Translation of the Saccharomyces cerevisiae tcm1 gene in the absence of a 5'-untranslated leader.

The role of eukaryotic 5'-untranslated messenger RNA leaders is not entirely clear, since they share little sequence similarity among each other. The importance of the leader in determining the efficiency of translation initiation was addressed here by examining the polyribosome distribution of several leader-deletion alleles of the yeast tcm1 gene (coding for ribosomal protein L3). Shortening of this 22-nucleotide leader, or complete removal of it (the first nucleotide of the mRNA becoming the A of the translation initiation codon AUG) permitted translation, albeit reduced. Further deletion of as few as the first two nucleotides of the initiation codon leads to a substantial reduction in ribosome loading, which is compatible with inefficient initiation at the next downstream, out-of-frame, AUG triplet. A second measure of translation initiation was obtained by assaying qualitatively for the production of biologically active L3 protein using growth-resistance to trichodermin. This experiment indicates that ribosomes can recognize the correct initiation codon even in the complete absence of a leader. We conclude that the 5'-untranslated leader of the yeast tcm1 gene is not essential for accurate translation initiation, but enhances its efficiency.

A sequence pattern that occurs at the transcription initiation region of yeast RNA polymerase II promoters.

Saccharomyces cerevisiae mRNA 5'-ends map at a variable distance from the TATA element. The mechanism for the choice of the transcription Initiation Region (IR) over other neighbouring sequences is not clearly understood. Sequences on the coding strand flanking the IR of 95 yeast RNA polymerase II promoters have been compared. They indicate the following pattern: statistically, a preponderance of T residues beginning as far as 30 nucleotides upstream and ending approximately 10 nucleotides upstream of the IR, and a preponderance of A residues from approximately 8 nucleotides upstream of the transcription initiation-site onward. The switch in base composition noted above thus occurs over a short region that is centered typically -9 nucleotides with respect to the major transcription start-site. We call this overall sequence pattern the locator. It is more evident among strong promoters than weak ones, suggesting a role in transcription initiation. The promoter of the TCM1 gene (coding for ribosomal protein L3) has a typical locator in the region of its IR. In an attempt to confirm the role of this sequence motif in defining the IR, deletions were introduced between the TATA element and the IR of the TCM1 gene. In most deletions, the new transcription start-sites are found within a recognizable locator, supporting the suggestion that this sequence pattern is important in defining the IR. These data appear to indicate that in yeast the IR is defined by a pattern of base composition situated at a suitable distance from the TATA element.

The accumulation of three yeast ribosomal proteins under conditions of excess mRNA is determined primarily by fast protein decay.

The suggestion that compensation for overabundant mRNA of the genes for Saccharomyces cerevisiae ribosomal protein (r-protein) L3, L29, or rp59 occurs by translation repression has been reinvestigated. First, analysis of the distribution of these three mRNAs in polysome profiles revealed no differences between normal and mRNA-overproducing strains, indicating that initiation of r-protein translation is not repressed under conditions of mRNA overaccumulation. Second, experiments involving radioactive pulse-labeling of proteins were done by using a modified method of data collection and analysis that allows quantitation and correction for fast decay during the pulse. These measurements revealed that the synthesis rate of the three r-proteins is increased when their mRNA levels are elevated and that their decay rate is also high, with half-lives ranging from a fraction of a minute to more than 10 min. We conclude that accumulation of excess r-protein mRNA has no effect on translation rate; rapid decay of protein during the course of the labeling period can account for the apparent discrepancy between mRNA levels and protein synthesis rates. Yeast r-proteins, when produced in excess, are among the most rapidly degraded proteins so far described.

Isolation of the SUP45 omnipotent suppressor gene of Saccharomyces cerevisiae and characterization of its gene product.

The Saccharomyces cerevisiae SUP45+ gene has been isolated from a genomic clone library by genetic complementation of paromomycin sensitivity, which is a property of a mutant strain carrying the sup45-2 allele. This plasmid complements all phenotypes associated with the sup45-2 mutation, including nonsense suppression, temperature sensitivity, osmotic sensitivity, and paromomycin sensitivity. Genetic mapping with a URA3+-marked derivative of the complementing plasmid that was integrated into the chromosome by homologous recombination demonstrated that the complementing fragment contained the SUP45+ gene and not an unlinked suppressor. The SUP45+ gene is present as a single copy in the haploid genome and is essential for viability. In vitro translation of the hybrid-selected SUP45+ transcript yielded a protein of Mr = 54,000, which is larger than any known ribosomal protein. RNA blot hybridization analysis showed that the steady-state level of the SUP45+ transcript is less than 10% of that for ribosomal protein L3 or rp59 transcripts. When yeast cells are subjected to a mild heat shock, the synthesis rate of the SUP45+ transcript was transiently reduced, approximately in parallel with ribosomal protein transcripts. Our data suggest that the SUP45+ gene does not encode a ribosomal protein. We speculate that it codes for a translation-related function whose precise nature is not yet known.