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.

Figure 1.

Structure of the archaeal transcription elongation factor Spt4/5. X-ray structure of M. jannaschii Spt4/5NGN complex (A). The interface between Spt4 and Spt5 (B). Alignment of Spt4 and Spt5 NGN homologs. Highly conserved residues are depicted in bold. The Spt5 residues that are mutated are indicated by asterisk. Mj, Methanocaldococcus jannaschii; Sc, Saccheromyces cerevisiae; Hs, Homo sapiens; Ec, Escherichia coli (C). Structural comparison of bacterial NusG NGN from E. coli (left, pdb 2K06), the archaeal structure of M. jannaschii Spt4/5 NGN complex (middle) and eukaryotic Spt4/5 NGN from S. cerevisiae (right, pdb 2EXU) (D). Domain architecture of Spt4/5 complexes in the three domains of life (E). A model of the complete M. jannaschii Spt4/5 complex (F).

Figure 2.

Archaeal Spt4/5 stimulates transcription elongation. Transcription assay using a synthetic elongation scaffold and recombinant 10-subunit M. jannaschii RNAP (200 nM). Transcript synthesis was monitored over a time course of 2, 5, 10 and 20 min in the absence of NTS (A), and 20, 40, 90 and 300 s in the presence of NTS (B). Recombinant Spt4/5 (10 µM) was added to the reaction in conjunction with RNAP. The full length ‘run off' transcript was quantitated in the absence (C) and presence of the NTS, and normalized to the reaction end point in the absence of Spt4/5 (20 min and 300 s w/o NTS, C, and plus NTS, D, respectively). The gels (A and B) are representative and all quantitations are based on at least three independent experiments (arbitrary units, AU).

Figure 3.

Spt4 stabilizes the Spt5 NGN domain. The Spt4/5, Spt4/5 NGN, Spt5 and Spt5 NGN variants were incubated at either 65 or 75°C. The heat stable and soluble fraction was resolved on SDS–PAGE and the gel subsequently stained with Coomassie.

Figure 4.

Domain deletion analysis identifies Spt5 NGN as effector domain. Transcription elongation assays using recombinant 10-subunit RNAP (200 nM) and Spt4/5, Spt4/5 NGN, Spt5, Spt5 KOW and Spt4 (each at 10 µM) in the absence of NTS. Samples were taken at 2, 5, 10 and 20 min (A and B). The full-length run-off transcript was quantitated and normalized to the end point (5 min) in the absence of any added factors (C).

Figure 5.

Recruitment of Spt4/5 to RNAP depends on the Spt5 NGN domain. Spt4/5 (50 nM) was radio-labelled and incubated with recombinant 10- (ΔF/E) or 12-subunit RNAP (250 and 500 nM) and the complexes were separated with native gel electrophoresis (A). Radio-labelled Spt4/5 deletion variants (50 nM) Spt4/5 NGN (B), Spt5 (C), Spt5 NGN (D) were incubated with 10-subunit RNAP (250 and 500 nM) and separated on native gels. The specificity of the RNAP–Spt4/5 interactions was ascertained by competition experiments (E). Labelled Spt4/5 (50 nM) was incubated with RNAP (500 nM) and 10- to 50-fold excess of unlabelled Spt4/5, Spt4/5 NGN, Spt5 KOW and Spt4 (500 nM and 2.5 µM) prior to separation with native gel electrophoresis.

Figure 6.

Site-directed mutagenesis of the Spt5 NGN domain identifies a hydrophobic cavity as RNAP binding site. The residues Ala-4, Tyr-42 and Leu-44 line a hydrophobic depression in the Spt5 NGN domain (A). Spt4/5-RNAP binding assays using labelled Spt4/5 mutants (Spt5-4^Arg, -42^Ala, -44^Ala and -44^Arg, 50 nM) and 10-subunit RNAP (50 and 400 nM) were separated with native gel electrophoresis (B). Transcription elongation assays using 10-subunit RNAP (200 nM) and Spt4/5 mutants (10 µM) in the absence of NTS (C).

Figure 7.

A deletion-substitution (CC-Gly₄) in the tip of the RNAP clamp coiled-coil domain abrogates Spt4/5 binding and stimulation. Transcription elongation assays with 10-subunit RNAP and RNAP CC-Gly₄ in the absence (A, 200 nM) and presence of NTS (C, 50 nM), ± Spt4/5 (10 µM). Run-off transcripts were quantitated and normalized to the end points of reactions carried out without Spt4/5 (B and D). The binding of Spt4/5 to RNAP and RNAP CC-Gly₄ was monitored with native gel electrophoresis (E). The location of the RNAP clamp coiled-coil (highlighted in red), the active site NADFDGD motif (green) and metal ion (magenta) in archaeal RNAP (Sulfolobus shibatae RNAP, pdb 2WAQ).