The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.

Life with 6000 Genes

The large ribosomal subunit catalyzes peptide bond formation and binds initiation, termination, and elongation factors. We have determined the crystal structure of the large ribosomal subunit from Haloarcula marismortui at 2.4 angstrom resolution, and it includes 2833 of the subunit's 3045 nucleotides and 27 of its 31 proteins. The domains of its RNAs all have irregular shapes and fit together in the ribosome like the pieces of a three-dimensional jigsaw puzzle to form a large, monolithic structure. Proteins are abundant everywhere on its surface except in the active site where peptide bond formation occurs and where it contacts the small subunit. Most of the proteins stabilize the structure by interacting with several RNA domains, often using idiosyncratically folded extensions that reach into the subunit's interior.

https://science.sciencemag.org/content/sci/289/5481/905.full.pdf

The complete atomic structure of the large ribosomal subunit at 2.4 A resolution.

The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.

/pdf/genomic-libraries-and-a-host-strain-designed-for-highly-1u4u4zfe67.pdf

Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast

All eukaryotic cells have an extensive membranous labyrinth network of branching tubules and flattened sacs called the endoplasmic reticulum (ER). Approximately one-third of all cellular proteins are translocated into the lumen of the ER where post-translational modification, folding, and oligomerization occurs. The ER provides a unique oxidizing compartment for the folding of membrane and secretory proteins that are destined to the cell surface, as well as for proteins destined to other intracellular organelles, such as lysosomes and the Golgi compartment. Numerous cellular proteins reside within the ER through a mechanism that requires their continuous vesicle-mediated retrieval from post-ER compartments within the early secretory pathway. These ERresident proteins are chaperones and catalysts of protein folding that form a matrix on which newly synthesized proteins attain their final conformation. The ER is also the site of synthesis of cellular lipids and sterols. In addition, the ER is the major signal-transducing organelle within the cell that continuously responds to environmental cues to release calcium. The ER is exquisitely sensitive to alterations in homeostasis, where, upon a variety of different stimuli, signals are transduced from the ER to the cytoplasm and the nucleus to eventually result in adaptation for survival or induction of apoptosis. The immediate response occurs at the translational apparatus, whereas changes in gene expression promote long-term adaptation or apoptotic cell death. Recent evidence supports findings that these signaling pathways influence pathogenesis associated with viral infection and genetic disease. The purpose of this review is to summarize what is presently known about the diversity of molecular signaling mechanisms that coordinate the complex ER stress response at the translational and transcriptional level in yeast and in higher eukaryotic cells.

/pdf/stress-signaling-from-the-lumen-of-the-endoplasmic-reticulum-50luw2lbp2.pdf

Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls

▪ Abstract The compartmentation of eukaryotic cells requires all nuclear proteins to be imported from the cytoplasm, whereas, for example, transfer RNAs, messenger RNAs, and ribosomes are made in the nucleus and need to be exported to the cytoplasm. Nuclear import and export proceed through nuclear pore complexes and can occur along a great number of distinct pathways, many of which are mediated by importin β-related nuclear transport receptors. These receptors shuttle between nucleus and cytoplasm, and they bind transport substrates either directly or via adapter molecules. They all cooperate with the RanGTPase system to regulate the interactions with their cargoes. Another focus of our review is nuclear export of messenger RNA, which apparently largely relies on export mediators distinct from importin β-related factors. We discuss mechanistic aspects and the energetics of transport receptor function and describe a number of pathways in detail.

Transport between the cell nucleus and the cytoplasm.

Ribosomal large and small subunits are organized in four general structural patterns. The four types are found in ribosomes from eubacteria, archaebacteria, eukaryotes, and a group of sulfur-dependent bacteria ( eocytes ), respectively. All four ribosomal types share a common structural core, but each type also has additional independent structural features. The independent features include the eukaryotic lobes and the archaebacterial bill on the smaller subunit. On the larger subunit, they include the eocytic lobe, eocytic gap, and eocytic bulge and a modified central protuberance. These data are most parsimoniously fit by a single unrooted evolutionary tree. In this tree eocytes are closely related to eukaryotes, while archaebacteria and eubacteria are closest neighbors. The tree is consistent with currently known molecular biological properties and indicates that eocytes have a phylogenetic importance equal to that of the three known kingdoms. When other properties and molecular mechanisms of these organisms are better defined, we suggest that an appropriate kingdom name for this group would be the Eocyta .

/pdf/eocytes-a-new-ribosome-structure-indicates-a-kingdom-with-a-43zz5mafyz.pdf

Eocytes: a new ribosome structure indicates a kingdom with a close relationship to eukaryotes.

Chromosome I from the yeast Saccharomyces cerevisiae contains a DNA molecule of approximately 231 kbp and is the smallest naturally occurring functional eukaryotic nuclear chromosome so far characterized. The nucleotide sequence of this chromosome has been determined as part of an international collaboration to sequence the entire yeast genome. The chromosome contains 89 open reading frames and 4 tRNA genes. The central 165 kbp of the chromosome resembles other large sequenced regions of the yeast genome in both its high density and distribution of genes. In contrast, the remaining sequences flanking this DNA that comprise the two ends of the chromosome and make up more than 25% of the DNA molecule have a much lower gene density, are largely not transcribed, contain no genes essential for vegetative growth, and contain several apparent pseudogenes and a 15-kbp redundant sequence. These terminally repetitive regions consist of a telomeric repeat called W', flanked by DNA closely related to the yeast FLO1 gene. The low gene density, presence of pseudogenes, and lack of expression are consistent with the idea that these terminal regions represent the yeast equivalent of heterochromatin. The occurrence of such a high proportion of DNA with so little information suggests that its presence gives this chromosome the critical length required for proper function.

https://www.pnas.org/content/pnas/92/9/3809.full.pdf

The nucleotide sequence of chromosome I from Saccharomyces cerevisiae.

The temperature-sensitive mutation prp20-1 of Saccharomyces cerevisiae exhibits a pleiotropic phenotype associated with a general failure to maintain a proper organization of the nucleus. Its mammalian homolog, RCC1, is not only reported to be involved in the negative control of chromosome condensation but is also believed to assist in the coupling of DNA replication to the entry into mitosis. Recent studies on Xenopus RCC1 have strongly suggested a further role for this protein in the formation or maintenance of the DNA replication machinery. To elucidate the nature of the various components required for this PRP20 control pathway in S. cerevisiae, we undertook a search for multicopy suppressors of a prp20 thermosensitive mutant. Two genes, GSP1 and GSP2, were identified that encode almost identical polypeptides of 219 and 220 amino acids. Sequence analyses of these proteins show them to contain the ras consensus domains involved in GTP binding and metabolism. The levels of the GSP1 transcript are about 10-fold those of GSP2. As for S. cerevisiae RAS2, GSP2 expression exhibits carbon source dependency, while GSP1 expression does not. GSP1 is an essential gene, and GSP2 is not required for cell viability. We show that GSP1p is nuclear, that it can bind GTP in an in vitro assay, and finally, that a mutation in GSP1p which activates small ras-like proteins by increasing the stability of the GTP-bound form causes a dominant lethal phenotype. We believe that these two gene products may serve in regulating the activities of the multicomponent PRP20 complex.

GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization.

We report on the characterization of the yeast prp20-1 mutant. In this temperature-sensitive mutant, multiple steps of mRNA metabolism are affected. The prp20-1 mutant strain showed alterations in mRNA steady-state levels, defective mRNA splicing and changes in transcription initiation or termination when shifted from the permissive to the non-permissive temperature. In addition, a change in the structure of the nucleus in these cells became apparent. Electron microscopy revealed an altered structure of the nucleoplasm of prp20-1 mutant cells when grown at the no-permissive temperature that was not observed in cells grown at the permissive temperature or in wild-type cells. The wild-type PRP20 gene was isolated and sequenced. The putative PRP20 protein has a molecular weight of 52 kDa. We found that the PRP20 gene is identical to the yeast SRM1 gene (Clark and Sprague 1989). In addition, the PRP20 protein sequence shows significant sequence similarity to the human RCC1 protein (Ohtsubo et al. 1987). This protein has been implicated in the control of chromosome condensation. Based on the phenotype of the prp20-1 mutant and the observed sequence similarity to the human RCC1 protein, we postulate that the yeast PRP20 protein is involved in the control of nuclear organization.

A yeast mutant, PRP20, altered in mRNA metabolism and maintenance of the nuclear structure, is defective in a gene homologous to the human gene RCC1 which is involved in the control of chromosome condensation.

Yeast tRNA ligase is an enzyme required for tRNA splicing. A study by indirect immune fluorescence shows that this enzyme is localized in the cell nucleus. At higher resolution, studies using indirect immune electron microscopy show this nuclear location to be primarily at the inner membrane of the nuclear envelope, most likely at the nuclear pore. There is a more diffuse, secondary location of ligase in a region of the nucleoplasm within 300 nm of the nuclear envelope. When the amount of ligase in the cell is increased, nuclear staining increases but staining of the nuclear envelope remains constant. This experiment indicates that there are a limited number of ligase sites at the nuclear envelope. Since the other tRNA splicing component, the endonuclease, has the characteristics of an integral membrane protein, we hypothesize that it constitutes the site for the interaction of ligase with the nuclear envelope.

/pdf/the-subnuclear-localization-of-trna-ligase-in-yeast-f1dsolldpt.pdf

Michael W. Clark

Papers

Eocytes: a new ribosome structure indicates a kingdom with a close relationship to eukaryotes.

The nucleotide sequence of chromosome I from Saccharomyces cerevisiae.

GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization.

A yeast mutant, PRP20, altered in mRNA metabolism and maintenance of the nuclear structure, is defective in a gene homologous to the human gene RCC1 which is involved in the control of chromosome condensation.

The subnuclear localization of tRNA ligase in yeast