Catabolite control of the elevation of PGK mRNA levels by heat shock in Saccharomyces cerevisiae.

Heat shock enhances the very high level of transcription of the phosphoglycerate kinase (PGK) gene in fermentative cultures of Saccharomyces cerevisiae. This response of PGK mRNA levels was not found on gluconeogenic carbon sources, and could be switched on or off subject to availability of fermentable carbon source. The addition of glucose to yeast growing on glycerol resulted in acquisition, within 30-60 min, of the ability to elevate PGK mRNA levels after heat shock. In addition, in aerobic cultures growing on glucose the exhaustion of the medium glucose coincided with a loss of the heat-shock effect on PGK mRNA and a switch-over to slower growth by aerobic respiration. Levels of hsp26 mRNA were analysed during these experiments. Contrasting with this requirement for fermentable catabolite for manifestation of a heat-shock response of PGK mRNA levels, the PGK enzyme was not synthesized at a greater level in heat-shocked fermentative than in gluconeogenic cultures. PGK is one of only a few proteins made efficiently after mild heat shock of yeast. Thus, heat-stress-induced elevation of PGK mRNA levels does not appreciably increase PGK synthesis during exposure to high temperatures and so its role may be to assist cells repressed in mitochondrial function during recovery following a heat shock.

A heat shock element in the phosphoglycerate kinase gene promoter of yeast.

The phosphoglycerate kinase (PGK) promoter is often employed in yeast expression vectors due to its very high efficiency. Its activity in unstressed cells has been shown to be due to an upstream activator site (UASPGK) at -402 to -479. Since levels of PGK mRNA can sometimes be elevated by heat shock of yeast cultures this investigation determined how specific deletions of PGK promoter sequences effect levels of PGK mRNA both before and after heat shock. A series of PGK promoter deletions was inserted on a high copy plasmid into cells having a TRP1 gene disruption of the solitary chromosomal PGK locus. This enabled PGK transcripts of plasmid and chromosomal origin to be distinguished by virtue of their different sizes. Certain deletions lacking UASPGK displayed activities that were very low in unstressed cells, but which increased fifty to one-hundred fold after heat shock. With UASPGK present heat shock had only a relatively small or negligible effect on PGK mRNA levels. Heat shock activation was abolished when the -256 to -377 region with homology to the heat shock element consensus of eukaryotes was deleted in addition to UASPGK, but was unaffected by the deletion of regions further downstream containing TATA- and CAAT- sequence motifs. This is the first demonstration of a heat shock element, an activator site normally found upstream of eukaryotic heat shock protein genes, as a natural constituent of a high efficiency glycolytic promoter. It is proposed that PGK may be one member of a small subset of yeast genes that are highly expressed in unstressed cells yet possess a heat shock element to ensure their continued transcription after heat shock.

The effects of protein synthesis inhibition, and of mutations rna1.1 and rna82.1, on the synthesis of small RNAs in yeast.

Strains of Saccharomyces cerevisiae have been constructed that possess temperature-sensitive defects in tRNA precursor (pre-tRNA) splicing and which also lack the processing endonuclease that acts at the 3'-terminus of 5 S rRNA and 35 S rRNA precursors (pre-rRNAs). The unspliced pre-tRNAs accumulated by such strains at the nonpermissive temperature are identical in structure to those accumulated by pre-tRNA splicing-defective strains with a functional pre-5 S RNA processing enzyme. The pre-RNA processing activity is therefore not obligatorily involved in maturation of several yeast tRNAs. However, gels of the pulse-labelled RNAs of RNA82+ and rna82.1 strains provide evidence that this enzyme acts upon a few small unstable transcripts that are not 5 S RNA forms. The most prominent of these transcripts on gels was, in wild-type strains, an RNA 145 +/- 2 nucleotides in length.

Transcription of the phosphoglycerate kinase gene of Saccharomyces cerevisiae increases when fermentative cultures are stressed by heat-shock.

The single gene for phosphoglycerate kinase (PGK) in the haploid genome of Saccharomyces cerevisiae is expressed to a very high level in cultures fermenting glucose. Despite this it responds to heat-shock. When S. cerevisiae growing exponentially on glucose media was shifted from 25 degrees C to 38 degrees C transient increases of 6-7-fold in cellular PGK mRNA were observed. This elevation in PGK mRNA still occurred in the presence of the protein-synthesis inhibitor cycloheximide, but was not observed in cells bearing the rna1.1 mutation. From the kinetics of continuous labelling of PGK mRNA, relative to the labelling of other RNAs in the same cultures whose levels do not alter with heat-shock, it was shown that the elevation in PGK mRNA in response to temperature upshift reflects primarily an increased synthesis of this mRNA and not an alteration of its half-life. PGK mRNA synthesis is therefore one target of a response mechanism to thermal stress. Synthesis of PGK enzyme in glucose-grown cultures is efficient after mild (25 degrees C to 38 degrees C) or severe (25 degrees C to 42 degrees C) heat-shocks. Following the severe shock, the synthesis of most proteins is abruptly terminated, but synthesis of PGK and a few other glycolytic enzymes continues at levels comparable to the levels of synthesis of most of those proteins dramatically induced by heat (heat-shock proteins). Cells that overproduce PGK due to the presence of multiple copies of the PGK gene on a high-copy-number plasmid continue their overproduction of this enzyme during severe thermal stress. Therefore PGK mRNA is both elevated in level in response to heat-shock and translated efficiently at supra-optimal temperatures.

Processing of the 3' sequence extensions upon the 5S rRNA of a mutant yeast in Xenopus laevis germinal vesicle extract.

A processing endonuclease acts to remove a short sequence from the 3' end of transcripts of the Saccharomyces cerevisiae 5S ribosomal RNA gene in generating the mature sequence of 5S RNA. Cells bearing the nuclear mutation rna82 .1 lack this activity and accumulate 5S forms with additional nucleotides at their 3' termini. 5S RNAs labelled during short pulse- labellings of the mutant are essentially primary transcripts that mostly have the sequence U-U-A-U-U-U-C[U-U-U-U(U-U)] added to the 3' end of normal yeast 5S RNA. They are subjected in vivo to a series of slow processing events whereby this sequence is ultimately replaced by: U-U(A)1-9 in a substantial proportion of the 5S RNA molecules of the mutant [Piper, P. W., Bellatin , J. A. and Lockheart , A. (1983) EMBO J. 2, 353-359]. In higher eukaryotes no endonuclease cleavage occurs during 5S RNA maturation, yet processing at the 3' ends of certain transcripts made by RNA polymerase III, most notably transfer RNA precursors, is still important. Since the enzymes involved in this processing have not been well characterised, we investigated how the additional sequences upon rna82 .1 yeast 5S RNA are processed in vitro in a system from a higher eukaryote that is often used for studying transcription by RNA polymerase III, the Xenopus laevis germinal vesicle extract. Our results are consistent with slow digestion of these 5S molecules by a 3'----5' exonuclease until they become 122-123 nucleotides in length, whereupon digestion ceases. This activity probably participates in the processing of certain Xenopus RNA polymerase III transcripts.

A minor class of 5S rRNA genes in Saccharomyces cerevisiae X2180-1B, one member of which lies adjacent to a Ty transposable element.

In Saccharomyces cerevisiae the majority of the genes for 5S rRNA lie within a 9kb rDNA sequence that is present as 100-200 tandemly-repeated copies on Chromosome XII. Following our observations that about 10% of yeast 5S rRNA exists as minor variant sequences, we screened a collection of yeast DNA fragments cloned in lambda gt for 5S rRNA genes whose flanking sequences differed from those adjacent to 5S rRNA genes of the rDNA repeat. Three variant 5S rRNA genes were isolated on the basis of such dissimilarity to rDNA repeat sequences. They display a remarkable conservation of their DNA in the vicinity of the 5S coding region, and are examples of a minor form of 5S rRNA coding sequence present in a small number of copies in the yeast genome. These variant sequences appear to be transcribed as efficiently as 5S rRNA genes of the rDNA repeat. In one of our isolates of the variant sequence a Ty transposable element is inserted 145bp upstream of the initiation point for 5S rRNA synthesis.

Altered maturation of sequences at the 3' terminus of 5S gene transcripts in a Saccharomyces cerevisiae mutant that lacks a RNA processing endonuclease.

Sequences at the immediate 3' terminus of several eukaryotic primary transcripts, synthesised just before the termination of transcription, are often lost during RNA processing. The rna82.1 mutation in Saccharomyces cerevisiae appears to result in a deficiency of the endonuclease that removes such sequences from certain yeast transcripts. Some small RNAs of rna82.1 cells are a few nucleotides longer than their counterparts in wild-type S. cerevisiae. The 5S rRNAs made during very short pulse-labellings of the mutant have, relative to the mature 121 nucleotide 5S RNA of wild-type cells, an additional 7, 11 or 13 nucleotides at their 3' terminus. These 5S forms reveal sites upon 5S genes where transcription probably terminates in vivo. The extra nucleotides upon 5S RNAs in rna82.1 cells are lost very slowly by sequential removal from the 3' terminus. Through this 3'-5' exonuclease action the total 5S RNA of the mutant possesses several 3'-terminal sequences yet is mostly only 0-3 nucleotides longer than in wild-type S. cerevisiae. Just one or two of these 3'-terminal sequences serve as a substrate in vivo for a poly(A) polymerase since a small proportion of rna82.1 5S RNAs terminate in the sequence: CAAUCUUU(A)n.