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Nucleic Acids Research Advance Access published online on December 10, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm1086
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Structure–function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding and RNA displacement

Souad Naji1, Michela G. Bertero2, Patrizia Spitalny1, Patrick Cramer2,* and Michael Thomm1,*

1Lehrstuhl für Mikrobiologie und Archaeenzentrum, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg and 2Gene Center Munich and Center for integrated Protein Science CiPS M, Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 München, Germany

*To whom correspondence should be addressed: Tel: 49-941-943-3160; Fax: 49-941-943-2403; Email: michael.thomm{at}biologie.uni-regensburg.de *Correspondence may also be addressed to: Tel: 49 89 2180 76951; Fax: 49 89 2180 76999; Email: cramer{at}lmb.uni-muenchen.de.

Received September 14, 2007. Revised November 14, 2007. Accepted November 19, 2007.

The active center clefts of RNA polymerase (RNAP) from the archaeon Pyrococcus furiosus (Pfu) and of yeast RNAP II are nearly identical, including four protruding loops, the lid, rudder, fork 1 and fork 2. Here we present a structure–function analysis of recombinant Pfu RNAP variants lacking these cleft loops, and analyze the function of each loop at different stages of the transcription cycle. All cleft loops except fork 1 were required for promoter-directed transcription and efficient elongation. Unprimed de novo transcription required fork 2, the lid was necessary for primed initial transcription. Analysis of templates containing a pre-melted bubble showed that rewinding of upstream DNA drives RNA separation from the template. During elongation, downstream DNA strand separation required template strand binding to an invariant arginine in switch 2, and apparently interaction of an invariant arginine in fork 2 with the non-template strand.


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