We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 M and 0.5 mM sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80°C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

Archaea have a eukaryotic type of transcriptional machinery containing homologues of the transcription factors TATA-binding protein (TBP) and TFIIB (TFB) and a pol II type of RNA polymerase, whereas transcriptional regulators identified in archaeal genomes have bacterial counterparts. We describe here a novel regulator of heat shock response, Phr, from the hyperthermophilic archaeon Pyrococcus furiosus that is conserved among Euryarchaeota. The protein specifically inhibited cellfree transcription of its own gene and from promoters of a small heat shock protein, Hsp20, and of an AAA ؉ ATPase. Inhibition of transcription was brought about by abrogating RNA polymerase recruitment to the TBP/ TFB promoter complex. Phr bound to a 29-bp DNA sequence overlapping the transcription start site. Three sequences conserved in the binding sites of Phr, TTTA at ؊10, TGGTAA at the transcription start site, and AAAA at position ؉10, were required for Phr binding and are proposed as consensus regulatory sequences of Pyrococcus heat shock promoters. Shifting the growth temperature from 95 to 103°C caused a dramatic increase of mRNA levels for the aaa ؉ atpase and phr genes, but expression of the Phr protein was only weakly stimulated. Our findings suggest that heat shock response in Archaea is negatively regulated by a mechanism involving binding of Phr to conserved sequences.All living organisms have developed molecular mechanisms to protect themselves from the harmful effects of elevated temperatures and other stress factors. The overwhelming majority of information on these heat-induced heat shock proteins (Hsps) 1 and heat shock genes comes from bacteria and eukaryotes. In bacteria, several independent mechanisms of regulation of heat shock promoters have been elucidated. Regulation in Escherichia coli is brought about by the use of alternative sigma factors that direct RNA polymerase to heat shock promoters differing from the standard consensus promoter sequence (1). A different mechanism operates in Grampositive bacteria, which involves binding and dissociation of a repressor to a DNA control element upstream of the groEL and dnaK operons (2). Recent analyses suggest that bacteria have evolved sophisticated regulatory networks often combining positive and negative control mechanisms to allow a timetuned expression of heat shock genes (3). In eukaryotes stressinduced transcription requires activation of a heat shock factor, HSF, which binds as a trimer to the heat shock DNA element, thereby stimulating transcription. The monomeric form of HSF lacks both promoter binding and transcriptional activity (4).Regulators of the heat shock response in the domain of Archaea have not yet been identified, but studies of stress genes in archaeal genomes revealed the presence of homologues of Hsp70/DnaK, Hsp60, Hsp40, GrpE, and of small heat shock proteins (5). Hsp70 is only present in about 50% of the archaeal species inspected; GroEL and GroES seem not to exist in Archaea (5). Their function in peptide folding is apparently performed by...

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