MUTANT T7 RNA-POLYMERASE AS A DNA-POLYMERASE

We have identified a T7 RNA polymerase (RNAP) mutant that efficiently utilizes deoxyribonucleoside triphosphates. In vitro this mutant will synthesize RNA, DNA or 'transcripts' of mixed dNMP/rNMP composition de pending on the mix of NTPs present in the synthesis reaction. The muta tion is conservative, changes Tyr639 within the active site to phenyla lanine and does not affect promoter specificity or overall activity. N on-conservative mutations of this tyrosine also reduce discrimination between deoxyribo- and ribonucleoside triphosphates, but these mutatio ns also cause large activity reductions. Of 26 mutations of other resi dues in and around the active site examined none showed marked effects on rNTP/dNTP discrimination. Mutations of the corresponding tyrosine in DNA polymerase (DNAP) I increase miscoding, though effects on dNTP/ rNTP discrimination for the DNAP I mutations have not been reported. T his conserved tyrosine may therefore play a similar role in many polym erases by sensing incorrect geometry in the structure of the substrate /template/product due to inappropriate substrate structure or mismatch es. T7 RNAP can use RNA templates as well as DNA templates and is capa ble of both primer extension and de novo initiation. The Y639F mutant retains the ability to use RNA or DNA templates. Thus this mutant can display de novo initiated or primed DNA-directed DNA polymerase, rever se transcriptase, RNA-directed RNA polymerase or DNA-directed RNA poly merase activities depending simply on the templates and substrates pre sented to it in the synthesis reaction.