A zinc-binding region in the beta' subunit of RNA polymerase is involved in antitermination of early transcription of phage HK022.
Antitermination of early transcription in phage HK022 requires no virus-encoded proteins and thus differs from antitermination by other lambdoid phages. It does require cis-acting phage sequences, which may be analogous to the lambdoid nut sites. To identify host proteins involved in antitermination, we isolated 14 Escherichia coli mutants that are specifically blocked in HK022 growth. The mutations are located in the rpoC gene, which encodes the beta' subunit of RNA polymerase. Each mutation alters one of three amino acid residues located within a cluster of four completely conserved cysteine residues that are believed to bind zinc. We examined the effect of one mutation on HK022 antitermination in vivo. rpoCY75N greatly reduced readthrough of a strong rho-independent transcription terminator placed downstream of the HK022 PL promoter and nutL analog, but did not decrease promoter activity. Purified enzyme had a similar effect on PL-directed transcription in vitro: wild-type but not mutant polymerase read through a strong rho-independent terminator located immediately downstream of the nutL analog with high efficiency. We suggest that interaction of the putative zinc-binding domain of the RNA polymerase beta' subunit with the HK022 antitermination sites suppresses transcription termination, and that this interaction can occur in the absence of other proteins.
Clerget
M
M
Section on Microbial Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Jin
D J
DJ
Weisberg
R A
RA
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics* / Q000254:growth & development; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000821:virology; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D008242:Lysogeny; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D013728:Terminator Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D015032:Zinc / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D012321:DNA-Directed RNA Polymerases; D015032:Zinc; D003545:Cysteine