Binding affinity of T7 RNA polymerase to its promoter in the supercoiled and linearized DNA templates.
A promoter competition assay was used to measure the stability of T7 RNA polymerase with its promoter. When T7 RNA polymerase was incubated with GTP for 5 minutes before the elongation of transcription in either the supercoiled or linearized template, the half-lives of T7 RNA polymerase-DNA complexes were reduced. The transcription product increased when T7 RNA polymerase preincubated with GTP in the supercoiled DNA but not in the linearized DNA template. On the other hand, preincubation of ATP with T7 RNA polymerase decreased the stability of T7 RNA polymerase with the supercoiled DNA, but did not affect the stability of T7 RNA polymerase with the linearized DNA. Furthermore, the production of RNA transcript was increased when T7 RNA polymerase was incubated with ATP in either supercoiled or linearized template before transcription elongation. This study is important to understand the relationship between the transcription initiation and the stability of the ternary complex, and to produce large quantities of RNA transcript in vitro.
Chen
Y C
YC
Department of Botany, National Taiwan University, Taipei, Republic of China.
Jeng
S T
ST
D000255:Adenosine Triphosphate / Q000378:metabolism; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014764:Viral Proteins
D004278:DNA, Superhelical; D004279:DNA, Viral; D014764:Viral Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases