NTP concentration effects on initial transcription by T7 RNAP indicate that translocation occurs through passive sliding and reveal that divergent promoters have distinct NTP concentration requirements for productive initiation.
The hypothesis that active site translocation during initial transcription occurs by a passive sliding mechanism which allows the pre- and post-translocated states to equilibrate on the time scale of bond formation was tested by evaluating the effects of NTP concentration on individual transcript extension steps in the presence of translocation roadblocks created by proteins bound immediately downstream of a T7 promoter, as well as by evaluating the effects of NTP concentration on competing transcript extension pathways (iterative synthesis and "normal" extension). Results are consistent with a passive sliding mechanism for translocation which is driven by NTP binding, and are inconsistent with mechanisms in which the pre- and post-translocated states fail to equilibrate with each other on the time scale of bond formation or in which translocation is driven by NTP hydrolysis. We also find, in agreement with many previous studies, that divergence from consensus in the ITS (initially transcribed sequence) of the T7 promoter decreases productive initiation. However, this appears to be largely due to increases in the NTP concentration requirements for efficient transcription on the divergent ITSs.
Guajardo
R
R
Dept. of Biochemistry, University of Texas Health Science Ctr., 7703 Floyd Curl Drive, San Antonio, TX 78284-7760, USA.
Lopez
P
P
Dreyfus
M
M
Sousa
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D004256:DNA Polymerase I / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation / Q000235:genetics; D006150:Guanine Nucleotides / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000378:metabolism; D056968:Lac Repressors; D009843:Oligoribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012313:RNA / Q000737:chemistry; D012097:Repressor Proteins / Q000235:genetics; D012265:Ribonucleotides / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*; D014544:Uridine Triphosphate / Q000378:metabolism; D014764:Viral Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides; D056968:Lac Repressors; D009843:Oligoribonucleotides; D012097:Repressor Proteins; D012265:Ribonucleotides; D014764:Viral Proteins; C036895:oligo(G); D012313:RNA; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004256:DNA Polymerase I; D014544:Uridine Triphosphate