Conformational switching of Escherichia coli RNA polymerase-promoter binary complex is facilitated by elongation factor GreA and GreB.
BACKGROUND
The initiation arrest at a modified lambdaPR promoter is caused by irreversible divergence of the reaction pathway into productive and arrested branches. Escherichia coli GreA and GreB induce cleavage of the nascent transcript and relieve arrest in elongation. They also reduce abortive synthesis at several promoters and relieve initiation arrest. Their mechanism of action during initiation, and its relationship to the branched initiation pathway are unknown.


RESULTS
The Gre factors mitigated initiation arrest only when they were added to the binary complex of the holoenzyme bound to the lambdaPR promoter, prior to RNA synthesis. They exerted little effect when they were added to ternary initiation complexes. They accelerated the exchange of the binary complex with its free components by 6-9-fold. When they are present, a high concentration of the initiating nucleotide increased yield of the full-length transcript, whereas a low concentration did not.


CONCLUSIONS
All the results presented above can be explained by a model where the productive and arrested pathways diverge at the binary complex stage. The Gre factors relieve the initiation arrest by introducing reversibility between subspecies of the binary complex that are precursors of the two pathways. RNA cleavage is unlikely to cause relief of initiation arrest.
Sen
R
R
Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka-411, Japan.
Nagai
H
H
Shimamoto
N
N
D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D066298:In Vitro Techniques; D008957:Models, Genetic; D009154:Mutation; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000378:metabolism; D013698:Templates, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
D029968:Escherichia coli Proteins; C079668:GreA protein, E coli; C079667:GreB protein, E coli; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D012321:DNA-Directed RNA Polymerases