Identification of bacteriophage N4 virion RNA polymerase-nucleic acid interactions in transcription complexes.
Bacteriophage N4 mini-virion RNA polymerase (mini-vRNAP), the 1106-amino acid transcriptionally active domain of vRNAP, recognizes single-stranded DNA template-containing promoters composed of conserved sequences and a 3-base loop-5-base pair stem hairpin structure. The major promoter recognition determinants are a purine located at the center of the hairpin loop (-11G) and a base at the hairpin stem (-8G). Mini-vRNAP is an evolutionarily highly diverged member of the T7 family of RNAPs. A two-plasmid system was developed to measure the in vivo activity of mutant mini-vRNAP enzymes. Five mini-vRNAP derivatives, each containing a pair of cysteine residues separated by approximately 100 amino acids and single cysteine-containing enzymes, were generated. These reagents were used to determine the smallest catalytically active polypeptide and to map promoter, substrate, and RNA-DNA hybrid contact sites to single amino acid residues in the enzyme by using end-labeled 5-iododeoxyuridine- and azidophenacyl-substituted oligonucleotides, cross-linkable derivatives of the initiating nucleotide, and RNA products with 5-iodouridine incorporated at specific positions. Localization of functionally important amino acid residues in the recently determined crystal structures of apomini-vRNAP and the mini-vRNAP-promoter complex and comparison with the crystal structures of the T7 RNAP initiation and elongation complexes allowed us to predict major rearrangements in mini-vRNAP in the transition from transcription initiation to elongation similar to those observed in T7 RNAP, a task otherwise precluded by the lack of sequence homology between N4 mini-vRNAP and T7 RNAP.
Davydova
Elena K
EK
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Kaganman
Irene
I
Kazmierczak
Krystyna M
KM
Rothman-Denes
Lucia B
LB
D025522:Bacteriophage N4 / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009696:Nucleic Acids / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D014771:Virion / Q000378:metabolism*
D009696:Nucleic Acids; D010455:Peptides; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases