An archaebacterial cell-free transcription system. The expression of tRNA genes fromMethanococcus vannieliiis mediated by a transcription factor

Abstract

Our understanding of the mechanism of RNA biosynthesis in archaebacteria is limited, due in part to the inability of purified RNA polymerases to transcribe purified genes accurately in vitro. In the present study, we show that cell extracts of Methanococcus vannielii and Methanococcus thermolithotrophicus purified by gradient centrifugation synthesize a distinct transcript from templates harboring a cloned homologous tRNAVal and tRNAArg gene. The in vitro tanscripts initiate with GTP at the same sites as in Methanococcus cells. About 60% of the sequence of the in vitro RNA products was analyzed by dideoxyterminated primer extension and found to be identical with that of the precursors of tRNAVal and tRNAArg. This findings indicate that this RNA polymerase fraction both initiates and terminates transcription faithfully in vitro. After purification of a cell-free extract (S-100) of M. thermolithotrophicus by phosphocellulose chromatography, the endogenous RNA polymerase has lost its ability to transcribe the tRNAVal gene accurately. The activity directing specific expression of this template was reconstituted by the addition of a protein-fraction devoid of RNA polymerase activity. Thus, a transcription factor appears to be required for accurate cell-free expression of tRNA genes from M. vannielii.