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. 2011 May 27;286(21):18816-24.
doi: 10.1074/jbc.M110.202101. Epub 2011 Apr 5.

The transcription elongation factor Spt5 influences transcription by RNA polymerase I positively and negatively

Susan J Anderson  1 , Martha L Sikes, Yinfeng Zhang, Sarah L French, Shilpa Salgia, Ann L Beyer, Masayasu Nomura, David A Schneider
Affiliations

The transcription elongation factor Spt5 influences transcription by RNA polymerase I positively and negatively

Susan J Anderson et al. J Biol Chem. .

Abstract

Spt5p is a universally conserved transcription factor that plays multiple roles in eukaryotic transcription elongation. Spt5p forms a heterodimer with Spt4p and collaborates with other transcription factors to pause or promote RNA polymerase II transcription elongation. We have shown previously that Spt4p and Spt5p also influence synthesis of ribosomal RNA by RNA polymerase (Pol) I; however, previous studies only characterized defects in Pol I transcription induced by deletion of SPT4. Here we describe two new, partially active mutations in SPT5 and use these mutant strains to characterize the effect of Spt5p on Pol I transcription. Genetic interactions between spt5 and rpa49Δ mutations together with measurements of ribosomal RNA synthesis rates, rDNA copy number, and Pol I occupancy of the rDNA demonstrate that Spt5p plays both positive and negative roles in transcription by Pol I. Electron microscopic analysis of mutant and WT strains confirms these observations and supports the model that Spt4/5 may contribute to pausing of RNA polymerase I early during transcription elongation but promotes transcription elongation downstream of the pause(s). These findings bolster the model that Spt5p and related homologues serve diverse critical roles in the control of transcription.

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Figures

FIGURE 1.
FIGURE 1.
Isolated mutations in SPT5 map to different faces of the protein and suppress cold sensitivity of rpa49Δ strains. A, ribbon diagram adapted from the published structure of Spt4p-Spt5 (NusG-like domain) fusion protein (Protein Data Bank code 2EXU) (36). Spt4p is colored yellow, and the NusG-like domain of Spt5p is pink. Residues in Spt5p implicated in binding Spt4p, Glu-338 and Ser-324, are shown in spacefill and colored blue and green, respectively. Cys-292 is colored red (also in spacefill) and is positioned on the opposite face of the domain. B, segregants resulting from dissection of tetrads of spt4 spt5 heterozygous diploids (constructed by mating NOY2167 to DAS540&541) are shown with the relevant genotypes of individual haploid strains indicated. The spt4 spt5 double mutants are indicated by 4,5. The plates were incubated 5 days at 27 °C. No viable spt4Δ spt5(C292R) double mutants were recovered. C, 10-fold dilutions of individual haploid segregants resulting from sporulation of DAS578, DAS579, and DAS581 were spotted onto YEPD plates and grown at 23 °C for 5 days before imaging. D, haploid segregants shown in C were grown in YEPD liquid culture at 27 °C with aeration and growth rates (doublings per hour) were calculated. The “expected” growth rate is the product of the growth rates of the parental haploid mutants as a percentage of the WT growth rate.
FIGURE 2.
FIGURE 2.
rDNA copy number is not reduced by mutation of SPT5. A, chromosomes from strains indicated (grown in YEPD at 27 °C) were separated by contour-clamped homogeneous field electrophoresis and transferred to a nylon membrane. Southern blot hybridization using an rDNA probe permitted detection of chromosome XII in the upper part of the blot. Control strains with known rDNA copy number (locked by deletion of FOB1) were included (left four lanes). The image was processed to delete lanes between the rightmost three lanes and the remainder of the gel. Contrast and position were not altered. B, rDNA copy number from control strains was plotted as a function of migration distance of chromosome XII. A linear regression was generated from these data, and the equation from the regression is shown. C, rDNA copy numbers in WT and mutant strains were estimated according to the equation in B.
FIGURE 3.
FIGURE 3.
Mutation of SPT5 reduces rRNA synthesis rate but not Pol I occupancy of rDNA. A, duplicate WT and spt5(C292R) cultures were grown in SD−Met at 27 °C with aeration to A600 = ∼0.3. The cells were pulse-labeled for 5 min with 25μCi/ml [methyl-3H]methionine and chased for 5 min with excess cold methionine (500 μg/ml). Isolated RNA from same number of cell equivalents (normalized to final A600 of culture) was subject to electrophoresis in a 1% formaldehyde:agarose gel, transferred to a nylon membrane, and visualized by autoradiography. The film was developed after 24 h of exposure. 25 and 18 S rRNA (and a background band) were excised from the membrane, and 3H incorporation was quantified by a scintillation counter. The counts were averaged and normalized to WT with 1 standard deviation ± shown. B, diagram of location of primer pairs used for quantitative PCR analysis of ChIP DNA. C, ChIP data demonstrate that Pol I occupancy of rDNA is not reduced in spt5(C292R)(DAS540) cells compared with WT (NOY396). A polyclonal anti-A190 antibody was used for immunoprecipitation. The data shown are the averages of three DNA dilutions from each of two independent cultures. The error bars represent one standard deviation.
FIGURE 4.
FIGURE 4.
rRNA is not overproduced in rrp6Δ spt5(C292R) double mutants. NOY396, DAS208, DAS570, and DAS604 were grown and labeled as described for Fig. 3, except that cells were harvested after a 4-min pulse (without chase) and after a 5-min pulse and a 5-min chase with cold methionine. P indicates pulse samples, and C indicates pulse-chase samples. RNA was loaded for equal A600 of the culture. Precursor and mature RNA species in the gel are labeled. The upper panel is a 24-h exposure of the film, and the lower panel is a 4-day exposure of film with the same blot.
FIGURE 5.
FIGURE 5.
EM analysis of rRNA gene transcription supports a role for Spt4/5 in pause and release of Pol I transcription elongation. A, representative rDNA repeats from WT (NOY396) and spt5(C292R) (DAS540) analyzed by EM of Miller chromatin spreads are shown. The 5′ end of each gene is oriented to the left. The straight arrows indicate individual transcripts near the 3′ end of the genes that are characteristic of the transcript processing status for that strain (cleaved at A2 for WT and uncleaved for spt5(C292R)) (41, 42). Bracketed arrows at the 5′ and 3′ ends of each gene indicate gene regions quantified for polymerase density for C. Scale bar, 0.5 μm. B, the frequency of detection of polymerase density was plotted as a function of the number of polymerases per gene for WT and spt5(C292R) spreads. The data were averaged with errors indicated (n = number of active genes analyzed). Analysis of “on” versus “off” rDNA repeats was performed as described previously (22), and the data are shown (n = number of rDNA repeats analyzed). The error in each case equals one standard deviation. By multiplying the rDNA copy number (Fig. 3), the average number of polymerases per gene, and the percentage of active genes, we calculated the approximate number of polymerase engaged in transcription in the WT and spt5(C292R) strains. C, polymerase density in the first and last 10% of each rDNA repeat (as shown by brackets in A) was quantified. For WT (NOY396) and spt5(C292R) (DAS540) strains, the frequency at which the density was greater at the 5′ end versus the 3′ end on individual genes was plotted as well as the frequency at which the density was lesser at the 5′ end versus the 3′ end. D, a model for the positive and negative effects of Spt4/5 on Pol I transcription is depicted using an idealized EM view of one rDNA repeat. Gray circles on the straight line indicate transcribing Pol I on rDNA. Increased polymerase density near 5′ end of the gene indicates a proposed Spt4/5-mediated pause of the transcription elongation complex. One or more modifications of Spt5p (depicted by star) led to pause release and enhancement of Pol I transcription elongation rate in the remaining portion of the gene. Black circles at the ends of rRNA transcripts represent formation of mature processomes, which are cleaved from nascent transcripts after compaction of the pre-18 S rRNA into the processome (42).
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