The basal transcription factors TBP and TFB from the mesophilic archaeon Methanosarcina mazeii: Structure and conformational changes upon interactionwith stress-gene promoters

Transcription of archaeal non-stress genes involves the basal factors TBP a nd TFB, homologs of the eucaryal TATA-binding protein and transcription fac tor IIB, respectively. No comparable information exists for the archaeal mo lecular-chaperone, stress genes hsp70(dnaK), hsp40(dnaJ), and grpE. These d o not occur in some archaeal species, but are present in others possibly du e to lateral transfer from bacteria, which provides a unique opportunity to study regulation of stress-inducible bacterial genes in organisms with euk aryotic-like transcription machinery. Among the Archaea with the genes, tho se from the mesophilic methanogen Methanosarcina mazeii are the only ones w hose basal (constitutive) and stress-induced transcription patterns have be en determined. To continue this work, tbp and tfb were cloned from M. mazei i, sequenced, and the encoded recombinant proteins characterized in solutio n, separately and in complex with each other and with DNA. M. mazeii TBP ra nks among the shortest within Archaea and, contrary to other archaeal TBPs, it lacks tryptophan or an acidic tail at the C terminus and has a basic N- terminal third. M. mazeii TFB is similar in length to archaeal and eucaryal homologs and all have a zinc finger and HTH motifs. Phylogenetically, the archaeal and eucaryal proteins form separate clusters and the M. mazeii mol ecules are closer to the homologs from Archaeoglobus fulgidus than to any o ther. Antigenically, M. mazeii TBP and TFB are close to archaeal homologs w ithin each factor family, but the two families are unrelated. The purified recombinant factors were functionally active in a cell-free in vitro transc ription system, and were interchangeable with the homologs from Methanococc us thermolithotrophicus. The M. mazeii factors have a similar secondary str ucture by circular dichroism (CD). The CD spectra changed upon binding to t he promoters of the stress genes grpE, dnaK, and dnaJ, with the changes bei ng distinctive for each promoter; in contrast, no effect was produced by th e promoter of a non-stress-gene. Factor(s)-DNA modeling predicted that modi fications of H bonds are caused by TBP binding, and that these modification s are distinctive for each promoter. It also showed which amino acid residu es would contact an extended TATA box with a B recognition element, and evo lutionary conservation of the TBP-TFB-DNA complex orientation between two a rchaeal organisms with widely different optimal temperature for growth (37 and 100 degreesC). (C) 2001 Academic Press.