Enzymatic properties of a proteolytically nicked RNA polymerase of bacteriophage T7.
The RNA polymerase of bacteriophage T7 is sensitive to cleavage by a protease associated with the membrane fraction of many strains of Escherichia coli. A major degradation product is a T7 RNA polymerase that is proteolytically cleaved between amino acids 172 (lysine) and 173 (arginine) (Tabor, S., and Richardson, C.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078). The cleavage splits the enzyme into a large fragment (Mr approximately 75,000) and a small fragment (Mr approximately 23,000) which remain tightly associated during the purification of nicked RNA polymerase. The protein retains RNA polymerase activity, but specific activity is reduced 3.5-fold. The proteolytic cleavage also reduces the Mg2+ requirements, increases the apparent Michaelis-Menten constants for the utilization of the ribonucleoside 5'-triphosphates, increases the temperature sensitivity, increases the sensitivity to inhibition by heparin, and increases the probability that a transcript will not be removed from the template. The reduced activity of nicked T7 RNA polymerase is apparently a consequence of inefficient initiation and premature termination. Nicked T7 RNA polymerase successfully initiates at the phi 10 promoter at half the efficiency of native T7 RNA polymerase. Transcripts synthesized by the nicked enzyme are also significantly shorter than transcripts synthesized by the native enzyme. In contrast, nicked T7 RNA polymerase and T7 RNA polymerase exhibit equivalent poly(dI).poly(dC)-dependent activity and equivalent polymerization velocities (60 bases/s at 25 degrees C).
D002462:Cell Membrane / Q000201:enzymology; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005796:Genes; D005814:Genes, Viral; D007700:Kinetics; D010447:Peptide Hydrolases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic
D012321:DNA-Directed RNA Polymerases; D010447:Peptide Hydrolases