Activation of T7 RNA polymerase in Xenopus oocytes and cell-free extracts.
Single-subunit bacteriophage T7 RNA polymerase (T7 RNAP) is universally employed for in vivo and in vitro transcription of genes put under control of the T7 promoter. The enzyme is capable of transcribing a complete gene without additional proteins. In this study, we reveal the presence of a low molecular weight factor, which induces several-fold activation of T7 RNAP in the cytoplasm of oocytes and eggs from Xenopus laevis. Cell-free reconstitution of the T7 RNAP activation allowed us to investigate the molecular properties of the activator, establish its peptide nature and suggest T7 RNAP activation mechanism. In contrast to the previously described nonspecific transcriptional activators, which interact with scattered ionic sites on nucleic acids, the peptide activator associates with T7 RNAP molecule, thus being a bona fide activator of the polymerase. To our knowledge, this is the first report concerning the specific activation of T7 RNAP by a factor of peptide or protein origin. Besides rather obvious merits in gaining more efficient transcription with T7 RNAP, this finding can provide additional insights into regulatory mechanisms of transcription. The study also introduces a novel highly sensitive luminescent assay of T7 RNAP activity.
Tokmakov
Alexander A
AA
Research Center for Environmental Genomics and Graduate School of Science, Kobe University, Nada, Kobe, Japan. tokmak@phoenix.kobe-u.ac.jp
Fukami
Yasuo
Y
D000818:Animals; D002457:Cell Extracts / Q000494:pharmacology; D002474:Cell-Free System / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D007700:Kinetics; D008845:Microinjections; D009865:Oocytes / Q000166:cytology*; D014764:Viral Proteins / Q000378:metabolism*; D014982:Xenopus laevis
D002457:Cell Extracts; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases