摘要：

A method was devised for directing RNA polymerase on a single promoter site on T7 DNA. Initiation complexes were formed on each of the three main promoter sites using one dinucleotide plus one nucleoside triphosphate. The ternary initiation complexes are resistant to rifampicin action, to inhibition by (rI) n at 0 °C and are stable at high salt concentrations. A minimum of a trinucleotide is required to form a stable ternary complex. To determine which promoter site was selected by RNA polymerase during initiation, the (rI) n -resistant RNA was digested by RNAse III to generate three characteristic initiator RNA fragments, resolved by gel electrophoresis. The three major promoter sites could be selected individually by using different primer and substrate combinations: ApC + ATP selected promoter A 3 , CpG + CTP selected A 2 and CpC + ATP specified preferentially A 1 . A number of primer-substrate combinations specified each site at low salt concentration but the substrate requirement became very stringent at high salt concentration, suggesting that the postulated local opening of the promoter site could be more or less extensive, depending on the ionic strength. The minimum opening observed at high salt concentration corresponded to the insertion of a leader trinucleotide sequence. The promoter region melted by RNA polymerase at low salt concentration was (G + C)-rich and corresponded to about 9 to 11 base pairs. Sequences of the melting recognition regions were tentatively inferred from the results.

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