Purification and characterization of a general transcription factor, aTFB, from the archaeon Methanococcus thermolithotrophicus.
We have recently shown that cell-free transcription of homologous templates from the archaeon Methanococcus thermolithotrophicus requires an archaeal transcription factor (aTFA) that separated from the RNA polymerase during phosphocellulose chromatography. We report here the identification and extensive purification of a second activity, aTFB, required for in vitro transcription. This activity copurified with RNA polymerase during initial chromatographic steps but was positively identified as a distinct transcription factor after Superdex 200 sizing chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the intensity of a M(r) = 28,000 polypeptide in silver-stained gels is correlated with transcription factor activity. The same polypeptide, when eluted from a denaturing polyacrylamide gel and subsequently renatured, showed the functional properties of the transcription factor. In conjunction with gel filtration and sedimentation studies, which indicated a molecular mass of 54,000 Da for the native protein, these results suggested that aTFB is a dimer with polypeptide chains of identical molecular mass. Functional studies with highly purified aTFB demonstrated that it is a general factor required for transcription of genes encoding tRNA and proteins.
Hausner
W
W
Institut für Allgemeine Mikrobiologie, Universität Kiel, Federal Republic of Germany.
Thomm
M
M
D002474:Cell-Free System; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D017017:Methanococcus / Q000378:metabolism*; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism
D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases