Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli.
The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Res. 16, 9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3-->ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.
Borukhov
S
S
Public Health Research Institute, New York, New York 10016.
Sagitov
V
V
Josaitis
C A
CA
Gourse
R L
RL
Goldfarb
A
A
D003080:Cold Temperature; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D009841:Oligonucleotides / Q000378:metabolism; D009994:Osmolar Concentration; D011401:Promoter Regions, Genetic; D012335:RNA, Ribosomal / Q000235:genetics*; D012808:Sigma Factor / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
D009841:Oligonucleotides; D012335:RNA, Ribosomal; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases