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J. Biol. Chem., Vol. 282, Issue 50, 36582-36592, December 14, 2007

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A Tunable Ratchet Driving Human RNA Polymerase II Translocation Adjusted by Accurately Templated Nucleoside Triphosphates Loaded at Downstream Sites and by Elongation Factors^*

From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319

When nucleoside triphosphate (NTP) substrates and -amanitin are added to a human RNA polymerase II elongation complex simultaneously, the reaction becomes stalled in the core of the bond synthesis mechanism. The mode of stalling is influenced by NTP substrates at the active site and at downstream sites and by transcription factor IIF (TFIIF) and TFIIS. NTP substrates templated at i+2, i+3, and i+4 downstream DNA sites can reverse the previously stable binding of an NTP loaded at the i+1 substrate site. Deoxy-(d)NTPs and NDPs (nucleoside diphosphates) do not substitute for NTPs at the i+2 and i+3 positions (considered together) or the i+4, i+5, and i+6 positions (considered together). The mode of stalling is altered by changing the number of downstream template sites that are accurately occupied by NTPs and by changing NTP concentration. In the presence of the translocation blocker -amanitin, a steady state condition is established in which RNA polymerase II stably loads an NTP substrate at i+1 and forms a phosphodiester bond but cannot rapidly complete bond synthesis by releasing pyrophosphate. These observations support a role for incoming NTP substrates in stimulating translocation; results appear inconsistent with the secondary pore being the sole route of NTP entry for human RNA polymerase II, and results indicate mechanisms of dynamic error avoidance and error correction during rapid RNA synthesis.

Received for publication, August 21, 2007 , and in revised form, September 10, 2007.

^* This work was supported by Grant GM 057461 from the National Institutes of Health (to Z. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental materials and Figs. S1-S3.

¹ Faculty member of the Agricultural Experimental Station and the College of Osteopathic Medicine, Michigan State University. To whom correspondence should be addressed. Tel.: 517-353-0859; Fax: 517-353-9334; E-mail: burton{at}msu.edu.

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