title: ADP-ribosylation and early transcription regulation by bacteriophage T4.
abstract: Bacteriophage T4 codes at least for two ADP-ribosylating activities, the 76 kDa Alt and the 24 kDa Mod gene products. The main target for both enzymes is the host RNA polymerase. We cloned and sequenced the alt gene and overexpressed the corresponding enzyme. The recombinant protein shows ADP-ribosylating activities in vitro, as had been described earlier for the native enzyme isolated from phage heads. The native as well as the recombinant protein ADP-ribosylate the alpha-subunit of RNA polymerase, but also subunits beta, beta' and sigma 70 and perform an autoribosylation reaction. Taking advantage of the pKWIII test system, constructed to measure promoter strengths in vivo, it was found that ADP-ribosylation of RNA polymerase leads to an increase of transcription from T4 early promoters up to a factor of two. In an infected host cell this should cause an enhanced expression of T4 genes. Depending on whether RNA polymerase was ADP-ribosylated or not, it initiated transcription at T4 promoters with different sequence characteristics: unribosylated RNA polymerase recognizes the early T4 promoters by an extended -10 region, whereas the ribosylated enzyme selects for T4 early promoters with an extended T4-specific and highly conserved -35 region. These results may reflect how the virus, step by step imposes its genetic program on the host cell, and in part they give a rationale for the extension of the consensus sequence observed with these promoters. We also sequenced the genomic region of the T4 mod gene and found two open reading frames coding both for proteins of approximately 24 kDa. Up to now none of the reading frames could be cloned into E. coli in an active form, making it highly probable that the ADP-ribosylation pattern inflicted by gene product Mod on host RNA polymerase is deleterious to these bacteria. Comparisons of the amino acid sequences showed significant homologies among the two reading frames. Computer analysis reveals that both Mod sequences and also the sequence of the Alt protein exhibit a structural concordance with the catalytic domains of other prokaryotic ADP-mono-ribosyltransferases such as the Pseudomonas aeruginosa exotoxin A, the cholera labile enterotoxin, the diphteria toxin, the heat labile enterotoxin A of E. coli, and pertussis toxin. We present a detailed model for T4 transcription regulation.
authors.affiliation: Lehrstuhl für Biologie der Mikoorganismen, Ruhr-Universität Bochum, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Wilkens
authors.forename: B
authors.initials: B
authors.lastname: Tiemann
authors.forename: F
authors.initials: F
authors.lastname: Bazan
authors.forename: W
authors.initials: W
authors.lastname: Rüger
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004279:DNA, Viral; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D004279:DNA, Viral; D000246:Adenosine Diphosphate Ribose; D000244:Adenosine Diphosphate; D011065:Poly(ADP-ribose) Polymerases