Characterization of T7 RNA polymerase transcription complexes assembled on nucleic acid scaffolds.
We have used synthetic oligomers of DNA and RNA to assemble nucleic acid scaffolds that, when mixed with T7 RNA polymerase, allow the formation of functional transcription complexes. Manipulation of the scaffold structure allows the contribution of each element in the scaffold to transcription activity to be independently determined. The minimal scaffold that allows efficient extension after challenge with 200 mm NaCl consists of an 8-nt RNA primer hybridized to a DNA template (T strand) that extends 5-10 nt downstream. Constructs in which the RNA-DNA hybrid is less than or greater than 8 bp are less salt-resistant, and the hybrid cannot be extended beyond 12-13 bp. Although the presence of a complementary nontemplate strand downstream of the primer does not affect salt resistance, the presence of DNA upstream decreases resistance. The addition of a 4-nt unpaired "tail" to the 5' end of the primer increases salt resistance, as does the presence of an unpaired nontemplate strand in the region that contains the 8-bp hybrid (thereby generating an artificial transcription "bubble"). Scaffold complexes having these features remain active for over 1 week in the absence of salt and exhibit many of the properties of halted elongation complexes, including resistance to salt challenge, a similar trypsin cleavage pattern, and a similar pattern of RNA-RNA polymerase cross-linking.
D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D014158:Transcription, Genetic; D014357:Trypsin / Q000494:pharmacology; D014764:Viral Proteins
D003432:Cross-Linking Reagents; D012492:Salts; D014764:Viral Proteins; D012965:Sodium Chloride; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; C034970:bacteriophage T7 induced DNA polymerase; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase; D014357:Trypsin