Molecular evidence for a positive role of Spt4 in transcription elongation

Abstract

We have previously shown that yeast mutants of the THO complex have a defect in gene expression, observed as an impairment of lacZ transcription. Here we analyze the ability of mutants of different transcription elongation factors to transcribe lacZ. We found that spt4Delta, like THO mutants, impaired transcription of lacZ and of long and GC-rich DNA sequences fused to the GAL1 promoter. Using a newly developed in vitro transcription elongation assay, we show that Spt4 is required in elongation. There is a functional interaction between Spt4 and THO, detected by the lethality or strong gene expression defect and hyper-recombination phenotypes of double mutants in the W303 genetic background. Our results indicate that Spt4-Spt5 has a positive role in transcription elongation and suggest that Spt4-Spt5 and THO act at different steps during mRNA biogenesis.

Fig. 1. Transcription analysis of a leu2Δ3′::lacZ fusion in different transcription elongation mutants and their respective isogenic wild-type strains. Northern analyses were performed from overnight cultures of yeast cells transformed with plasmid pSCh204. Strains used were W303-1A (WT), ela1Δ (ela1Δ), JSY102 (elp1Δ), elp2Δ (elp2Δ), JSY130 (elp3Δ), MH236H-1B (WT), MH236H-1D (spt4-3), MH137H-1B (WT), MH137H-4A (spt6-140), MF348-5B (WT), MF348-1A (spt16-197), ADW11-7C (WT), ADW11-11A (rpb2-10), ADW11-4B (dst1), ALY95-4A (WT), ALY95-1B (rpb2-10 dst1Δ). The DNA probes used were the 3 kb BamHI 5′-end fragment of lacZ and a 589 bp 28S rDNA internal fragment obtained by PCR. The relative amount of lacZ mRNA values normalized with the corresponding WT value for each mutant analyzed is shown at the bottom.

Fig. 2. Transcription analysis of lacZ in wild-type, spt4Δ and spt5 mutant cells. (A) Scheme of the lacZ-URA3 translational fusion under the control of the tet promoter. (B) Northern analyses of lacZ-URA3 expression in wild-type (SPT4TW-1A or OY 97), spt4Δ (SPT4TW-1B), spt5-194 (GHY 94) and spt5-242 (GHY 828) strains transformed with plasmid pCM184-LAUR. DNA probes used were the 3 kb BamHI 5′-end fragment of lacZ fragment and a 589 bp 28S rDNA internal fragment obtained by PCR. For more details, see Figure 1. Shown values are the mean of two independent experiments.

Fig. 3. Transcription analysis of GAL1p::lacZ, GAL1p::PHO5, GAL1p::klLAC4, GAL1p::LYS2 and GAL1p::YAT1 in wild-type and spt4Δ cells. Northern analyses of lacZ, PHO5, klLAC4, LYS2 and YAT1 mRNAs driven from the GAL1 promoter in the strains BY4741 (WT) and YGR063c (spt4Δ) transformed with plasmids p416GAL1lacZ, pSCh202, pSCh255, pSCh227 and pSCh247 are shown. Mid-log phase cells grown in 2% glucose SC medium lacking uracil were washed and diluted into SC 3% glycerol-2% lactate medium lacking uracil to an OD₆₀₀ of 0.8 and incubated for 14 h. Galactose was then added and samples were taken for northern analyses at different times, as specified. DNA probes used were the 3 kb BamHI 5′-end fragment of lacZ, a 0.9 kb EcoRV PHO5 internal fragment, 3 kb XbaI fragment containing klLAC4 ORF, a 2 kb XhoI–XbaI fragment containing YAT1, LYS2 internal fragment obtained by PCR and a 589 bp 28S rDNA internal fragment obtained by PCR. Plotted mRNA values are given in arbitrary units (AU) with respect to rRNA levels. For more details, see Figure 1. The YAT1 and LYS2 expression levels observed at 0 min is due to transcription of the endogenous genes copies. Other details are as in Figure 1.

Fig. 4. In vitro transcription assays of wild-type and spt4Δ WCEs. (A) Scheme of double-G-less cassette system. RNase T1-treatment of mRNA driven from GAL4-CYC1 promoter render two fragments corresponding to the G-less cassettes. (B) In vitro transcription assay using 400 ng of the double-G-less cassette-containing fragments as template, 100 µg of WCE from W303-1A (WT) and MGSC339 (spt4Δ) strains and 100 ng of Gal4-VP16. Reactions were stopped at 0, 5, 10, 20 and 40 min, treated with 160 U of RNaseT1 and run in a 6% PAGE. Two bands from each G-less cassette were obtained probably due to incomplete action of RNase T1. (C) Percentage of total transcripts that reach the 376 bp G-less cassette in both wild-type and spt4Δ cells with respect to the transcripts that cover the 84 bp cassette, taken as 100%. Each result is the mean value of two independent in vitro transcription assays. Radioactivity incorporated into the G-less cassettes was quantified in a Fuji FLA3000 and normalized with respect to the C content of each G-less cassette.

Fig. 5. Genetic analysis of the growth phenotype conferred by spt4Δ in combination with mutations of the THO complex. Tetrad analyses of the diploid strains obtained by crossing spt4Δ strain with either tho2Δ, hpr1Δ and mft1Δ null mutants are shown in the W303 genetic background. Whereas tho2Δ spt4Δ and hpr1Δ spt4Δ were synthetically lethal (double-mutant spores were able to germinate but died after several divisions), mft1Δ spt4Δ were able to grow. hpr1Δ spt4Δ double-mutant spores were rescued by the YEpHPR1 plasmid containing a wild-type HPR1 copy. The THO null mutations, which contained the KAN cassette, were scored by G418 resistance and the spt4Δ::URA3 by the Ura⁺ phenotype. Double-mutant spores are indicated with circles.

Fig. 6. Transcription analysis of GAL1 in spt4Δ mft1Δ cells. Northern blot analyses of GAL1 mRNA in the strains W303-1A (WT), MGSC339 (spt4Δ), WMS-1C (mft1Δ) and WMS-7C (spt4Δ mft1Δ). DNA probes used were the 750-bp AvaI–PvuII fragment of GAL1 and a 589 bp 28S rDNA internal fragment obtained by PCR. Other details are as in Figure 1.

Fig. 7. Recombination and plasmid instability analyses in wild-type, mft1Δ, spt4Δ and spt4Δ mft1Δ cells. (A) Scheme of the deletion resulting from a recombination event between the direct repeats. Transcripts driven from the external LEU2 promoter are indicated by arrows. Recombination frequencies were determined in the strains W303-1A, MGSC339 (spt4Δ), WMK-2A (mft1Δ) and WMS-7C (spt4Δ mft1Δ) transformed with pRS314-LA (LA). The median frequency of the six colonies is given. (B) Scheme of pRS313GZ used to assay plasmid instability. The frequencies of plasmid loss were determined in the same strains mentioned above under high or low transcription of the GAL4-CYC1p::lacZ construct. For this, six colonies were isolated in SC with either 2% glucose (low) or with 2% galactose (high). The frequency of colonies that have retained the plasmid were determined as the number of colonies growing in SC-his.