Multiple roles for the T7 promoter nontemplate strand during transcription initiation and polymerase release.
Transcription initiation begins with recruitment of an RNA polymerase to a promoter. Polymerase-promoter interactions are retained until the nascent RNA is extended to 8-12 nucleotides. It has been proposed that accumulation of "strain" in the transcription complex and RNA displacement of promoter-polymerase interactions contribute to releasing the polymerase from the promoter, and it has been further speculated that too strong a promoter interaction can inhibit the release step, whereas a weak interaction may facilitate release. We examined the effects of partial deletion of the nontemplate strand on release of T7 RNA polymerase from the T7 promoter. T7 polymerase will initiate from such partially single-stranded promoters but binds them with higher affinity than duplex promoters. We found that release on partially single-stranded promoters is strongly inhibited. The inhibition of release is not due to an indirect effect on transcription complex structure or loss of specific polymerase-nontemplate strand interactions, because release on partially single-stranded templates is recovered if the interaction with the promoter is weakened by a promoter base substitution. This same substitution also appears to allow the polymerase to escape more readily from a duplex promoter. Our results further suggest that template-nontemplate strand reannealing drives dissociation of abortive transcripts during initial transcription and that loss of interactions with either the nontemplate strand or duplex DNA downstream of the RNA lead to increased transcription complex slippage during initiation.
Guo
Qing
Q
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Sousa
Rui
R
D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D009692:Nucleic Acid Heteroduplexes; D011401:Promoter Regions, Genetic / Q000502:physiology*; D024363:Transcription Initiation Site / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology*; D014764:Viral Proteins
D009692:Nucleic Acid Heteroduplexes; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases