Site-directed mutagenesis of an amino acid residue in the bacteriophage P2 ogr protein implicated in interaction with Escherichia coli RNA polymerase.
The P2 ogr gene encodes a 72-amino-acid protein required for P2 late gene expression. This gene was defined originally by a class of compensatory mutations which overcome the block to P2 late transcription imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of Escherichia coli RNA polymerase. Spontaneous compensatory ogr mutations substitute a Cys for a Tyr residue at amino acid 42 in the Ogr polypeptide. Using suppression of an ogr amber mutation and site-directed oligonucleotide mutagenesis, we have studied the effect of amino acid substitutions at this position in Ogr. Substitution of charged residues at this site renders Ogr protein inactive, in rpoA+ and rpoA109 strains. While 11 different amino acids are capable of replacing the wild-type Tyr-42 to allow P2 growth to varying degrees in a wild-type E. coli strain, only three of these allow phage growth in strains carrying the rpoA109 mutation. Phages carrying Cys or Ala in place of Tyr-42 gave burst sizes at least as high as P2 ogr+ in a rpoA+ strain; a Gly substitution also allowed P2 to grow in either a rpoA+ or rpoA109 background, but markedly reduced the burst size. These results are consistent with a direct interaction between Ogr and the alpha subunit of E. coli RNA polymerase in positive control of P2 late transcription, and indicate that the block imposed by the rpoA109 mutation is due to steric hindrance.
King
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RA
Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond 23298-0678.
Anders
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Christie
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012318:RNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D001426:Bacterial Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C074897:ogr protein, Enterobacteria phage P2; D012318:RNA Polymerase I