Extended upstream A-T sequence increases T7 promoter strength.
Bacteriophage T7 promoters contain a consensus sequence from -17 to +6 relative to the transcription start site, +1. In addition, the strong class III promoters are characterized by an extended AT-rich region upstream of -17, which is often interrupted by one or more GC base pairs in the weaker class II promoters. Herein we studied the role of the AT-rich region upstream of -17 in transcription regulation of T7 RNA polymerase. Equilibrium DNA binding studies with promoter fragments of consensus sequence truncated at various positions between -17 and -27 showed that the polymerase-promoter complex is significantly stabilized as the upstream AT-rich sequence is extended to and beyond -22. Similarly, promoters in which the AT-rich region from -17 to -22 is interrupted by several GC base pairs showed weak binding. Kinetic studies indicated that the presence of extended AT-rich sequence slows the dissociation rate constant of the polymerase-promoter complex and slightly stimulates the association rate constant, thereby increasing the stability of the complex. Measurement of the transcription activity revealed that the extended AT-rich region does not affect the kinetics of abortive synthesis up to the formation of 8-nucleotide RNA but causes accumulation of longer abortive products between 9 and 13 nucleotides. The observed effects of the upstream DNA region were AT sequence-specific, and the results suggested a larger role for the extended AT-rich sequence that has been unappreciated previously. We propose that the AT-rich DNA sequence upstream of -17 plays a role in modulating the efficiency of transcription initiation by affecting both the affinity of T7 RNA polymerase for the promoter and the efficiency of promoter clearance.
Tang
Guo-Qing
GQ
Department of Biochemistry, University of Medicine and Dentistry of New Jersey (UMDNJ) Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Bandwar
Rajiv P
RP
Patel
Smita S
SS
D020856:AT Rich Sequence / Q000502:physiology*; D017123:Bacteriophage T7 / Q000235:genetics*; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D020862:GC Rich Sequence; D015967:Gene Expression Regulation, Viral; D007700:Kinetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D012235:Rhodamines; D014764:Viral Proteins; C005358:tetramethylrhodamine; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases