Events during Initiation of Archaeal Transcription: Open Complex Formation and DNA-Protein Interactions

Transcription initiation in all three domains of life requires the assembly of large multiprotein complexes at DNA promoters before RNA polymerase (RNAP)-catalyzed transcript synthesis. Core RNAP subunits show homology among the three domains of life, and recent structural information supports this homology. General transcription factors are required for productive transcription initiation complex formation. The archaeal general transcription factors TATA-element-binding protein (TBP), which mediates promoter recognition, and transcription factor B (TFB), which mediates recruitment of RNAP, show extensive homology to eukaryal TBP and TFIIB. Crystallographic information is becoming available for fragments of transcription initiation complexes (e.g. RNAP, TBP-TFB-DNA, TBP-TFIIB-DNA), but understanding the molecular topography of complete initiation complexes still requires biochemical and biophysical characterization of protein-protein and protein-DNA interactions. In published work, systematic site-specific protein-DNA photocrosslinking has been used to define positions of RNAP subunits and general transcription factors in bacterial and eukaryal initiation complexes. In this work, we have used systematic site-specific protein-DNA photocrosslinking to define positions of RNAP subunits and general transcription factors in an archaeal initiation complex. Employing a set of 41 derivatized DNA fragments, each having a phenyl azide photoactivable crosslinking agent incorporated at a single, defined site within positions ؊40 to ؉1 of the gdh promoter of the hyperthermophilic marine archaea, Pyrococcus furiosus (Pf), we have determined the locations of PfRNAP subunits PfTBP and PfTFB relative to promoter DNA. The resulting topographical information supports the striking homology with the eukaryal initiation complex and permits one major new conclusion, which is that PfTFB interacts with promoter DNA not only in the TATA-element region but also in the transcription-bubble region, near the transcription start site. Comparison with crystallographic information implicates the PfTFB N-terminal domain in the interaction with the transcription-bubble region. The results are discussed in relation to the known effects of substitutions in the TFB and TFIIB N-terminal domains on transcription initiation and transcription start-site selection.Extensive structural and biochemical characterization of transcription initiation complexes has revealed similarity of transcription systems across the three domains of life (1-4). Transcription initiation in archaea closely resembles eukaryal class II transcription initiation and is mediated by a single RNA polymerase (RNAP) 1 and two general transcription factors, TATA-element binding protein (TBP) and transcription factor B (TFB) (5, 6). In vitro transcription initiation has been demonstrated with RNAP, TBP, and TFB in Pyrococcus (7-10), Methanococcus (11), Methanosarcina (12), and Sulfolobus (13) cell-free transcription systems.Recent crystallographic structures of bacterial RNAP and eukary...

Section: Resultssupporting

confidence: 89%

Section: Ment Both In Thesupporting

confidence: 65%

Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

Renfrow

Naryshkin

Lewis

et al. 2004

“…5B). This DNA segment downstream of the TBP/TFB binding site has been shown to be bound by the archaeal RNAP (25). This finding suggests that Phr and TBP/TFB can bind simultaneously to the same DNA molecule.…”

Section: Phr Is a Specific Regulator Of Archaeal Heat Shockmentioning

confidence: 86%

A Novel Archaeal Transcriptional Regulator of Heat Shock Response

Vierke¹,

Engelmann²,

Hebbeln³

et al. 2003

Self Cite

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...

“…Thus, transcription initiation requires three promoter elements, the TATA box centered at Ϫ26/Ϫ27 from the start point of transcription, a transcription factor B recognition element (BRE) 1 comprising two adenines upstream of the TATA box, and an initiation element around the transcription initiation site. Correspondingly, the minimal factors needed to facilitate binding of the eukaryotic polymerase II-like archaeal RNA polymerase to the promoter are TATA box-binding protein (TBP) and transcription factor binding protein (21)(22)(23). One would therefore expect that regulation of transcription in Archaea would follow eukaryotic rather than prokaryotic schemes (24).…”

mentioning

confidence: 99%

TrmB, a Sugar-specific Transcriptional Regulator of the Trehalose/Maltose ABC Transporter from the Hyperthermophilic Archaeon Thermococcus litoralis

Lee¹,

Engelmann²,

Horlacher³

et al. 2003

Self Cite

105

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.