Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif

Abstract

Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA–RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5—the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.

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

“…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).…”

“…This difference in FRET values is likely due to structural adjustments of RNAP by TBP and TFB in the PICs. Bulk transcription assays have demonstrated the role of the elongation factor Spt4/5, the NTS, and the RNAP Rpo4/7 stalk for facilitating high transcription processivity (19,32). To characterize the influence of Spt4/5 and the NTS on the RNAP clamp conformation, we added them to or omitted them from the TEC and recorded FRET efficiencies of the resulting complexes.…”

“…Unlabeled transcription factors TBP, TFB, TFE, and Spt4/5 from M. jannaschii were produced as described previously (15,19,29,31).…”

“…EC complexes were formed by incubating 0.1 μM labeled RNAP with 3 μM of the elongation scaffold and 6.7 mM DTT and 0.07 mg/mL BSA in 1× TMNE buffer (32). To measure the influence of the elongation factor Spt4/5 or NTPs on the RNAP clamp position, 20 μM Spt4/5 was added to the EC and further incubated for 10 min at 65°C (15,19). To reduce unspecific binding of the RNAP to the DNA, 0.8 mg/mL heparin was added to the PIC or TEC mixture and incubated for another 10 min at 65°C before addition of TFE, Spt4/5, or NTPs.…”

“…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). Both the binding of TFE and Spt4/5 to RNAP and their modus operandi have been speculated to involve and depend on a repositioning of the clamp.…”