Hpr1 is preferentially required for transcription of either long or G+C-rich DNA sequences in Saccharomyces cerevisiae

Abstract

Hpr1 forms, together with Tho2, Mft1, and Thp2, the THO complex, which controls transcription elongation and genome stability in Saccharomyces cerevisiae. Mutations in genes encoding the THO complex confer strong transcription-impairment and hyperrecombination phenotypes in the bacterial lacZ gene. In this work we demonstrate that Hpr1 is a factor required for transcription of long as well as G+C-rich DNA sequences. Using different lacZ segments fused to the GAL1 promoter, we show that the negative effect of lacZ sequences on transcription depends on their distance from the promoter. In parallel, we show that transcription of either a long LYS2 fragment or the S. cerevisiae YAT1 G+C-rich open reading frame fused to the GAL1 promoter is severely impaired in hpr1 mutants, whereas transcription of LAC4, the Kluyveromyces lactis ortholog of lacZ but with a lower G+C content, is only slightly affected. The hyperrecombination behavior of the DNA sequences studied is consistent with the transcriptional defects observed in hpr1 cells. These results indicate that both length and G+C content are important elements influencing transcription in vivo. We discuss their relevance for the understanding of the functional role of Hpr1 and, by extension, the THO complex.

FIG. 1

Expression patterns of serial deletions of GAL1pr::PHO5-lacZ fusion constructs. Acid phosphatase activities under induced conditions of wild-type (W303-1A) and hpr1 (U768-4C) strains transformed with lacZ-deleted variants of plasmids pSCh212 (A) or pSCh211 (B) that contain the entire lacZ coding sequence fused to PHO5 in the same and opposite orientations, respectively, under the GAL1 promoter. The average value and standard deviation of four different transformants is shown for each strain. Vertical lines across the lacZ sequences indicate the end points of the deletion constructs analyzed.

FIG. 2

Transcription analysis of GAL1pr::PHO5, three different GAL1pr::PHO5-lacZ fusion constructs, and a GAL1pr::PHO5-GAL1 fusion in wild-type (W303-1A) and hpr1 (U768-4C) cells. (A) Northern blot analyses of PHO5-containing mRNAs driven from the GAL1 promoter. Plasmids used were pSCh229, pSCh226, and pSCh211Δ17-1 (carrying ≈0.4 kb of the 5′ end, middle part, and 3′ end of lacZ fused to PHO5, respectively), pSCh202 (carrying the PHO5 gene), or pSCh251 (carrying ≈0.4 kb of the 5′ end of GAL1 fused to PHO5). Mid-log phase cells were cultured in 3% glycerol–2% lactate synthetic complete (SC)-Ura medium and diluted into identical fresh media to an OD₆₀₀ of 0.3 and incubated for 16 h. Galactose (Gal) was then added and samples were taken for Northern analysis at different times, as specified. A 0.9-kb EcoRV PHO5 internal fragment and a 589-bp 28S rDNA internal fragment obtained by PCR (rRNA) were used as DNA probes. (B) Kinetics of induction of mRNAs as determined by quantification of Northern blots in a Fuji FLA3000. The mRNA values are given in arbitrary units (A.U.) with respects to rRNA levels. For any given construct, RNA levels are related to the wild-type (wt) levels at 90 min, which was set at 100 for each panel.

FIG. 3

Transcription analysis of three different GAL1pr::lacZΔ-PHO5 fusion constructs in wild-type (W303-1A) and hpr1 (U768-4C) cells. (A) Northern blot analyses of PHO5-containing mRNAs driven from the GAL1 promoter. Plasmids used were pSCh218, pSCh220, and pSCh219 (carrying ≈0.4 kb of the 5′ end, middle part, and 3′ end of lacZ fused to PHO5, respectively). As a control we used pSCh202 (carrying the PHO5 gene; data not shown), which gave identical results as those shown in Fig. 2. The transcript levels of each construct with respect to PHO5 were similar to those of the wild type shown in Fig. 2. Other details were as described for Fig. 2. (B) Quantification of Northern analyses.

FIG. 4

Transcription and recombination analyses of several GAL1pr::lacZ fusion constructs in wild-type (W303-1A) and hpr1 (U768-4C) cells. (A) Northern analyses of lacZ-containing mRNAs transcribed from the GAL1 promoter. Plasmids used were pSCh215, pSCh213, and pSCh216 (carrying ≈0.4 kb of the 5′ end, middle part, and 3′ end of lacZ, respectively) or p416GAL1-lacZ (carrying the entire lacZ ORF). Other details were as described for Fig. 2. (B) Recombination frequencies of leu2-based direct-repeat systems containing the same short lacZ fragments used in the previous Northern experiments. Plasmids used were pSCh221, pSCh222, and pSCh223 (carrying ≈0.4 kb of the 5′ end, middle part, and 3′ end of lacZ, respectively) or pSCh205 (carrying the entire lacZ ORF). A schematic diagram of the recombination products obtained with the direct-repeat LEU2 recombination systems used is shown at the top of panel B. The LEU2 promoter (Prm) and transcriptional terminator (Ter) as well as the RNA (arrow) produced by the system are indicated. The median recombination frequency of six independent values is given in each case. All median frequencies were calculated in duplicate with two independent transformants. Recombinants were selected in SC-Leu-Trp. Data from the L-lacZ system containing the entire lacZ gene (bottom) are taken from Chávez and Aguilera (11).

FIG. 5

Transcription and recombination analyses of LYS2 sequences in wild-type and hpr1 cells. (A) Northern blot analyses of LYS2 mRNAs in strains transformed with plasmid pSCh227 containing a 3.7-kb fragment of the LYS2 coding sequence under the control of the GAL1 promoter. (B) Recombination frequencies of strains transformed with plasmid pSCh230 harboring a leu2-based direct repeat system containing as intervening sequence the same 3.7 kb fragment of LYS2 used for the transcription assays. Other details are as described for Fig. 4.

FIG. 6

Transcription analyses of five yeast endogenous genes, EGT2, CDC48, KAR2, OLE1, and GOG5, having high levels of expression and different transcript sizes, in wild-type and hpr1 cells. Total RNA was isolated from mid-log phase cells, grown in YEPD broth, and used for Northern analyses. Internal fragments of each gene and of the 23S rDNA, obtained by PCR, were used as DNA probes. The hpr1:wild-type transcript ratio was obtained from the mRNA levels that were quantified in a Fuji FLA3000 and normalized with respect to the rRNA levels.

FIG. 7

Transcription and recombination analyses of LAC4 in wild-type and hpr1 cells. (A) Northern analyses of LAC4 mRNAs in strains transformed with the plasmid pSCh255, which contains the entire LAC4 coding sequence under the control of the GAL1 promoter. (B) Recombination frequencies of cells transformed with plasmid pSCh254, which harbors the leu2-based direct-repeat L-LAC4 construct containing LAC4 as the intervening region. (C) Northern analyses of the L-LAC4 repeat construct in wild-type and hpr1 cells. Other details are as described for Fig. 4.

FIG. 8

Transcription and recombination analyses of YAT1 in wild-type and hpr1 cells. (A) Northern blot analyses of YAT1 mRNAs in cells transformed with plasmid pSCh247 containing the entire YAT1 coding sequence under the control of the GAL1 promoter. (B) Recombination analyses of cells transformed with plasmid pSCh248, which harbors the leu2-based direct-repeat system containing the entire YAT1 gene as intervening sequence. Other details are as described for Fig. 4.

FIG. 9

MNase digestion pattern of the GAL1 gene and the GAL1pr::lacZ fusion in wild-type and hpr1 strains under repression and activation conditions. (A) Northern blot analysis of GAL1 mRNAs. (B) Nucleosome positioning over the GAL1 gene. (C) MNase digestion pattern of the GAL1pr::lacZ fusion. A scheme of the analyzed regions of GAL1 and lacZ indicating the position of nucleosomes and the most relevant regulatory elements is shown. Asterisks indicate the MNase hypersensitive sites associated with the activation of transcription of GAL1.