T7 RNA polymerase elongation complex structure and movement.
We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single template using a combination of digestion with exonuclease III, lambda exonuclease, RNAse T1, and treatment with KMnO(4). Our results indicate that the transcription bubble is approximately nine bases long and that the RNA:DNA hybrid is 7-8 bp in size. An additional four to six bases of RNA immediately 5' to the hybrid interact with the RNAP, probably with a site on the N-terminal domain. When ECs with transcripts of different length were probed in the presence or absence of the incoming NTP we found that the position of the EC on the template and the RNA shifted downstream upon NTP binding. NTP binding also restricted the lateral mobility of the complex on the template. Our results indicate that, in the absence of bound NTP, the RNAP is relatively free to slide on the template around a position that usually lies one to two bases upstream of the position from which NTP binding and bond formation occur. NTP binding stabilizes the RNAP in the post-translocated position and keeps it from sliding upstream, either due directly to RNAP:NTP:template interactions, or to an isomerization which causes the fingers subdomain of the RNAP to clamp down on the downstream end of the template strand.
Huang
J
J
Department of Biochemistry, University of Texas Health Sciences Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7760, USA.
Sousa
R
R
D017123:Bacteriophage T7 / Q000201:enzymology*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007536:Isomerism; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism; D009068:Movement; D023282:Nuclease Protection Assays; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000378:metabolism; D009711:Nucleotides / Q000235:genetics / Q000378:metabolism; D011196:Potassium Permanganate / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012313:RNA / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D006163:Ribonuclease T1 / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins
D020409:Molecular Motor Proteins; D009692:Nucleic Acid Heteroduplexes; D009711:Nucleotides; D014764:Viral Proteins; D011196:Potassium Permanganate; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C043586:exo protein, Bacteriophage lambda; D006163:Ribonuclease T1