Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae

Abstract

The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt(-)) phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS(+) phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt(-) phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6 We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt(-) mutants. We determine that the lys2-128∂ Spt(-) phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the ∂ element as the position of Spt(-) transcription in tested Spt(-) mutants.

Keywords: Ty1 element; cryptic transcription; gene expression; transcription elongation; transcription initiation.

Figure 1

Allele-specific synthetic lethality between Pol II activity mutants and alleles of SPT4 and SPT5. Yeast strains containing mutants of SPT4 or SPT5 and rpo21∆ were transformed with LEU2-marked CEN plasmids containing rpo21/rpb1 alleles. Transformants were patched and replica-plated to media allowing rpo21/rpb1 complementation by a URA3-marked CEN RPO21/RPB1 plasmid (SC-Leu) or media toxic to cells maintaining the URA3-marked CEN RPO21/RPB1 plasmid (SC-Leu+5FOA). Resulting growth of replica-plated patches was documented after 1 and 5 d of growth. Color bars indicate genetically predicted/known reduction of function (blue) or increase of function (green) rpo21/rpb1 alleles. H1085Q/E1103G has mixed LOF/GOF properties based on genetic assays. GOF, gain of function; LOF, loss of function; Vec, vector only; WT, wild-type.

Figure 2

Allele-specific synthetic lethality between Pol II activity mutants and the SPT6 allele, spt6-1004. Yeast strain containing spt6-1004 and rpo21∆ was transformed with LEU2-marked CEN plasmids containing rpo21/rpb1 alleles. Transformants were patched and replica-plated to media allowing rpo21/rpb1 complementation by a URA3-marked CEN RPO21/RPB1 plasmid (SC-Leu) or media toxic to cells maintaining the URA3-marked CEN RPO21/RPB1 plasmid (SC-Leu 5FOA). Resulting growth of replica-plated patches was documented after 1 and 5 d of growth. Color bars indicate genetically predicted/known reduction of function (blue) or increase of function (green) rpo21/rpb1 alleles. Vec, vector only; WT, wild-type.

Figure 3

Conditional genetic interactions between Pol II activity mutants and spt4∆ and spt5-194. Phenotypes of viable double mutants between spt4∆ or spt5-194 combined with rpo21/rpb1 alleles (see Materials and Methods). Serial dilutions (10-fold) of control or double mutant strains spotted on various media for phenotyping of genetic interactions (general growth, temperature sensitivity, Spt^-, MPA^S, and Gal^R phenotypes). Color bars indicate genetically predicted/known reduction of function (blue) or increase of function (green) rpo21/rpb1 alleles. Gal, galactose; MPA, mycophenolic acid; WT, wild-type; YPD, yeast extract peptone dextrose.

Figure 4

Conditional genetic interactions between Pol II activity mutants and spt5-242. Phenotypes of viable double mutants between spt5-242 combined with rpo21/rpb1 alleles (see Materials and Methods). Serial dilutions (10-fold) of control or double mutant strains spotted on various medial for phenotyping of genetic interactions (general growth, temperature sensitivity, Spt^-, MPA^S, and Gal^R phenotypes). Color bars indicate genetically predicted/known reduction of function (blue) or increase of function (green) rpo21/rpb1 alleles. Raf, raffinose; Gal, galactose; MPA, mycophenolic acid; WT, wild-type; YPD, yeast extract peptone dextrose.

Figure 5

Conditional genetic interactions between Pol II activity mutants and spt6-1004. Phenotypes of viable double mutants between spt6-1004 combined with rpo21/rpb1 alleles (see Materials and Methods). Serial dilutions (10-fold) of control or double mutant strains spotted on various medial for phenotyping of genetic interactions (general growth, temperature sensitivity, Spt^-, MPA^S, and Gal^R phenotypes). Color bars indicate genetically predicted/known reduction of function (blue) or increase of function (green) rpo21/rpb1 alleles. Raf, raffinose; Gal, galactose; MPA, mycophenolic acid; WT, wild-type; YPD, yeast extract peptone dextrose.

Figure 6

Summary of genetic interactions between Pol II activity mutants and Spt elongation factor alleles. Phenotypes of viable Spt-Pol II double mutants are shown as a heatmap with qualitative determinations of growth defects for the relevant media. Dark gray indicates inviable double mutants. Light gray indicates conditions/strains not tested. For YPD, YPD 37°, YPRaf, and SC-Leu media, single and double mutant growth levels were normalized to WT. Decreased growth relative to WT is shown in shades of blue. Red indicates increased growth relative to WT. For SC-Leu+MPA, growth differences were normalized to those on SC-Leu to account for general effects on growth vs. those specific to MPA treatment. Similarly, YPRafGal was normalized to defects on YPD, and SC-Lys to those on SC-Leu. See Materials and Methods for additional explanation of heatmaps. (A) Interactions between Spt gene alleles and Pol II mutants. Orange and purple outlines indicate examples of clusters of allele-specific interactions. (B) Temperature-dependent genetic interactions between spt5-242 and Pol II alleles. Purple outline highlights suppression of spt5-242 at lower temperatures (but apparent at 30°). Orange outline highlights decreased growth at 37° for double mutants. Raf, raffinose; Gal, galactose; MPA, mycophenolic acid; WT, wild-type; YPD, yeast extract peptone dextrose.

Figure 7

Pol II LOF alleles suppress the Spt^- phenotype of high copy SPT6. Strains containing rpo21/rpb1 alleles were transformed by introduction of high copy (2µ) empty vector (pRS423) or SPT6 (pRS423 SPT6) and general growth (SC-Leu-His medium) or Spt^- phenotypes (SC-Leu-His-Lys medium) were assessed. Color bars indicate genetically predicted/known reduction of function (blue) or increase of function (green) rpo21/rpb1 alleles. LOF, loss of function; WT, wild-type.

Figure 8

Derepression of the FLO8 intragenic cryptic promoter occurs in an extreme Pol II allele, G1097D, and requires FLO8 transcription. (A) Schematic of GAL1promoter::FLO8::HIS3 construct allowing analysis of HIS3-dependent growth due to transcription from a cryptic FLO8 intragenic promoter (denoted by “TATA”) driving HIS3 expression. (B) Assay of rpo21/rpb1 allele activation of the FLO8 cryptic intragenic promoter dependent on activation of the GAL1 promoter by presence of galactose in growth medium (+Gal). Gal, galactose; WT, wild-type.

Figure 9

Expression levels of FLO8 and STE11 cryptic transcripts are sensitive to reduced Pol II activity mutants. (A) Northern blotting for FLO8-derived RNAs (left) or STE11-derived RNAs (right) in various mutant strains. Northern blotting for SED1 provided a normalization control. spt6 indicates presence of spt6-1004 allele. (B) Quantification of mRNA levels for FLO8-derived transcripts (full-length, top; major cryptic, middle; and minor cryptic, bottom) normalized to SED1. Full-length FLO8 expression was normalized relative to the level in WT. Levels of cryptic transcripts were normalized to full-length and then relative to those in spt6-1004. Error bars represent standard deviation of the mean for at least three independent biological replicates. (C) Quantification of mRNA levels for STE11-derived transcripts (full-length, top; cryptic 1, middle; and cryptic 2, bottom) normalized to SED1. Full-length STE11 expression was normalized relative to the level in WT. Levels of cryptic transcripts were normalized relative to full-length then to those in spt6-1004. Error bars as in (B). WT, wild-type.

Figure 10

Suppression of lys2-128∂ by spt6-1004 or rpo21/rpb1 alleles requires upstream transcription and can be modulated by an ectopic promoter. (A) Analysis of spt6-1004 effects on lys2-128∂ promoter derivatives. lys2-128∂ was modified in the promoter region for LYS2 in SPT6 and spt6-1004 strains containing either GAL10 or gal10∆56. Modifications included deletion of the LYS2 promoter by homologous recombination-based insertion of a drug resistance cassette (in either orientation denoted “Ori 1” or “Ori 2”) or by replacement of the LYS2 promoter with the GAL1 promoter. Strains were assayed for the Spt^- phenotype on media containing galactose (+Gal). All other media used glucose as carbon source. Ten-fold serial dilutions of each strain spotted onto appropriate medium. (B) Analysis of rpo21/rpb1 allele effects on lys2-128∂ promoter derivatives. The LYS2 promoter was replaced with the GAL1 promoter as in (B) for a strain allowing transformation with different rpo21/rpb1 alleles. Analysis and conditions as in (B). Color bars indicate genetically predicted/known reduction of function (blue) or increase of function (green) rpo21/rpb1 alleles. Gal, galactose; WT, wild-type; YPD, yeast extract peptone dextrose.

Figure 11

Expression levels of LYS2 and lys2-128∂ transcripts in Spt^- phenotype-modulating mutants. (A) Schematic of lys2-128∂ (top). Northern blotting for LYS2/lys2-128∂-derived RNAs under different media conditions in rpo21/rpb1 and/or spt6-1004 (spt6) mutant strains. SED1 mRNA provides a normalization control. (B) Quantification of LYS2/lys2-128∂-derived RNA levels under different media conditions in rpo21/rpb1 and/or spt6 mutant strains. Error bars indicate standard deviation of the mean of three independent biological replicates. Left: full- or near-full-length LYS2 mRNA levels normalized to level in WT strain grown in SD comparing LYS2 with lys2-128∂ and effects of spt6-1004. Note that scale of y-axis is logarithmic. Center: effects on full- or near-full-length LYS2 mRNA levels in rpo21/rpb1 and/or spt6 mutant strains. Right: Effects on truncated (short) LYS2 mRNA levels in rpo21/rpb1 and/or spt6 mutant strains. SD, synthetic defined; WT, wild-type; YPD, yeast extract peptone dextrose.

Figure 12

Detection of lys2-128∂ transcript 5′ ends in an Spt^- Pol II allele rpb1-E1103G and spt6-1004. (A) Primer extension analysis detecting transcripts emanating from the Ty1 ∂ element at lys2-128∂ for rpo21/rpb1 and spt6-1004 strains (left). Right side of panel shows nmd2∆ and rrp6∆ versions of strains for detection of possible unstable transcripts in the region targeted by nonsense-mediated decay (nmd2∆) or by the nuclear exosome (rrp6∆). Numbered asterisks are bands more apparent in mutant strains but are not specific to LYS2 (see B). Asterisk indicates nonspecific band in all strains, present even upon deletion of LYS2 (not shown) “G A T C” represents Sanger DNA sequencing reactions using same primer as primer extension reactions, for identification of putative lys2-128∂ transcripts. (B) Primer extension analysis for strains with or without the LYS2 promoter driving lys2-128∂. Deletion of the LYS2 promoter (spt6-1004 strain) or inhibition of transcription upstream of lys2-128∂ (GAL1p:lys2-128∂, rpb1-E1103G strain under glucose growth) suppresses the Spt^- phenotypes of spt6-1004 and rpo21/rpb1 alleles (Figure 10) and is predicted to abolish transcripts conferring Lys2 function. (C) Schematic showing RNA 5′ ends emanating from the downstream end of the Ty1 ∂ element from lys2-128∂ detected by 5′ RACE in WT, spt6-1004, and rpo21/rpb1-E1103G strains. The +1 position is defined as the A in the LYS2 ATG. Lower case letters indicate ∂ element sequence. Yellow highlighting indicates ∂ ATG in-frame with downstream LYS2 sequence. Underlined sequence is a 5 bp duplication created upon Ty1 retrotransposition (Farabaugh and Fink 1980). Blue dots indicate number of clones detected by sequencing and the positions of their 5′ ends. RACE, rapid amplification of cDNA ends; WT, wild-type.