Bacteriophage N4 virion RNA polymerase interaction with its promoter DNA hairpin

doi:10.1073/pnas.0610627104

Bacteriophage N4 virion RNA polymerase interaction with its promoter DNA hairpin

*Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637; and
^†Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853

Edited by Carol A. Gross, University of California, San Francisco, CA, and approved March 6, 2007 (received for review November 30, 2006)

Abstract

Bacteriophage N4 minivirion RNA polymerase (mini-vRNAP), the RNA polymerase (RNAP) domain of vRNAP, is a member of the T7-like RNAP family. Mini-vRNAP recognizes promoters that comprise conserved sequences and a 3-base loop–5-base pair (bp) stem DNA hairpin structure on single-stranded templates. Here, we defined the DNA structural and sequence requirements for mini-vRNAP promoter recognition. Mini-vRNAP binds a 20-nucleotide (nt) N4 P2 promoter deoxyoligonucleotide with high affinity (K _d = 2 nM) to form a salt-resistant complex. We show that mini-vRNAP interacts specifically with the central base of the hairpin loop (−11G) and a base at the stem (−8G) and that the guanine 6-keto and 7-imino groups at both positions are essential for binding and complex salt resistance. The major determinant (−11G), which must be presented to mini-vRNAP in the context of a hairpin loop, appears to interact with mini-vRNAP Trp-129. This interaction requires template single-strandedness at positions −2 and −1. Contacts with the promoter are disrupted when the RNA product becomes 11–12 nt long. This detailed description of vRNAP interaction with its promoter hairpin provides insights into RNAP–promoter interactions and explains how the injected vRNAP, which is present in one or two copies, recognizes its promoters on a single copy of the injected genome.

T7-like RNA polymerase

Footnotes

^§To whom correspondence should be addressed at:
Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, CLSC 613, Chicago, IL 60637.
E-mail: lbrd{at}uchicago.edu
Author contributions: E.K.D., T.J.S., and L.B.R.-D. designed research; E.K.D. performed research; T.J.S. contributed new reagents/analytic tools; E.K.D. and L.B.R.-D. analyzed data; and L.B.R.-D. wrote the paper.
↵ ^‡Present address: Department of Microbiology, Ohio State University, Columbus, OH 43210.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations:

bATP,

hydroxybenzaldehyde ester of ATP;

bGTP,

hydroxybenzaldehyde ester of GTP;

5-IdU,

5-iododeoxyuracil;

mini-vRNAP,

mini-virion RNA polymerase;

NTCB,

2-nitro-5-thiocyanobenzoic acid;

RNAP,

RNA polymerase;

SC,

solution competition;

SPR,

surface plasmon resonance.