Inhibition of T7 RNA polymerase: transcription initiation and transition from initiation to elongation are inhibited by T7 lysozyme via a ternary complex with RNA polymerase and promoter DNA.
The mechanism of transcription repression of T7 RNA polymerase by T7 lysozyme was investigated using a combination of kinetic and equilibrium methods. HPLC gel-filtration experiments demonstrated complex formation between T7 lysozyme, T7 RNA polymerase, and promoter DNA. The interactions between the two proteins were quantitated by measuring in real time the changes in protein fluorescence upon binary complex formation using stopped-flow kinetics. Complex formation between T7 lysozyme and the RNA polymerase was found to occur by a one-step process, with a bimolecular association rate constant of 38 microM-1 S-1 and a dissociation rate constant of 3.5 S-1. These constants provided an equilibrium dissociation constant, Kd, of 92 nM for the polymerase lysozyme complex. The interactions of the polymerase with the DNA were studied by stopped-flow kinetics and nitrocellulose equilibrium DNA binding experiments in the absence and in the presence of T7 lysozyme. The results showed that T7 lysozyme did not prevent or change the kinetic or thermodynamic interactions of the RNA polymerase with the DNA. T7 lysozyme by itself did not bind to the DNA, but since it bound to the RNA polymerase as well as to the polymerase DNA complex, transcription repression must involve the formation of the ternary complex between T7 lysozyme, T7 RNA polymerase and the promoter DNA. The effect of T7 lysozyme was most striking on runoff product synthesis which was greatly inhibited whereas the steady-state synthesis of abortive products, limited by polymerase cycling or RNA dissociation, was relatively unaffected by the presence of T7 lysozyme. Investigation of the pre-steady-state kinetics of transcription in the presence and absence of T7 lysozyme indicated that the inhibition of runoff product synthesis was largely due to inhibition of transcription initiation and transition from initiation to elongation.
Kumar
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A
Department of Biochemistry, Ohio State University, Columbus 43210, USA.
Patel
S S
SS
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D004279:DNA, Viral / Q000378:metabolism / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects*; D014764:Viral Proteins
D004279:DNA, Viral; D046911:Macromolecular Substances; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D009238:N-Acetylmuramoyl-L-alanine Amidase