Dynamic architecture of a minimal RNA polymerase II open promoter complex.

The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ single-molecule Förster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture of a minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and general transcription factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.

Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry.

Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes.

Structure of a bifunctional DNA primase-polymerase.

Genome replication generally requires primases, which synthesize an initial oligonucleotide primer, and DNA polymerases, which elongate the primer. Primase and DNA polymerase activities are combined, however, in newly identified replicases from archaeal plasmids, such as pRN1 from Sulfolobus islandicus. Here we present a structure-function analysis of the pRN1 primase-polymerase (prim-pol) domain. The crystal structure shows a central depression lined by conserved residues. Mutations on one side of the depression reduce DNA affinity. On the opposite side of the depression cluster three acidic residues and a histidine, which are required for primase and DNA polymerase activity. One acidic residue binds a manganese ion, suggestive of a metal-dependent catalytic mechanism. The structure does not show any similarity to DNA polymerases, but is distantly related to archaeal and eukaryotic primases, with corresponding active-site residues. We propose that archaeal and eukaryotic primases and the prim-pol domain have a common evolutionary ancestor, a bifunctional replicase for small DNA genomes.