Complete architecture of the archaeal RNA polymerase open complex from single-molecule FRET and NPS

Abstract

The molecular architecture of RNAP II-like transcription initiation complexes remains opaque due to its conformational flexibility and size. Here we report the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and the 12-subunit RNA polymerase (RNAP) from Methanocaldococcus jannaschii. By combining single-molecule Förster resonance energy transfer and the Bayesian parameter estimationbased Nano-Positioning System analysis, we model the entire archaeal OC, which elucidates the path of the non-template DNA (ntDNA) strand and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH.

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

“…It has not been possible to crystallize the open PIC for structure determination. However, single-molecule FRET mapping and cryo-EM approaches have provided high-confidence models of the PIC in archaea and eukaryotes (13,33,39). To characterize the conformational state of the RNAP clamp in the context of closed and open complexes, we assembled PICs using recombinant TBP, TFB, and RNAP on promoter templates that were either complementary (homoduplex, closed) or contained a short region of noncomplementarity (heteroduplex, open) proximal to the transcription start site (TSS) ( Fig.…”

“…S1). Labeled RNAPs were incubated with the respective transcription factors, nucleic acids, and/or nucleoside triphosphates (NTPs) at 65°C to allow transcription complex formation (15,19,29,(31)(32)(33). The FRET efficiency between the donor and acceptor dye was measured at room temperature (21°C) using a PRISM total internal reflection fluorescence (TIRF) microscope with alternating laser excitation (ALEX), which allowed us to spectroscopically select and analyze the complexes that contain both donor and acceptor dyes (34).…”

“…PIC complexes were assembled by incubating 0.13 μM promoter DNA, 5 μM TBP, 5 μM TFB, 0.1 μM RNAP, 6.7 mM DTT, and 0.07 mg/mL BSA in 1× TMNE buffer (40 mM Tris/HCl, pH 7.3, 250 mM sodium chloride, 2.5 mM magnesium chloride, 0.1 mM EDTA, and 5% glycerol) at 65°C for 15 min (31,33). To measure the PIC in the presence of the third basal transcription factor TFE, 15 μM TFE was added to the PIC and further incubated at 65°C for 10 min (15, 33).…”

Molecular Mechanisms of Transcription Initiation—Structure, Function, and Evolution of TFE/TFIIE-Like Factors and Open Complex Formation

“…Pioneering work from Wolfram Zillig's laboratory identified an AT-rich sequence upstream of the TSS important for the promoter activity [36]. Permanganate foot-printing experiments on the Sso and Mja OC have revealed similar boundaries for the initially melted region (IMR) of the promoter DNA extending up to position -12 relative to the TSS [11,37,38].…”

“…However, despite heroic efforts it has yet not been possible to crystallise any complete archaeal or eukaryotic PIC, while structural information of partial complexes and biochemical-and biophysical proximity analyses have enabled structural models of PICs [13,30,38,45]. We have recently prepared a solution model of the Mja OC, shown in Figure 4.…”