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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.