A Model for the Initiation of Transcription by DNA‐Dependent RNA Polymerase from Escherichia coli

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Abstract
The stable initiation complexes formed by interaction of DNA‐dependent RNA polymerase from Escherichia coli containing initiation factor σ with native DNAs of bacteriophages T4, T5 and T7 have been differentiated into a “labile‐starter” and a “stable‐starter” class. The former exhibits a half‐life of 1 min with T5‐DNA and of 10 min with T4‐DNA under standard conditions, the latter a half‐life of more than 100 min for the first‐order decay in the presence of the strong template‐competitor heparin. The labile class is competed out by core enzyme, the stable is not. The relative amounts of both classes of complexes depend on ionic strength.The stable‐starter class has been subdivided into a “delayed‐starter” complex formed rapidly and already possessing high stability in the presence of heparin but unable to directly initiate and an “immediate‐starter” complex as a first‐order transition product of the delayed‐starter state. From the immediate‐starter complex initiation can proceed immediately. The time for transition of half of the former into the latter complex is 3.5 min with T5‐DNA. The temperature dependence of the equilibrium concentration of the stable‐starter complex reveals a sigmoid shape indicating a highly cooperative event, probably a conformational change in the transition from delayed to immediate starter.From the length‐average chain length of the transcripts of T5‐DNA in vitro and the length of the transcribed fraction of the DNA an approximate number of 14 promoters per DNA molecule was estimated. For T4‐DNA, calculation yields about 6. In excess of RNA polymerase about 27 copies of transcript per T5‐ “promoter” were produced from the stable complex under conditions excluding consecutive initiation by free enzyme and reinitiation. The corresponding value for T4‐DNA is between 1 and 4.On the basis of these results, a model for the initiation of transcription is postulated, in which an initiation site on the DNA (promoter) is subdivided into an clement for primary recognition, the entry site R, an element for the storage of enzyme for immediate initiation, termed storage stretch S, which furnishes space for the stable alignment of a certain number of polymerase molecules, and an element for actual initiation, termed initiator I.