PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing.

We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing. PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity. pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame. The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect. Episome-borne PTA1 directs recombination to the pta1-1 locus. PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base. Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA1 gene product are discussed.     

Structures of ribosomal subunits from Saccharomyces cerevisiae

The large (60S) and small (40S) ribosomal subunits were isolated from yeast (Saccharomyces cerevisiae), and preparations stained with uranyl acetate were imaged by transmission electron microscopy. Averages of three different projections of each subunit were obtained by computerized image processing with reproducible spatial resolutions approaching 3.0 and 3.5 nm, respectively. Similarities between the structures of these particles and those of other eukaryotic ribosomal subunits contribute to the development of a concensus model for the eukaryotic ribosome.     

Binding of proteins from the large ribosomal subunits to 5.8 S rRNA of Saccharomyces cerevisiae

Specific binding of purified proteins from the large ribosomal subunits of Saccharomyces cerevisiae to 5.8 S rRNA was examined by three different methods: nitrocellulose membrane filtration, sucrose density gradient centrifugation, and RNA-Sepharose column chromatography. RNA-protein complex formation was proportional to the amount of proteins added to the reaction mixture. The binding of proteins to the RNA could be saturated. Such RNA-protein complexes were isolated on sucrose density gradients. Protein species present in these complexes were isolated, iodinated, and analyzed by two-dimensional polyacrylamide gel electrophoresis. Eleven proteins, L13, L14, L17, L19, L21, L24, L25, L29, L30, L33, and L39, were identified. By comparison, only six proteins interacted with the 5.8 S rRNA-Sepharose under similar ionic conditions. They were proteins L14, L21, L24, L27, L29, and L30. To better characterize these binding proteins, the interaction of individual proteins with 5.8 S rRNA was studied by nitrocellulose membrane filtration. Proteins L14, L19, L21, L29, L33, and L39 were observed to bind individually with 5.8 S rRNA. Binding of each protein to the RNA could be saturated. The apparent association constants (K'a), measured at 4 degrees C and in 30 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 330 mM KCl, and 6 mM beta-mercaptoethanol, ranged from 1.05 to 3.70 X 10(6) M-1.     

RRP20, a component of the 90S preribosome,is required for pre-18S rRNA processing in Saccharomyces cerevisiae

A strain of Saccharomyces cerevisiae, defective in small subunit ribosomal RNA processing, has a mutation in YOR145c ORF that converts Gly235 to Asp. Yor145c is a nucleolar protein required for cell viability and has been reported recently to be present in 90S pre-ribosomal particles. The Gly235Asp mutation in YOR145c is found in a KH-type RNA-binding domain and causes a marked deficiency in 18S rRNA production. Detailed studies by northern blotting and primer extension analyses show that the mutant strain impairs the early pre-rRNA processing cleavage essentially at sites A₁ and A₂, leading to accumulation of a 22S dead-end processing product that is found in only a few rRNA processing mutants. Furthermore, U3, U14, snR10 and snR30 snoRNAs, involved in early pre-rRNA cleavages, are not destabilized by the YOR145c mutation. As the protein encoded by YOR145c is found in pre-ribosomal particles and the mutant strain is defective in ribosomal RNA processing, we have renamed it as RRP20.     

Defective processing of ribosomal precursor RNA in Saccharomyces cerevisiae.

Saccharomyces cerevisiae (strain A224A) has an abnormal distribution of cytoplasmic ribosomal subunits when grown at 36 degrees C, with sucrose-gradient analysis of extracts revealing an apparent excess of material sedimenting at 60 S. This abnormality is not observed at either 23 degrees C or 30 degrees C. At 36 degrees C the defect(s) is expressed as a slowed conversion of 20 S ribosomal precursor RNA to mature 18 S rRNA, although the corresponding maturation of 27 S ribosomal precursor RNA to mature 25 S rRNA is normal. Studies on this yeast strain and on mutants derived from it may help to elucidate the role(s) of individual ribosomal components in controlling ribosome biogenesis in eukaryotes.     

Temperature sensitive nop2 alleles defective in synthesis of 25S rRNA and large ribosomal subunits in Saccharomyces cerevisiae

Using molecular genetic techniques, we have generated and characterized six temperature sensitive (ts) alleles of nop2. All failed to support growth at 37°C and one was also formamide sensitive (fs) and failed to grow on media containing 3% formamide. Conditional lethality is not due to rapid turnover of mutant Nop2p proteins at 37°C. Each allele contains between seven and 14 amino acid substitutions and one possesses a nonsense mutation near the C-terminus. Mapping experiments with one allele, nop2-4, revealed that a subset of the amino acid substitutions conferred the ts phenotype and that these mutations have an additive effect. All six mutants exhibited dramatic reductions in levels of 60S ribosome subunits under non-permissive conditions as well as some reduction at permissive temperature. Processing of 27S pre-rRNA to mature 25S rRNA was defective in all six mutants grown under non-permissive conditions. Levels of the 40S ribosomal subunit and 18S rRNA were not significantly affected. Amino acid substitutions in nop2 conditional alleles are discussed in the context of the hypothesis that Nop2p functions both as an RNA methyltransferase and a trans-acting factor in rRNA processing and large ribosomal subunit biogenesis.     

Isolation and characterization of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae

The unsaturated fatty acid (ufa) requiring ole1 mutant of Saccharomyces cerevisiae appears to produce a defective delta-9 fatty acid desaturase. This enzyme catalyzes double bond formation between carbons 9 and 10 of palmitoyl and stearoyl coenzyme A. A DNA fragment isolated by complementation of an ole1 strain repairs the ufa requirement in mutant cells. Genetic analysis of the cloned DNA fragment indicates that it is allelic to the OLE1 gene. Disruption of a single copy of the wild type gene in a diploid strain produces both wild type and nonreverting ufa-requiring haploid progeny upon sporulation. Membrane lipids of the disrupted haploid strains contain only ufas supplied in the growth medium. The recovery of activity in both wild type and disrupted segregants was examined after removal of ufas from the growth medium. Following ufa deprivation disruptant cells grew normally for about three generations and then at a slower rate for at least 0.6 generations. During that time cellular ufas dropped from 63 to 7.3 mol % of the total fatty acids. No production of the 16:1 and 18:1 products of the desaturase was observed in disruptant cells, whereas desaturation in wild type control cells was evident 2 h after deprivation. These results indicate that 1) the OLE1 gene is essential for production of monounsaturated fatty acids and is probably the structural gene for the delta-9 desaturase enzyme. 2) A large part of membrane ufas present under normal culture conditions are not essential for growth and cell division.     

Location of the 5.8S rRNA gene of Saccharomyces cerevisiae.

Direct DNA sequence analysis of Saccharomyces cerevisiae ribosomal DNA cloned in an Escherichia coli plasmid revealed part of the structural gene for 5.8S rRNA at one end of a 700-base-pair EcoRI fragment. Taken with the previously established EcoRI restriction map of the ribosomal repeat unit, this sequence establishes that the yeast 5.8S RNA segment is located between the 18S and 28S segments in the 42S rRNA precursor and in the DNA which codes for it.     

The KRR1 gene encodes a protein required for 18S rRNA synthesis and 40S ribosomal subunit assembly in Saccharomyces cerevisiae

The newly discovered Saccharomyces cerevisiae gene KRR1 (YCL059c) encodes a protein essential for cell viability. Krr1p contains a motif of clustered basic amino acids highly conserved in the evolutionarly distant species from yeast to human. We demonstrate that Krr1p is localized in the nucleolus. The KRR1 gene is highly expressed in dividing cells and its expression ceases almost completely when cells enter the stationary phase. In vivo depletion of Krr1p leads to drastic reduction of 40S ribosomal subunits due to defective 18S rRNA synthesis. We propose that Krr1p is required for proper processing of pre-rRNA and the assembly of preribosomal 40S subunits.     

gcr2, a new mutation affecting glycolytic gene expression in Saccharomyces cerevisiae.

Screening of a mutagenized strain carrying a multicopy ENO1-'lacZ fusion plasmid revealed a new mutation affecting most glycolytic enzyme activities in a pattern resembling that caused by gcr1: levels in the range of 10% of wild-type levels on glycerol plus lactate but somewhat higher on glucose. The recessive single nuclear gene mutation, named gcr2-1, was unlinked to gcr1, and GCR1 in multiple copies did not restore enzyme levels. GCR2 was obtained by complementation from a YCp50 genomic library; the complemented strain had normal enzyme levels, as did a strain with GCR2 in multiple copies. GCR2 in multiple copies did not suppress gcr1. A chromosomal gcr2 null mutant was constructed; its pattern of enzyme activities resembled that of the gcr2-1 mutant and, like the gcr2-1 mutant, its growth defect on glucose was only partial (in contrast to the glucose negativity of the gcr1 mutant). Northern (RNA) analysis showed that gcr2 and gcr1 affect ENO1 mRNA levels.     

DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae.

The mating pheromone alpha-factor arrests Saccharomyces cerevisiae MATa cells in the G1 phase of the cell cycle. Size control is also exerted in G1, since cells do not exit G1 until they have attained a critical size. A dominant mutation (DAF1-1) which causes both alpha-factor resistance and small cell size (volume about 0.6-fold that of the wild type) has been isolated and characterized genetically and by molecular cloning. Several alpha-factor-induced mRNAs were induced equivalently in daf1+ and DAF1-1 cells. The DAF1-1 mutation consisted of a termination codon two-thirds of the way through the daf1+ coding sequence. A chromosomal deletion of DAF1 produced by gene transplacement increased cell volume about 1.5-fold; thus, DAF1-1 may be a hyperactive or deregulated allele of a nonessential gene involved in G1 size control. Multiple copies of DAF1-1 also greatly reduced the duration of the G1 phase of the cell cycle.     

Assembly of Saccharomyces cerevisiae 60S ribosomal subunits: role of factors required for 27S pre-rRNA processing

The precise functions of most of the ～200 assembly factors and 79 ribosomal proteins required to construct yeast ribosomes in vivo remain largely unexplored. To better understand the roles of these proteins and the mechanisms driving ribosome biogenesis, we examined in detail one step in 60S ribosomal subunit assembly-processing of 27SA(3) pre-rRNA. Six of seven assembly factors required for this step (A(3) factors) are mutually interdependent for association with preribosomes. These A(3) factors are required to recruit Rrp17, one of three exonucleases required for this processing step. In the absence of A(3) factors, four ribosomal proteins adjacent to each other, rpL17, rpL26, rpL35, and rpL37, fail to assemble, and preribosomes are turned over by Rat1. We conclude that formation of a neighbourhood in preribosomes containing the A(3) factors establishes and maintains stability of functional preribosomes containing 27S pre-rRNAs. In the absence of these assembly factors, at least one exonuclease can switch from processing to turnover of pre-rRNA.     

Similarity between the association factor of ribosomal subunits and the protein Stm1p from Saccharomyces cerevisiae

A ribosome association factor (AF) was isolated from the yeast Sacchharomyces cerevisiae. Partial amino acid sequence of AF was determined from its fragment of 25 kDa isolated by treating AF with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-Bromoindolenine (BNPS-skatole). This sequence has a 86% identity to the product of the single-copy S. cerevisiae STM1 gene that is apparently involved in several events like binding to quadruplex and triplex nucleic acids and participating in apoptosis, stability of telomere structures, cell cycle, and ribosomal function. Here we show that AF and Stm1p share some characteristics: both bind to quadruplex and Pu triplex DNA, associates ribosomal subunits, and are thermostable. These observations suggest that these polypeptides belong to a family of proteins that may have roles in the translation process.