Bacteriophage N4 virion RNA polymerase interaction with its promoter DNA hairpin.
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.
Davydova
Elena K
EK
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA.
Santangelo
Thomas J
TJ
Rothman-Denes
Lucia B
LB
D025522:Bacteriophage N4 / Q000201:enzymology*; D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006147:Guanine; D006860:Hydrogen Bonding / Q000187:drug effects; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009711:Nucleotides; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D012492:Salts / Q000494:pharmacology; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects; D014364:Tryptophan; D014771:Virion / Q000201:enzymology*
D004279:DNA, Viral; D009711:Nucleotides; D012492:Salts; D006147:Guanine; D014364:Tryptophan; D012321:DNA-Directed RNA Polymerases