Overexpression, purification, and characterization of the ADP-ribosyltransferase (gpAlt) of bacteriophage T4: ADP-ribosylation of E. coli RNA polymerase modulates T4 "early" transcription.
The bacteriophage T4 Alt gene product is a component of the phage head and enters the host cell in the process of infection together with the phage DNA. It immediately ADP-ribosylates host RNA polymerase, presumably at only one of the two alpha-subunits. Transcription from T4 "early" promoters, therefore, might be catalyzed, at least in part, by an altered RNA polymerase. The T4 alt gene was cloned into the expression vector pBluescript. E. coli cells, transformed with this recombinant vector, overexpressed the 76 kDa Alt gene product, which was purified to homogeneity. The purified enzyme not only ADP-ribosylates the alpha-subunit of RNA polymerase, but also subunits beta and beta', as well as the sigma 70-factor. The recombinant enzyme behaved like the native enzyme isolated from mature phage particles. The effect of the ribosylation reaction on the transcription activity of host RNA polymerase was investigated in vivo. It results in a modulation of T4 "early" promoter strengths, presumably, in a number of cases, leading to an overexpression of T4 "early" genes. The degree of overexpression, in some cases, should reach 50%, and seems to be well dosed for each promoter, controlling an individual transcription unit.
Koch
T
T
Arbeitsgruppe Molekulare Genetik, Lehrstuhl Biologie der Mikroorganismen, Ruhr-Universität Bochum, Germany.
Raudonikiene
A
A
Wilkens
K
K
Rüger
W
W
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism*; D002874:Chromosome Mapping; D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D011065:Poly(ADP-ribose) Polymerases; D012321:DNA-Directed RNA Polymerases