PURIFICATION AND CHARACTERIZATION OF A GENERAL TRANSCRIPTION FACTOR, ATFB, FROM THE ARCHAEON METHANOCOCCUS-THERMOLITHOTROPHICUS

We have recently shown that cell-free transcription of homologous temp lates from the archaeon Methanococcus thermolithotrophicus requires an archaeal transcription factor (aTFA) that separated from the RNA poly merase during phosphocellulose chromatography. We report here the iden tification and extensive purification of a second activity, aTFB, requ ired for in vitro transcription. This activity copurified with RNA pol ymerase during initial chromatographic steps but was positively identi fied as a distinct transcription factor after Superdex 200 sizing chro matography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the intensity of a M(r) = 28,000 polypeptide in silver-s tained gels is correlated with transcription factor activity. The same polypeptide, when eluted from a denaturing polyacrylamide gel and sub sequently renatured, showed the functional properties of the transcrip tion factor. In conjunction with gel filtration and sedimentation stud ies, which indicated a molecular mass of 54,000 Da for the native prot ein, these results suggested that aTFB is a dimer with polypeptide cha ins of identical molecular mass. Functional studies with highly purifi ed aTFB demonstrated that it is a general factor required for transcri ption of genes encoding tRNA and proteins.