The Initiation Factor TFE and the Elongation Factor Spt4/5 Compete for the RNAP Clamp during Transcription Initiation and Elongation

Abstract

SummaryTFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP “clamp” region as a regulatory hot spot for both transcription initiation and transcription elongation.

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

“…For immobilization, a biotin moiety was incorporated into the Rpo11 subunit. RNAPs were assembled from the 12 subunits resulting in single-, double-, or triple-labeled RNAP variants (15,(28)(29)(30) (Fig. 1C).…”

“…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).…”

“…Recruitment of the RNAP to the DNA-TBP-TFB complex leads to the formation of the preinitiation complex (PIC), the promoter DNA is melted, and the template strand is loaded into the active site of the RNAP, which is collectively referred to as open complex formation. This process is not very efficient but is stimulated by a third factor, TFE (TFIIEα) (15,18,31). It has not been possible to crystallize the open PIC for structure determination.…”

“…The RNAP clamp provides binding sites for the transcription initiation factor TFE (homologous to TFIIEα) and the elongation factor Spt4/5, both of which bind competitively to the clamp coiled coil motif (15)(16)(17) of archaeal-eukaryotic RNAPs. TFE facilitates open complex formation by interacting with the RNAP clamp and stalk and the nontemplate strand (NTS) (15,18).…”

“…TFE facilitates open complex formation by interacting with the RNAP clamp and stalk and the nontemplate strand (NTS) (15,18). The transcription elongation factor Spt4/5 bridges the gap across the DNA binding channel and stabilizes the elongation complex by preventing its dissociation, thereby stimulating the processivity of RNAP (15,19,20).…”