Abstract
Transcription initiation has been assumed to be a multi-step sequential process, although additional steps could exist. Initiation from the T7A1 promoter, in particular, apparently behaves in vitro in a manner that can be fully explained by the sequential pathway. However, initiation from the lambda P(R)AL promoter has been shown to follow a branched pathway from which a part of the enzyme-promoter complex is arrested at the promoter raising the question as to which mechanism is general. We found that a moribund complex, characteristic of the arrested branch, is formed at the T7A1 promoter, especially in low salt condition indicating that the initiation mechanism for this promoter is also branched. The results of DNA footprinting suggested that holoenzyme in the moribund complex is dislocated on DNA from the position of productive complex. However, only a small fraction of the binary complex becomes arrested at this promoter, and the interconversion between subspecies of binary complex is apparently more reversible than at the lambda P(R)AL promoter, which explains why the reaction pathway appears to be sequential. These findings suggest a generality of the branched pathway mechanism, which would resolve contradictory observations that have been reported for various promoters.
Highlights
Transcription initiation in prokaryotes includes at least four events: 1) the binding of holoenzyme to a promoter; 2) the isomerization of the resulting complex accompanied by strand opening; 3) the iterative synthesis and release of abortive transcripts; and 4) the achievement of continuous elongation accompanied by the escape of the enzyme from the promoter
We found that a moribund complex, characteristic of the arrested branch, is formed at the T7A1 promoter, especially in low salt condition indicating that the initiation mechanism for this promoter is branched
Promoter is the occurrence of persistent abortive synthesis. Because this phenomenon disappears in a high salt condition [3], lower salt conditions might favor the formation of the moribund complex at the T7A1 promoter
Summary
Synthesized in single-round transcription and production of abortive transcripts should cease before the full-length synthesis is completed. This situation occurs at the PRAL promoter in the presence of the Gre factors and a high concentration of initiating nucleotide [6]. We consistently observe that the interconversion among subspecies of binary complex at the T7A1 promoter is more reversible than at the PRAL promoter. These findings suggest a generality of the branched pathway mechanism that can explain the seemingly contradictory characteristics of various promoters
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