Efficient signal transduction by a chimeric yeast-mammalian G protein alpha subunit Gpa1-Gsalpha covalently fused to the yeast receptor Ste2.

Saccharomyces cerevisiae uses G protein-coupled receptors for signal transduction. We show that a fusion protein between the alpha-factor receptor (Ste2) and the Galpha subunit (Gpa1) transduces the signal efficiently in yeast cells devoid of the endogeneous STE2 and GPA1 genes. To evaluate the function of different domains of Galpha, a chimera between the N-terminal region of yeast Gpa1 and the C-terminal region of rat Gsalpha has been constructed. This chimeric Gpa1-Gsalpha is capable of restoring viability to haploid gpa1Delta cells, but signal transduction is prevented. This is consistent with evidence showing that the C-terminus of the homologous Galpha is required for receptor-G protein recognition. Surprisingly, a fusion protein between Ste2 and Gpa1-Gsalpha is able to transduce the signal efficiently. It appears, therefore, that the C-terminus of Galpha is mainly responsible for bringing the G protein into the close proximity of the receptor's intracellular domains, thus ensuring efficient coupling, rather than having a particular role in transmitting the signal. To confirm this conclusion, we show that two proteins interacting with each other (such as Snf1 and Snf4, or Ras and Raf), each of them fused either to the receptor or to the chimeric Galpha, allow efficient signal transduction.

TAP1, a yeast gene that activates the expression of a tRNA gene with a defective internal promoter.

We developed a genetic selection system based on nonsense suppression in Saccharomyces cerevisiae to identify mutations in proteins involved in transcription initiation by RNA polymerase III. A SUP4 tRNA(Tyr) internal promoter mutation (A53T61) that was unable to suppress ochre mutations in vivo and was incapable of binding TFIIIC in vitro was used as the target for selection of trans-acting compensatory mutations. We identified two such mutations in the same gene, which we named TAP1 (for transcription activation protein). The level of the SUP4A53T61 transcript was threefold higher in the tap1-1 mutant than in the wild type. The tap1-1 mutant strain was also temperature sensitive for growth. The thermosensitive character cosegregated with the restorer of suppression activity, as shown by meiotic linkage analysis and coreversion of the two traits. At 1 to 2 h after a shift to the restrictive temperature, RNA synthesis was strongly inhibited in the tap1-1 mutant, preceding any effect upon protein synthesis or growth. A marked decrease in tRNA and 5S rRNA synthesis was seen, and shortly after that, rRNA synthesis was inhibited. By complementation of the ts- growth defect, we cloned the wild-type TAP1 gene. It is essential for yeast growth. We show in the accompanying report (T. L. Aldrich, G. Di Segni, B. L. McConaughy, N. J. Keen, S. Whelen, and B. D. Hall, Mol. Cell. Biol. 13:3434-3444, 1993) that TAP1 is identical to RAT1, a yeast gene implicated in poly(A)+ RNA export and that the TAP1/RAT1 gene product has extensive sequence similarity to the protein encoded by another yeast gene (variously named DST2, KEM1, RAR5, SEP1, or XRN1) having exonuclease and DNA strand transfer activity (reviewed by Kearsey and Kipling [Trends Cell Biol. 1:110-112, 1991]).

Structure of the yeast TAP1 protein: dependence of transcription activation on the DNA context of the target gene.

Sequence data are presented for the Saccharomyces cerevisiae TAP1 gene and for a mutant allele, tap1-1, that activates transcription of the promoter-defective yeast SUP4 tRNA(Tyr) allele SUP4A53T61. The degree of in vivo activation of this allele by tap1-1 is strongly affected by the nature of the flanking DNA sequences at 5'-flanking DNA sequences as far away as 413 bp from the tRNA gene and by 3'-flanking sequences as well. We considered the possibility that this dependency is related to the nature of the chromatin assembled on these different flanking sequences. TAP1 encodes a protein 1,006 amino acids long. The tap1-1 mutation consists of a thymine-to-cytosine DNA change that changes amino acid 683 from tyrosine to histidine. Recently, Amberg et al. reported the cloning and sequencing of RAT1, a yeast gene identical to TAP1, by complementation of a mutant defect in poly(A) RNA export from the nucleus to the cytoplasm (D. C. Amberg, A. L. Goldstein, and C. N. Cole, Genes Dev. 6:1173-1189, 1992). The RAT1/TAP1 gene product has extensive sequence similarity to a yeast DNA strand transfer protein that is also a riboexonuclease (variously known as KEM1, XRN1, SEP1, DST2, or RAR5; reviewed by Kearsey and Kipling [Trends Cell Biol. 1:110-112, 1991]). The tap1-1 amino acid substitution affects a region of the protein in which KEM1 and TAP1 are highly similar in sequence.

Comparative mutational analysis of wild-type and stretched tRNA3(Leu) gene promoters.

We demonstrate that, when the yeast tRNA(3Leu) gene is stretched so that the distance between the two portions of the intragenic promoter is increased to 365 base pairs, the A and B blocks remain functional. Mutations in the A block, which show a weak phenotype when inserted in the wild type, exert a dramatic effect when inserted into the stretched gene. Experiments with extensively purified transcription factor tau indicate that the tau B-B block interaction is not influenced by A-B distance; only the ability of tau A to interact with A block sequences is affected, possibly because of the additional free-energy cost of forming a large loop of the intervening DNA.

Translational control by messenger RNA competition for eukaryotic initiation factor 2.

Translation of globin mRNA in a micrococcal nuclease-treated reticulocyte lysate was studied in the presence of increasing amounts of Mengovirus RNA, under conditions in which the number of translation initiation events remains constant as judged by the transfer of label from N-formyl[35S]methionyl-tRNAf into protein. The translation of globin mRNA is progressively inhibited by low concentrations of Mengovirus RNA, free of detectable traces of double-stranded RNA, concomitant with the increasing synthesis of Mengovirus RNA-directed products. On a molar basis, Mengovirus RNA apparently competes about 35 times more effectively than globin mRNA for a critical component in translation. The competition is relieved by the addition of highly purified eukaryotic initiation factor 2 (eIF-2). Addition of eIF-2 does not stimulate overall protein synthesis, but shifts it in favor of globin synthesis. No stimulation of globin mRNA translation by eIF-2 is seen when Mengovirus RNA is absent. These experiments show that Mengovirus RNA competes, directly or indirectly, with globin mRNA for eIF-2. In direct binding experiments using isolated mRNA and eIF-2, Mengovirus RNA is shown to compete with globin mRNA for eIF-2 and to exhibit a 30-fold higher affinity for this factor. The binding of Mengovirus RNA to eIF-2 is much more resistant to increasing salt concentrations than is the binding of globin mRNA, again reflecting its high affinity. These results reveal a direct correlation between the ability of these mRNA species to compete in translation and their ability to bind to initiation factor eIF-2. They suggest that the affinity of a given mRNA species for eIF-2 is essential in determining its translation, relative to that of other mRNA species. Messenger RNA competition for eIF-2 may contribute significantly to the selective translation of viral RNA in infected cells.

Deletion of the 3' half of the yeast tRNA-Leu3 gene does not abolish promotor function in vitro.

The promotor function of the 3' and 5' half tRNA sequences in the yeast tRNA-Leu3 gene has been studied by in vitro transcription in Xenopus laevis germinal vesicle (GV) extracts. Truncation of the DNA template within the tRNA intervening sequence by Hpa I abolishes transcription. However, separation of the tRNA gene halves by insertion of a 300 bp DNA fragment at the Hpa I site does not affect the promoter efficiency. Further, the complete sequence of the 3' half of the tRNA is not necessary for promoter function, because removal of the 3' half of the gene by cleavage with Pvu II, within the DNA inserted at the Hpa I site, does not inhibit transcription.

Selective in vitro transcription of one of the two Alu family repeats present in the 5' flanking region of the human epsilon-globin gene.

The sequence of 2965 nucleotides 5' of the human epsilon-globin gene has been completed. It includes two Alu family repeats present in an inverted configuration. Only the one located farthest from the gene was active as template for RNA polymerase III in a transcription system prepared from nuclei of Xenopus laevis oocytes. This selective transcription may be explained by the lack of homology of the first 45 nucleotides of the non transcribed repeat with other members of the Alu family. In fact this region includes one of the homology blocks described for other RNA polymerase III templates.

Absence of functional beta-globin messenger RNA in Kurdish Jews with beta0-thalassemia.

Human globin messenger RNA was isolated from reticulocytes of four Jewish patients of Kurdish origin with homozygous beta0-thalassemia. On translation in the wheat-germ cell-free system, messenger RNA from these patients directed extensive synthesis of alpha- and gamma-globin chains, but synthesis of beta-globin chains was not detectable. In contrast, nonthalassemic human globin messenger RNA directed the synthesis of essentially equimolar amounts of alpha- and beta-globin. The patterns of globin synthesized by beta0-thalassemic messenger RNA in the cell-free system were virtually identical to the patterns of globin synthesized in peripheral blood cells of these patients. beta0-thalassemic messenger RNA similarly failed to direct any detectable beta-globin synthesis in a micrococcal nuclease-treated rabbit reticulocyte lysate, even in the presence of an excess of purified eukaryotic initiation factor 2. These results strongly suggest that functional messenger RNA for beta-globin chains is absent in Kurdish Jews with homozygous beta0-thalassemia.