Synthesis of complementary RNA on RNA templates using the DNA-dependent RNA polymerase of Escherichia coli.
It is shown that the DNA-dependent RNA polymerase of Escherichia coli can synthesize complementary RNA (cRNA) directly on rRNA and mRNA templates. Synthesis occurred preferentially in the presence of Mn2+ and at relatively high substrate and enzyme concentrations. No primer was required, and addition of oligo-U to a mRNA-dependent reaction gave no marked stimulation. Sedimentation analysis of cRNA made on different templates indicated that the products were mainly 2-4 S, but a fraction of the product was larger. Fingerprints of 32P-labelled cRNA made on 5 S rRNA and 18 S rRNA indicated that the complexity of the cRNAs was related to the size of the template, suggesting that a substantial portion of the templates were copied. This reaction provides a simple method for preparing cRNA of high specific activity for use in hybridisation studies, and possibly in sequence analysis. 32P-labelled cRNA made on 18 S and 28 S rRNA was a sensitive hybridisation probe for detection of the specific fragments of mouse DNA containing the rRNA genes.
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D066298:In Vitro Techniques; D008345:Manganese; D009693:Nucleic Acid Hybridization; D011072:Poly U / Q000494:pharmacology; D012313:RNA / Q000138:chemical synthesis*; D012333:RNA, Messenger / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic
D012333:RNA, Messenger; D012335:RNA, Ribosomal; D011072:Poly U; D008345:Manganese; D012313:RNA; D012321:DNA-Directed RNA Polymerases