Topology of the product binding site in RNA polymerase revealed by transcript slippage at the phage lambda PL promoter.
In the presence of transcription substrates ATP, CTP, and UTP, a stable ternary complex containing tetranucleotide AUCA is formed on the phage lambda PL promoter (starting sequence C-3A-2C-1A+1U+2C+3A+4G+5). We show that in the absence of GTP or at undersaturating GTP concentrations the AUCA transcript synthesized at the +1 to +4 segment slips back by 3 nucleotides and is stabilized in the ternary complex in such a way that only its 2 3'-proximal bases remain paired to the -1/+1 positions of the template DNA. The slipped transcript can be extended in a template-directed manner into longer chains that can be cleaved by the GreA or GreB proteins at the +1/+2 junction. The slipped stabilized tetranucleotide delineates the "tight product binding site" of RNA polymerase responsible for stable holding of the transcript in the ternary transcription complex. The results suggest that the tight product binding site encompasses the locality within the complex where the nascent transcript detaches from the template strand of DNA.
Severinov
K
K
Public Health Research Institute, New York, New York 10016.
Goldfarb
A
A
D001426:Bacterial Proteins / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C079667:GreB protein, E coli; D046911:Macromolecular Substances; D010445:Peptide Elongation Factors; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C075886:GREA protein, Rickettsia prowazekii; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases