RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes.
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 moves away from the start site. The enzyme undergoes a sequence of structural changes during initiation and transition to an elongating complex. Complexes with five to eight nucleotide transcripts, designated initial transcribing complexes (ITC), have identical footprints; they all retain the sigma factor and have a slightly extended DNase I footprint (-57 to +24) as compared to the open promoter complex (-57 to +20). ITC complexes all show a region of marked DNase I hypersensitivity in the -25 region that may reflect bending or distortion of the DNA template. Complexes with 10 or 11 nucleotide transcripts, designated initial elongating complexes (IEC), have lost the sigma factor and have a slightly reduced and shifted DNase I footprint (-32 to +30). However, these IEC have not yet achieved the much smaller footprint (approximately 30 bp) reported as characteristic of elongating ternary complexes bearing longer RNA chains. During the initial phase of transcription, the RNA polymerase does not move monotonically along the DNA template as RNA chains are extended, but instead, the upstream and downstream contacts remain more or less fixed as the nascent transcript is elongated up to about eight nucleotides in length. Only after incorporation of 10 nucleotides is there significant movement of the enzyme away from the promoter region and a commitment to elongation.
Krummel
B
B
Department of Biochemistry, University of California, Berkeley 94720.
Chamberlin
M J
MJ
D001483:Base Sequence; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012313:RNA / Q000096:biosynthesis*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D012313:RNA; D012321:DNA-Directed RNA Polymerases