Search

The yeast poly(A) binding protein Pab1p mediates the interactions between the 5' cap structure and the 3' poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. The PAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes.

The PPR1 gene encodes the positive regulator of the URA1 and URA3 genes in yeast. Its transcription product is a 2.9‐kb polyadenylated RNA with an extremely short half‐life of 1 min. The induced or non‐induced cell contains approximately 0.1 molecules of PPR1 RNA, a constitutive level which is not altered by changing the number of structural genes to be regulated. The DNA sequence of a 399‐bp AccI‐Bg/II fragment including 180 nucleotides of the 5′‐flanking region of the gene PPR1 has been determined. By S1 mapping we present evidence that the 5′ non‐coding region of the PPR1 mRNA is heterogeneous in length. The 5′ termini of the different RNAs map 20, 36, 45 and 50 nucleotides upstream from the translation start codon. The sequence indicates that the only open translation phase begins with an AUG codon that is preceded by two out‐of‐frame AUG triplets. This particular structure of the 5′‐terminal sequence of the transcripts of PPR1 is discussed in relation to both their stability and translational efficiency.

A cDNA encoding the Arabidopsis thaliana uridine 5'-monophosphate (UMP)/cytidine 5'-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [Km] = 29 microM when UMP is the other substrate and Km = 292 microM when CMP is the other substrate) as a phosphate donor. However, both UMP (Km = 153 microM) and CMP (Km = 266 microM) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P1, P5-di(adenosine-5') pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.

The yeast arg3 gene, coding for ornithine carbamoyltransferase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3), has been cloned on a hybrid pBR322-2-micrometers plasmid. The cloned gene gives a normal regulatory response in yeast. It is not expressed at 35 degrees C when a mutation preventing mRNA export from the nucleus at this temperature is included in the genetic make-up of the carrier strain. In Escherichia coli, no functional expression can be observed from the native yeast arg3 gene. The study of a mutant plasmid (M1) producing low levels of yeast carbamoyltransferase in E. coli has permitted the localization and orientation of arg3 on the plasmid. The mutation involved is a deletion that alters the regulatory response of arg3 in yeast. The plasmid bla gene produces detectable amounts of beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) in yeast: the data provide an estimate of the beta-lactamase activity associated with one exemplar of the plasmid expressing arg3 (0.6 units).

From the protein and RNA content of Saccharomyces cerevisiae growing in different media we calculate that ribosome efficiency is changed: incorporation of amino acids into protein decreases from 8.8 amino acids/s per ribosome in fast-growing cells (0.54 doubling/h) to 5.2 amino acids/s per ribosome in slow-growing cells (0.30 doubling/h). We could not detect significant protein turnover in either fast-or slow-growing cultures, so the lower ribosome efficiency does not seem to be an artifact caused by changes in unstable protein production at different growth rates. Nor is the lower ribosome efficiency due to slower migration of ribosomes along mRNA: the times required to complete polypeptides of known molecular weights are the same in slow-growing cells as those previously determined for fast-growing cells [Waldron, Jund & Lacroute (1974) FEBS Lett. 46, 11-16]. We therefore deduce that ribosome efficiency changes in yeast because the fraction of ribosomes engaged in protein synthesis falls (from 84% in fast-growing cells to 50% in slow-growing cells.

The fine structure map of the yeast URA 3 gene was established by meiotic recombination, and amber nonsense mutations were located at different points on the map. The effect of the length of the labeling time on the specific radioactivity of ura 3 messenger RNA and on its repartition between poly(A)-RNA and RNA not containing poly(A) has been followed in nonsense mutants. Nonsense mutations reduce the messenger level without lowering its instantaneous rate of synthesis. The strength of the reduction depends on the position of the nonsense codon within the locus and concerns essentially the accumulation of polyadenylylated ura 3 mRNA.

S1 mapping on the VAS1 structural gene indicates the existence of two classes of transcripts initiating at distinct in-frame translation start codons. The longer class of VAS1 transcripts initiates upstream of both ATG codons located 138 base pairs away and the shorter class downstream of the first ATG. A mutation that destroys the first AUG on the long message results in respiratory deficiency but does not affect viability. Mutation of the ATG at position 139 leads to lethality because the initiating methionine codon of the essential cytoplasmic valyl-tRNA synthetase has been destroyed. N-terminal protein sequence data further confirm translation initiation at ATG-139 for the cytoplasmic valyl-tRNA synthetase. From these results, we conclude that the VAS1 single gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. The presequence of the mitochondrial valyl-tRNA synthetase shows amino acid composition but not the amphiphilic character of imported mitochondrial proteins. From mutagenesis of the ATG-139 we conclude that the presequence specifically targets the cytoplasmically synthesized mitochondrial valyl-tRNA synthetase to the mitochondrial outer membrane and prevents binding of the enzyme core to cytoplasmic tRNAVal.

From a large population of strains of Escherichia coli carrying shear fragments of yeast (Saccharomyces cerevisiae) DNA attached by in vitro recombination to the plasmid vector pMB9, two hybrid plasmids were selected that relieve the pyrimidine requirement of nonreverting pyrF mutants of E. coli. An 1100-base-pair DNA fragment common to the two complementing plasmids was recloned into another plasmid vector, pBR322; these new hybrids retained the ability to specify orotidine-5'-phosphate decarboxylase (orotidine-5'-phosphate carboxy-lyase, EC 4.1.1.23) synthesis in E. coli. Evidence is presented that this common fragment is yeast DNA and thus apparently carried the structural information for yeast orotidine-5'-phosphate decarboxylase, the product of yeast gene ura3. A hybrid plasmid containing the 1100-base-pair fragment was used to measure levels of putative ura3 mRNA from yeast cultures labeled with [3H]adenine, ura3 mRNA was unstable with an apparent half-life of 10.5 min. Under different circumstances previously shown to alter the level of orotidine-5'-phosphate decarboxylase in yeast, a coordinate variation in proportion of labeled RNA complementary to the hybrid plasmid was found. These data support the hypothesis that regulation of the ura3 gene in yeast is at the level of transcription.