RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes.

Biochemistry
B Krummel, M J Chamberlin

Abstract

We have studied the properties and structures of a series of Escherichia coli RNA polymerase ternary complexes formed during the initial steps of RNA chain initiation and elongation. Five different templates were used that contained the bacteriophage T7 A1 promoter or the E. coli Tac or the lac UV5 promoter, as well as variant templates with alterations in the initial transcribed regions. The majority of ternary complexes bearing short transcripts (from two to nine nucleotides) are highly unstable and cannot be easily studied. This includes transcripts from the phage T7 A1 promoter, for which the stability of complexes bearing transcripts as short as four nucleotides has previously been postulated. However, with one Tac promoter template, RNA polymerase forms ternary complexes with transcripts as short as five nucleotides that are stable enough for biochemical study. We describe several approaches to identifying and isolating such stable complexes and show that stringent criteria are needed in carrying out such experiments if the results are to be meaningful. Deoxyribonuclease I (DNase I) footprinting has been used to probe the general structure of the stable ternary complexes formed as the polymerase begins transcription and m...Continue Reading

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