of RNAP II to certain transcriptional activator proteins (14)(15)(16) ITo whom reprint requests should be addressed. 11920The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
TFIIH is a multifunctional RNA polymerase II transcription factor that possesses DNA-dependent ATPase, DNA helicase, and protein kinase activities. Previous studies have established that TFIIH enters the preinitiation complex and fulfills a critical role in initiation by catalyzing ATP-dependent formation of the open complex prior to synthesis of the first phosphodiester bond of nascent transcripts. In this report, we present direct evidence that TFIIH also controls RNA polymerase II activity at a postinitiation stage of transcription, by preventing premature arrest by very early elongation complexes just prior to their transition to stably elongating complexes. Unexpectedly, we observe that TFIIH is capable of entering the transcription cycle not only during assembly of the preinitiation complex but also after initiation and synthesis of as many as four to six phosphodiester bonds. These findings shed new light on the role of TFIIH in initiation and promoter escape and reveal an unanticipated f lexibility in the ability of TFIIH to interact with RNA polymerase II transcription intermediates prior to, during, and immediately after initiation.TFIIH is a multifunctional protein (1) that was originally identified by its requirement in transcription initiation by RNA polymerase II. In addition to its role in initiation, TFIIH has been shown to play a key role in nucleotide excision repair (NER) of damaged DNA and to possesses DNA-dependent ATPase and DNA helicase activities, as well as a protein kinase activity capable of phosphorylating the carboxylterminal domain (CTD) of the largest RNA polymerase II subunit. The two largest subunits of TFIIH are ATPdependent DNA helicases encoded by the NER XP-B and XP-D genes, and the TFIIH-associated CTD kinase is composed of the kinase͞cyclin pair cdk7͞cyclin H.Substantial evidence argues that TFIIH enters the RNA polymerase II preinitiation complex (2, 3) and activates it just prior to initiation, in a reversible reaction that is catalyzed by the TFIIH DNA helicase and involves ATP-dependent formation of an open complex that decays to a closed complex with a half-life of Ϸ40 s (4-11). In subsequent studies, our laboratory identified (12) a distinct postinitiation role for ATP in preventing premature arrest by very early RNA polymerase II elongation complexes prior to their transition to stably elongating complexes. We observed that, in the absence of a hydrolyzable ATP cofactor, a significant fraction of very early RNA polymerase II elongation complexes suffers arrest at promoter-proximal sites 10-14 bp downstream of the transcriptional start site. Further investigation revealed (i) that addition of ATP to transcription reactions prior to arrest by polymerase at these sites is sufficient to suppress arrest and (ii) that a fraction of arrested elongation complexes can be reactivated by addition of ATP.In this report, we present direct evidence that TFIIH mediates ATP-dependent suppression of arrest by very early RNA polymerase II elongation complexes. Characterization...
Isolated transcription complexes contain a protein kinase that phosphorylates the heptapeptide repeats of the carboxy-terminal domain (CTD) of the RNA polymerase II (RNAP II) large subunit in an apparently promoter-dependent manner. We now show that the essential features of this reaction can be reproduced in a reconstituted system containing three macromolecular components: a fusion piotein consisting of the CTD of RNAP II fused to a heterologous DNA-binding domain, an activating DNA fragment containing the recognition sequence for the fusion protein, and a protein kinase that binds nonspecifically to DNA. This kinase closely resembles a previously known DNA-dependent protein kinase. Evidently, the association of the CTD with DNA provides a key signal for phosphorylation. There appears to be no absolute requirement for specific contacts with other DNA-bound transcription factors.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.