A Novel Archaeal Transcriptional Regulator of Heat Shock Response

Abstract

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...

The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation

“…Our KMnO 4 footprints at 70°C (Fig. 5A) demonstrated that TFE leads to an increased complex stability by promoting DNA melting similar to the properties reported for eukaryotic TFIIE␣ (12,24,42). The roles of EЈ and TFE in open complex formation were better resolved when initiation by the Pyrococcus core enzyme was analyzed at 60°C.…”

“…The ⌬EЈF RNAP is therefore also designated as core enzyme here. The most active fractions of the core enzyme were eluting later (fractions [23][24][25][26] indicating that the lower activity of fractions 19 -21 containing higher amounts of RNAP (see SDS-PAGE of RNAP fractions in Fig. 2B) was not caused by a lower content of subunits EЈ and F in these fractions.…”

“…We are exploring the mechanism and regulation of transcription in the hyperthermophilic Archaea Pyrococcus furiosus (23)(24)(25)(26)(27). In this contribution we report the reconstitution of a highly active RNAP from 11 bacterially expressed subunits.…”

Comparison of modes of DNA-binding and bending by archaeal transcription factors, Phr, TrmB, GvpE, GvpD, NrpR, Lrs14 and MDR1

“…7,10). Apart from such genome-based approaches targeting FFRPs, a small number of archaeal transcription factors were experimentally identified in the past few years by other groups while focusing mainly on biological phenomena such as heat-shock responses 28) and metabolic changes induced by particular nutrients. 29) The aim of this paper Apparent similarity between the amino acid sequences of FL-FFRPs is low.…”

“…E. coli Lrp, has been reported. 31) In this study five archaeal transcription factors, Phr, 28) TrmB, 29) GvpE, 32) MDR1 33) and NrpR, 34) are analyzed, which were almost all the experimentally identified transcription factors I was able to find by searching literature. These form a random selection of archaeal transcription factors, for none of which the authors of the original papers claimed significant similarity to FFRPs.…”