TRANSCRIPTION FACTORS AND TERMINATION OF TRANSCRIPTION IN METHANOCOCCUS

We have recently shown that specific transcription in cell extracts of Methanococcus thermolithotrophicus depends upon a transcription facto r that was separated from the RNA polymerase by phosphocellulose chrom atography. Here, we provide evidence for a second transcription factor . This factor copurified with RNA polymerase during initial chromatogr aphic steps, but it was resolved as a distinct activity required for c ell-free transcription after centrifugation in sucrose density gradien ts. The native molecular weight of this factor was estimated by gel fi ltration to be 56000. After electrophoresis under denaturing condition s, a 28 kDa polypeptide was correlated with factor activity. Thus, the native transcription factor appeared to be a dimer composed of two po lypeptides of identical molecular mass. Oligo-dT sequences at the 3'-e nd of a tRNA(Val) gene and internal sequences of this tRNA were modifi ed by DNA deletions in order to investigate the nature of archaeal tra nscription terminators. Deletion of two residues of the decameric sequ ence 5'-TTTTAATTTT-3' reduced termination efficiency to about 27 perce nt of wild-type levels. Deletion of two additional residues from the 3 '-end completely abolished terminator function. Deletions in the DNA r egion encoding the T Psi C stem and loop of tRNA also significantly re duced termination efficiency. In addition a Rho-independent terminator of Escherichia coli perfectly replaced the decameric Methanococcus te rminator sequence. These findings suggested that transcription termina tion sequences in archaea are similar to the terminator sequences in b acteria.