Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest

Abstract

RNA polymerase II (RNAPII) undergoes structural changes during the transitions from initiation, elongation, and termination, which are aided by a collection of proteins called elongation factors. NusG/Spt5 is the only elongation factor conserved in all domains of life. Although much information exists about the interactions between NusG/Spt5 and RNA polymerase in prokaryotes, little is known about how the binding of eukaryotic Spt4/5 affects the biochemical activities of RNAPII. We characterized the activities of Spt4/5 and interrogated the structural features of Spt5 required for it to interact with elongation complexes, bind nucleic acids, and promote transcription elongation. The eukaryotic specific regions of Spt5 containing the Kyrpides, Ouzounis, Woese domains are involved in stabilizing the association with the RNAPII elongation complex, which also requires the presence of the nascent transcript. Interestingly, we identify a region within the conserved NusG N-terminal (NGN) domain of Spt5 that contacts the non-template strand of DNA both upstream of RNAPII and in the transcription bubble. Mutating charged residues in this region of Spt5 did not prevent Spt4/5 binding to elongation complexes, but abrogated the cross-linking of Spt5 to DNA and the anti-arrest properties of Spt4/5, thus suggesting that contact between Spt5 (NGN) and DNA is required for Spt4/5 to promote elongation. We propose that the mechanism of how Spt5/NGN promotes elongation is fundamentally conserved; however, the eukaryotic specific regions of the protein evolved so that it can serve as a platform for other elongation factors and maintain its association with RNAPII as it navigates genomes packaged into chromatin.

Keywords: DNA Protein Interactions; RNA polymerase II; Spt4/5; gene transcription; transcription elongation factor; transcription factor; transcription regulation.

FIGURE 1.

Characterization of ECs. A, schematic diagram of the formation of RNAPII ECs using 3′ end initiated templates. Radiolabeled UTP is incorporated into the transcript during the transcription of the G-less cassette. B, KMnO₄ footprint of the non-template strand. ECs (EC70) were prepared on immobilized RNAPII (see under “Experimental Procedures”). DNA sequence is indicated on the right of the gel. The four G bases (+1 to +4) are equivalent to the G-tract shown in A. The reactive Ts are marked with an asterisk. Heparin and TFIIS was added to the samples shown in lanes 4 and 5, respectively. All samples were run on the same gel, but irrelevant lanes were removed between lanes 4 and 5. C, RNase I footprinting of the transcript in ECs. ECs were formed on a template that produced a 35-nucleotide (NT) transcript with radiolabeled GTP incorporated into the 3′ end of the transcript in the active site of RNAPII. Left, schematic representation of RNA protection experiment. Right, gel image of the labeled RNA produced with and without RNase I digestion.

FIGURE 2.

Biochemical analysis of recombinant Spt4/5. A, Coomassie Blue-stained SDS-PAGE of recombinant of Spt4/5 and Spt4/5(ΔCTR). An arrow shows Spt4. The asterisk indicates a contaminating protein migrating just above Spt4 found in the preparation of full-length Spt4/5. B, EMSA of Spt4/5 and Spt4/Spt5 (ΔCTR) binding to RNAPII EC prepared on end-labeled DNA templates. The ratio of Spt4/5 to RNAPII was 0.75, 1.5, and 3.0. Free DNA migrated at the bottom of the gel and was cropped out of the image. C, RNAPII arrest assay comparing intact Spt4/5 and Spt4/5(ΔCTR). Saturating amounts of Spt4/5 were used in the assay, which was determined by shifting all of the EC in EMSA. D, RNAPII arrest assays were quantified as described under “Experimental Procedures” and are plotted as a fraction of active RNAPII as a function of time. The data were fit to an exponential decay curve. The data represent the averages and standard deviations of three independent assays.

FIGURE 3.

Spt4/5 contacts the emerging transcript. A, EMSA analysis of EC42 treated with (lanes 7–12) and without RNase I (lanes 1–6). Spt4/5 ΔCTR was titrated in and complexes were resolved on native polyacrylamide gels. B, RNase I footprint of the emerging transcript. Spt4/5 (ΔCTR) was titrated in prior to digestion. The experiment was conducted as described in the legend of Fig. 1C. C, ECs were prepared with body-labeled transcripts using [³²P]CTP and bromo-UTP. After exposure to UV, where indicated, samples were digested with DNase I and RNase A and separated on SDS-PAGE. Labeled proteins were visualized by exposing dried gels to a phosphorimager screen. Two different amounts (1- and 3-fold molar excess) of Spt4/5 were added. The locations of the bands corresponding to Rpb1, Rpb2, and Spt5(ΔCTR) are indicated on the right.

FIGURE 4.

C-terminal KOW domains of Spt5 are required for the binding to RNAPII ECs. A, schematic representation of Spt5 deletion mutants that were used in the figure. The area shaded in black is an acidic region in the N terminus; the white box is the NGN domain, and the diamonds are the KOW domains. B, Coomassie Blue-stained gel of the mutant complexes. C, EMSA comparing the binding of Spt4/5(ΔCTR), Spt4/5(Δ666–901), and Spt4/5(Δ419–1063) to ECs formed from 10 nm RNAPII. Increasing amounts of complex were titrated in and then resolved by native PAGE. D, quantification of EMSA experiments. Percent binding was plotted on the y axis as a function of Spt4/5 concentration. The data were then fit to a logarithmic binding curve, and a K_d value was estimated. Values represent the averages and standard deviations of three independent experiments (E). RNA cross-linking assay. The assay was conducted as described in Fig. 3C. A sample of RNAPII alone was untreated with UV (lane 1) (−UV) and all others were exposed to UV (lanes 2–10). Two amounts of each of the Spt4/5 complexes were titrated in. “+” indicates the amount of Spt4/5 required to shift ECs in EMSA assays and “++” is twice that amount (3- and 6-fold molar excess relative to RNAPII). Lane 1, −UV; lane 2, +UV; lanes 3 and 4, Spt4/5(ΔCTR); lanes 5 and 6, Spt4/5(Δ666–901); lanes 7 and 8, Spt4/5(284–931); and lanes 9 and 10, Spt4/5(Δ419–1063). The asterisk (Spt5*) marks a breakdown product in the Spt4/5 preparation. The dots mark the migration of the mutants in the gel.

FIGURE 5.

Footprinting of Spt4/5 containing ECs. A, DNase I footprinting experiment. ECs were formed on RNAPII immobilized on 8WG16 antibody beads. Washed ECs were eluted with recombinant GST-CTD; Spt4/5 was added or not and then treated with DNase I. The samples were resolved native gels, and the DNA in the bands was gel-purified and analyzed by denaturing PAGE. Letters above the panel in the denaturing PAGE (right) correspond to lettered bands in the native gel (left). Numbers on the left of the denaturing polyacrylamide gel indicate the sizes of the DNA marker (GA) in base pairs. The numbers on the right mark the location of the bases in the template relative to the first G (+1). B, quantitative analysis of the DNase I footprinting gel plotted as intensity versus distance migrated on the gel. Analysis and normalization are described under “Experimental Procedures.” The scan of the entire gel is show in the inset, and the region on the upstream side of RNAPII is shown in the main panel. Free DNA trace is shown in gray; RNAPII alone is shown in black, and RNAPII+Spt4/5 is shown in red.

FIGURE 6.

Footprinting elongation complexes with permanganate and exonuclease III. A, flow diagram explaining the experimental design. Details are given under “Experimental Procedures.” Briefly, immobilized RNAPII ECs were provided ATP and CTP (AC, lanes 3 and 4 in B) or provided CTP and GTP to allow transcription through the 4-base G-tract (CG, lanes 5 and 6 in B). Spt4/5(ΔCTD) was added where indicated. TFIIS was added after nucleotide addition to reduce the potential effects of backtracking on bubble progression. B, denaturing polyacrylamide gel showing the end-labeled non-template strand. Ts in the sequence are indicated on the right of the gel and labeled relative to the first G. C, ExoIII protection experiment to map the leading edge of RNAPII along the DNA. Denaturing PAGE comparing 6 and 9 min of cleavage by ExoIII. Free DNA (lanes 1–3) was compared with DNA + RNAPII-Spt4/5 (lanes 4 and 5) and DNA + RNAPII with Spt4/5(ΔCTD) (lanes 6 and 7). Positions of the bands are labeled to the right of the gel and represent the distance downstream from G+1. D, quantification of the ExoIII data from four independent experiments presented as the averages and S.E. The numbers on the left indicate the size of the band in nucleotides and correspond to those indicated in C.

FIGURE 7.

Spt5 cross-links to the non-template strand in ECs. A, schematic outline of the strategy used to generate site-specific labeled DNA probes on the non-template strand of the EC42 DNA template. A short oligo complementary to the template strand was annealed and used to prime synthesis of the NTS. In the first step, [³²P]dATP and 5-iodo-dCTP were incorporated using the Klenow (−exo) fragment of DNA polymerase. In a second step, cold dNTPs were added to complete the top strand (B). A model derived from overlaid crystal structures of RNAPII with the non-template strand, yeast Spt4/5, and archaeal RpoA fused to Spt5 (PDB codes 5C4X, 2EXU, and 3QQC) using PyMOL (version 1.7.4 Schrödinger, LLC). Highlighted in red are bases representing the position of photoreactive 5-iodo-dCTP nucleotide analog in the non-template strand of RNAPII. +1 is the site of NTP incorporation. C, SDS-PAGE analysis of cross-linked products. The location of the photoreactive nucleotide in each template is indicated above. The presence of Spt4/5(ΔCTD) (3-fold molar excess relative to RNAPII) is indicated in the panel. Arrows on the left of the gel indicate the migration of Rpb1, Rpb2, and Spt5(ΔCTD). Spt5* indicates the position of a breakdown product of Spt5 that cross-links to DNA. The band above Rpb1 marked with an asterisk may result from incomplete digestion of the nucleic acid template or products formed by protein-protein cross-links. D, SDS-PAGE comparing cross-linking of RNAPII only (lane 2), Spt4/5 (ΔCTR) (lane 3), and Spt4/5(Δ666–901) (lane 4) mutant to the −12 probe.

FIGURE 8.

Conserved KOW1-linker region is dispensable for the biochemical activities of Spt4/5. A, EMSA comparing the binding of Spt4/5(ΔCTR) and Spt4/5ΔK1L1 to ECs. Assays were conducted as described in the legend of Fig. 2B. The Spt4/5ΔK1L1 mutants also have the CTR removed, but it is labeled more simply in the figure. B, RNAPII arrest assay comparing the activity of RNAPII alone or RNAPII plus Spt4/5(ΔCTR) or Spt4/5ΔK1L1. Data are plotted as the fraction of active ECs as a function of time. The data were fit to an exponential decay curve. Error bars represent the standard deviation of three independent experiments. C, DNA photo-cross-linking of Spt4/5 to the −5 (lanes 1–4) and −21/−23 (lanes 5–8) positions.

FIGURE 9.

Charged surface on the NGN domain is required for the anti-arrest activity of Spt4/5. A, model derived as in Fig. 7B of Spt4/5(NGN) with the transcription bubble. Spt4 is colored in yellow and Spt5-NGN in blue. The region of Spt5-NGN encompassing the conserved basic patch (302–308) is colored in green, and the region Spt5-NGN containing the non-conserved patch (316–321) is colored in orange. B, SDS-PAGE of DNA cross-linking products using a template with a probe at positions −5 (lanes 1–8) and −12 (lanes 9–16). Note, each of the point mutations contained the CTR removed like the “wild type” Spt4/5 but is labeled only to indicate the amino acid changes in the NGN. C, EMSA testing binding of Spt4/5(ΔCTR) and Spt4/5(302–308A) to RNAPII ECs. D, RNAPII arrest assay comparing RNAPII alone and RNAPII plus the Spt4/5 derivatives indicated in the panel. The assay was conducted with saturating amounts of Spt4/5, estimated by the results of the EMSA. Data were plotted as the fraction of active RNAPII as a function of time and fit to an exponential decay curve for comparison. Error bars represent the standard deviation of four independent experiments.

FIGURE 10.

Analysis of the growth properties of Spt5 mutants. A, anchor-away yeast strains (SPT5-FRB) containing either pRS313, pRS313-SPT5-HA₃ (WT), pRS313-spt5(302–308A)-HA₃, or pRS313-spt5(ΔK1L1)-HA₃ were spotted onto synthetic media-His ± rapamycin (1 μg/ml) and incubated at either 30 or 37 °C for 3 days. B, Western blot analysis of yeast extracts from cells grown in the absence or presence of 1 μg/ml rapamycin. Cells were grown to an A₆₀₀ of 0.7, and then rapamycin was added for 1.5 h. Yeast bearing a plasmid containing an SPT5 mutant with the phosphorylated serine residues changed to alanines (spt5-CTR Ser → Ala) was used to discriminate the phosphorylated from the un-phosphorylated forms of Spt5 in the blot. C, analysis of H2B ubiquitylation. Western blot of cell extracts using an antibody to yeast H2B. A light exposure panel (below) serves as a control for total histone levels. Extracts from a rad6Δ mutant was run on the gel as a control to identify the ubiquitylated form of H2B.

FIGURE 11.

Bubble chaperone model of Spt4/5 action on ECs. A model illustrating the bubble chaperone mechanism. Model was generated by overlaying crystal structures of the archaeal clamp fused to archaeal Spt5 (data not shown, PDB code 3QQC), RNAPII with template and non-template strand (PDB code 5C4X) (18), and yeast Spt4/5 (PDB code 2EXU) (74). Spt4 and Spt5 are shown in electrostatic space-filling mode. Gray indicates all subunits of RNAPII except for Rpb2. Rpb2 was omitted from this structure except the Arch domain, which is shown in black. The template and non-template strand are shown as orange, and the RNA is in yellow. Hypothetical DNA path in the presence of Spt4/5 was drawn manually in red over the existing structure.