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J. Biol. Chem., Vol. 281, Issue 27, 18677-18683, July 7, 2006

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Kinetic Investigation of Escherichia coli RNA Polymerase Mutants That Influence Nucleotide Discrimination and Transcription Fidelity^*

Shannon F. Holmes¹, Thomas J. Santangelo², Candice K. Cunningham, Jeffrey W. Roberts, and Dorothy A. Erie^¶3

From the Department of Chemistry and ^¶Curriculum in Applied and Materials Sciences, University of North Carolina, Chapel Hill, North Carolina 27599 and the Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853

Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue Asp⁶⁷⁵, which is >10Å away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues Arg⁶⁷⁸ and Asp⁸¹⁴ do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the Asp⁶⁷⁵ mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site.

Received for publication, January 18, 2006 , and in revised form, April 7, 2006.

^* This work was supported by National Institutes of Health Grants GM 54316 (to D. A. E.) and GM 21941 (to J. W. R.). 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.

¹ Current address: NIEHS/NIH, P. O. Box 12233, Research Triangle Park, North Carolina 27709.

² Current address: Dept. of Microbiology, Ohio State University, 468 Biological Sciences Bldg., 484 West 12th Ave., Columbus, OH 43210-1292.

³ To whom correspondence should be addressed. Tel.: 919-962-6370; Fax: 919-966-3675; E-mail: derie{at}unc.edu.

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