Initiation of gene transcription by RNA polymerase (Pol) III requires the activity of TFIIIB, a complex formed by Brf1 (or Brf2), TBP (TATA-binding protein), and Bdp1. TFIIIB is required for recruitment of Pol III and to promote the transition from a closed to an open Pol III pre-initiation complex, a process dependent on the activity of the Bdp1 subunit. Here, we present a crystal structure of a Brf2–TBP–Bdp1 complex bound to DNA at 2.7 Å resolution, integrated with single-molecule FRET analysis and in vitro biochemical assays. Our study provides a structural insight on how Bdp1 is assembled into TFIIIB complexes, reveals structural and functional similarities between Bdp1 and Pol II factors TFIIA and TFIIF, and unravels essential interactions with DNA and with the upstream factor SNAPc. Furthermore, our data support the idea of a concerted mechanism involving TFIIIB and RNA polymerase III subunits for the closed to open pre-initiation complex transition.

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.

The DNA helicase Lhr is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared to DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the ‘parental’ DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than 0.06, corresponding to an interdye distance precision of less than 0.2 nm and accuracy of less than 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to less than 10% of the Foerster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

In all domains of life, the regulation of transcription by DNA-dependent RNA polymerases (RNAPs) is achieved at the level of initiation to a large extent. Whereas bacterial promoters are recognized by a σ-factor bound to the RNAP, a complex set of transcription factors that recognize specific promoter elements is employed by archaeal and eukaryotic RNAPs. These initiation factors are of particular interest since the regulation of transcription critically relies on initiation rates and thus formation of pre-initiation complexes. The most conserved initiation factor is the TATA-binding protein (TBP), which is of crucial importance for all archaeal-eukaryotic transcription initiation complexes and the only factor required to achieve full rates of initiation in all three eukaryotic and the archaeal transcription systems. Recent structural, biochemical and genome-wide mapping data that focused on the archaeal and specialized RNAP I and III transcription system showed that the involvement and functional importance of TBP is divergent from the canonical role TBP plays in RNAP II transcription. Here, we review the role of TBP in the different transcription systems including a TBP-centric discussion of archaeal and eukaryotic initiation complexes. We furthermore highlight questions concerning the function of TBP that arise from these findings.