The influence of an alternate template conformation on elongating phage T7 RNA polymerase.
We investigated the effect of left-handed Z-DNA on transcription by bacteriophage T7 RNA polymerase in vitro and, surprisingly, found that the enzyme can efficiently utilize a template containing a stretch of left-handed DNA close to the promoter. Analysis of transcription products revealed that only a small fraction of elongating polymerases abort transcription either at the promoter proximal or at the distal B-to-Z junction and, even less frequently, within the stretch of left-handed DNA. Our results indicate that, unlike E. coli RNA polymerase, T7 RNA polymerase can utilize a template with a CG stretch in an alternate conformation. In contrast, polymerases are completely blocked at the promoter proximal junction by a monoclonal antibody directed against Z-DNA. This blockage remains stable over a remarkable time, even when negative supercoiling is released by linearization of the template. Together with our recent finding of transcription-induced formation of Z-DNA (3), our data provide an example for a possible auto-regulatory mechanism that employs a change in DNA conformation.
Dröge
P
P
Institute for Molecular Biology, Hannover Medical School, FRG.
Pohl
F M
FM
D000911:Antibodies, Monoclonal; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins
D000911:Antibodies, Monoclonal; D004278:DNA, Superhelical; D004279:DNA, Viral; D011089:Polydeoxyribonucleotides; D014764:Viral Proteins; C019530:poly(dC-dG); C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases