Production of RNA strands containing nucleotide analogs in only one section.
RNA molecules containing randomly placed 4-thio-U residues only in the 3' one-fifth of the molecule were produced using E. coli RNA polymerase. To achieve this distribution of residues, an EcoRI site was engineered into the template at a position four-fifths of the way from the promoter to the end of the template. A tight-binding EcoRI(gln111) mutant protein was bound to this site. The presence of this protein resulted in paused transcription complexes that were purified using a Centricon 100 filtration device. The nucleotide composition was changed after filtration, and transcription was allowed to continue to the end of the template in buffer containing 0.5 M KCl, which caused the EcoRI(gln111) protein to be released from the DNA template. Analogous RNA molecules for other studies of protein-RNA interactions could be produced using this procedure.
Walstrom
K M
KM
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.
von Hippel
P H
PH
D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D015394:Molecular Structure; D011485:Protein Binding; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D012234:Rho Factor / Q000378:metabolism; D013698:Templates, Genetic; D013891:Thiouridine / Q000737:chemistry
D012234:Rho Factor; D013891:Thiouridine; D012313:RNA; D012321:DNA-Directed RNA Polymerases; D015246:Deoxyribonuclease EcoRI