Weakening of the T7 promoter-polymerase interaction facilitates promoter release.
During transcription initiation, RNA polymerases retain interactions with their promoters until the RNA is extended to 8-13 nucleotides, at which point the polymerase releases the promoter and moves downstream. It has been shown that release of the T7 promoter is inhibited when the T7 RNA polymerase-promoter interaction is strengthened. Here we asked whether release would be facilitated when the T7 promoter-polymerase interaction is weakened by the introduction of promoter mutations known to reduce promoter activity. Using chemical and enzymatic probes to monitor the position of the polymerase as a function of RNA length, we found that promoter mutations upstream of -4 facilitated release of the polymerase from the promoter, but more downstream mutations did not have such effects. We also found that released complexes turn over more slowly than promoter-bound complexes, indicating that retention of promoter interactions contributes to the dissociation of short RNAs during initial transcription.
Guo
Qing
Q
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Sousa
Rui
R
D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D034741:RNA, Small Interfering; D014764:Viral Proteins; D017665:Hydroxyl Radical; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003851:Deoxyribonucleases