Inhibition of T7 RNA polymerase by T7 lysozyme in vitro.
The in vivo observation that the expression of bacteriophage T7 gene 3.5 (T7 lysozyme) inactivates T7 class II transcription and the in vitro observation that T7 lysozyme inhibits T7 RNA polymerase lead to the hypothesis that T7 lysozyme might preferentially inhibit transcription from T7 class II promoters. T7 lysozyme was cloned into a lambda pL expression vector, overproduced in Escherichia coli, and purified. The ability of purified T7 lysozyme to inhibit transcription from T7 DNA, the cloned T7 class II promoters, phi 2.5 and phi 4.7, and the cloned class III promoter, phi 10, was measured in vitro. It was observed that the effectiveness of T7 lysozyme as an inhibitor of T7 RNA polymerase is inversely related to the concentration of Mg2+; T7 lysozyme inhibits T7 RNA polymerase most effectively at low Mg2+ concentrations. In addition, no preferential inhibition of transcription from cloned T7 class II promoters was observed, nor was a strong T7 class III promoter preferred when transcriptional capacity was reduced by T7 lysozyme. These observations contradict the hypotheses that the temporal control of T7 gene expression is either due to direct and selective inhibition of the T7 class II promoters by T7 lysozyme or to preferential transcription of the strong T7 class III promoters when transcriptional capacity is reduced by T7 lysozyme. It appears that alternative mechanisms such as the involvement of additional proteins and/or cellular conditions to enhance transcription from T7 class III promoters or to inhibit transcription from T7 class II promoter are necessary to explain the temporal control of transcription of bacteriophage T7.
Ikeda
R A
RA
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400.
Bailey
P A
PA
D017123:Bacteriophage T7 / Q000201:enzymology*; D003001:Cloning, Molecular; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005814:Genes, Viral; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins
D004279:DNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D009238:N-Acetylmuramoyl-L-alanine Amidase