Effect of heat stress on promoter binding by transcription factors in the cytosol of the archaeon Methanosarcina mazeii.
Regulation of archaeal stress genes is not yet fully understood. This work is part of a research effort aimed at elucidating the molecular mechanisms of transcription initiation and regulation of the stress genes in the hsp70(dnaK) locus of the mesophilic, methanogenic archaeon Methanosarcina mazeii. The locus has the stress genes 5'-grpE-hsp70(dnaK)-hsp40(dnaJ)-3' encoding the chaperone machine components GrpE, Hsp70(DnaK), and Hsp40(DnaJ), respectively, flanked by non-heat shock inducible genes, orf16 and orf11-trkA. Thus, the M. mazeii hsp70(dnaK) locus offers the opportunity for studying heat shock and non-heat shock inducible genes side by side. The objectives of the work reported here were to develop procedures for studying basal transcription factors in the cytosol of M. mazeii and their interaction with these genes' promoters in stressed cells for comparison with unstressed counterparts. The preparation of non-radioactive DNA probes for electrophoretic mobility shift assay (EMSA), and the combination of EMSA with Western blotting for DNA-binding protein identification were standardized for this investigation. DNA probes bearing the genes' promoter regions were used for detecting and identifying DNA-binding proteins in the cytosol of unstressed and heat-shocked cells. Cytosolic TATA-binding protein (TBP) was found to bind the stress-gene promoters in both unstressed and heat-shocked cells but more strongly in the latter. Likewise, in stressed cells TBP-transcription factor B (TFB)(TFIIB) association was increased by comparison with unstressed controls. The level of cytosolic TBP assessed by its DNA-binding activity using EMSA remained unchanged during the various phases of culture growth in the absence of heat stress. The results indicate that heat stress of cells in culture modulates the level and/or the stress-gene promoter-binding activity of the M. mazeii TBP, and enhances TBP-TFB association in the cytosol and DNA binding.
De Biase
Andrea
A
Wadsworth Center, New York State Department of Health, Division of Molecular Medicine, Albany, NY 12201, USA.
Macario
Alberto J L
AJ
Conway de Macario
Everly
E
D001483:Base Sequence; D001667:Binding, Competitive; D003600:Cytosol / Q000378:metabolism; D015342:DNA Probes; D019641:DNA, Archaeal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006358:Hot Temperature; D017020:Methanosarcina / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000378:metabolism*
D015342:DNA Probes; D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; D010761:Phosphorus Radioisotopes; D035181:TATA-Box Binding Protein; D014157:Transcription Factors