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

Abstract

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...

Quantitative Analysis of in Vivo Initiator Selection by Yeast RNA Polymerase II Supports a Scanning Model

“…The points of closest contact included nucleotides Ϫ6 to Ϫ8 (relative to the nucleotide addition site at ϩ1) and B-finger residues 62-66. These structurally predicted contacts are consistent with results showing that the archaeal TFIIB homologue cross-links to template DNA near the transcription start site (37)(38). We tested whether mutations in SUA7 genetically interact with mutations in the initiator element at positions that are required for accurate start site selection.…”

Direct Modulation of RNA Polymerase Core Functions by Basal Transcription Factors

“…The nonspecific transcription assays reveal only an overall stimulation of RNAP activity which could be due to increased rates of transcription initiation, elongation, or less frequent termination. Two observations favor transcription initiation as the most likely target for the stimulation: the S. cerevisiae TFIIB-B-finger penetrates deeply into the active site of RNAPII (9, 13), and archaeal TFB can be cross-linked to promoter DNA around the transcription start site (2,41). It is therefore likely that the B-finger productively interacts with the template DNA, the DNA/RNA hybrid, and/or the substrate rNTPs during transcription initiation.…”

Archaeal Minichromosome Maintenance (MCM) Helicase Can Unwind DNA Bound by Archaeal Histones and Transcription Factors

“…4, E-H). Within a pre-initiation complex, there are specific TFB-DNA, TBP-DNA, and limited RNAP-DNA contacts upstream of the site of transcription initiation, extensive RNAP-template strand DNA contacts within the main channel of RNAP, and extensive RNAP-DNA and limited TFB-DNA contacts downstream of the site of initiation (24,25). Given this organization, when the direction of helicase unwinding and transcription were the same, helicase would first approach and presumably be required to disrupt TFB-DNA contacts, whereas helicase unwinding in the direction opposite to transcription would presumably first encounter the interactions of RNAP with downstream DNA.…”