Specific binding of monomeric bacteriophage T3 and T7 RNA polymerases to their respective cognate promoters requires the initiating ribonucleoside triphosphate (GTP).
Bacteriophage T3 and T7 RNA polymerases are monomeric proteins of Mr of about 100,000. Each polymerase has stringent specificity for its own promoters that is present only on the homologous phage DNA template. Neither enzyme recognizes the heterologous phage promoters or Escherichia coli RNA polymerase promoters. In the present study, the interaction of T3 and T7 RNA polymerases with their respective cognate promoters was studied by DNase I footprinting techniques. These studies revealed an absolute requirement for the initiating nucleotide (GTP) for each phage RNA polymerase to bind specifically to and protect its cognate promoter from DNase I digestion. In the absence of the initiating nucleotide, both enzymes randomly bind DNA with lower affinity. No other nucleotide can substitute for GTP; however, the addition of GTP + ATP, which causes the synthesis of a hexamer RNA (pppGpGpGpApGpA), makes the DNA-RNA-protein complex highly stable. Nitrocellulose filter binding studies confirmed these observations. On the basis of these results we propose that the binding of the initiating nucleotide (in this case, GTP) drives the phage RNA polymerase into an "initiation conformation" in which the random DNA-binding property of the enzyme is converted to a promoter-specific recognition, and the polymerase is primed to initiate transcription.
D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D004279:DNA, Viral; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I