Isolation and characterization of PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae.

Keierleber, C; Wittekind, Michael; Qin, Shiliang; McLaughlin, Calvin S.

doi:10.1128/mcb.6.12.4419

Cited by 27 publications

(15 citation statements)

References 48 publications

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“…This location is consistent with the results of Keierleber et al (31), who mapped the prt1-1 mutation upstream of position 2220 by integrative transformation. The same study also suggested that, in the absence of gene conversion, the prt1-1 mutation lies upstream of position 1052; our findings indicate that gene conversion may indeed have occurred during that work.…”

Section: Discussionsupporting

confidence: 82%

“…Transcription of the PRT1 gene was found to initiate downstream of the first in-frame ATG of the ORF. This result is consistent with earlier work that localized PRT1 transcription initiation to a region 0.57 kbp upstream of the internal XbaI site (31). The first 39 codons of the PRT1 ORF are therefore not translated; the translation start site is most likely encoded by the second in-frame ATG.…”

Section: Discussionsupporting

confidence: 81%

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Mutational Analysis of the Prt1 Protein Subunit of Yeast Translation Initiation Factor 3

Evans

Rasmussen

Hanic-Joyce

et al. 1995

Molecular and Cellular Biology

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The Saccharomyces cerevisiae PRT1 gene product Prt1p is a component of translation initiation factor eIF-3, and mutations in PRT1 inhibit translation initiation. We have investigated structural and functional aspects of Prt1p and its gene. Transcript analysis and deletion of the PRT1 5 end revealed that translation of PRT1 mRNA is probably initiated at the second in-frame ATG in the open reading frame. The amino acid changes encoded by six independent temperature-sensitive prt1 mutant alleles were found to be distributed throughout the central and C-terminal regions of Prt1p. The temperature sensitivity of each mutant allele was due to a single missense mutation, except for the prt1-2 allele, in which two missense mutations were required. In-frame deletion of an N-terminal region of Prt1p generated a novel, dominant-negative form of Prt1p that inhibits translation initiation even in the presence of wild-type Prt1p. Subcellular fractionation suggested that the dominant-negative Prt1p competes with wild-type Prt1p for association with a component of large Prt1p complexes and as a result inhibits the binding of wild-type Prt1p to the 40S ribosome.Mutations in the PRT1 gene of the yeast Saccharomyces cerevisiae, as exemplified by the temperature-sensitive prt1-1 mutant allele, cause a conditional impairment of translation initiation in vivo (26, 27; see also reference 18); in vitro, prt1-1 mutant cell extracts are defective for the interaction of the eIF-2 GTP Met-tRNA i ternary complex with the 40S ribosomal subunit (16), suggesting that the PRT1 polypeptide (Prt1p) may be a component of the yeast translation initiation factor 3 (eIF-3) complex (37, 38). Recently, this suggestion has been validated by the identification of Prt1p in purified yeast .Mutant forms of the PRT1 gene have been found several times by selection for temperature-sensitive growth mutants (26,27,58) and also through searches for more-specific effects. The prt1-63 allele (formerly cdc63 [22]) was obtained by selection for mutants conditionally unable to perform the G 1 cell cycle regulatory step (2), whereas the prt1-26 allele (formerly dna26 [15]) was identified in a mutant defective for DNA synthesis (13). The identification of prt1 mutant alleles by the latter criteria can be understood from the effects of prt1 mutations on the cell cycle. At appropriate restrictive temperatures, all temperature-sensitive prt1 mutations can cause regulated cell cycle blockage (23), and the DNA synthesis impairment caused by the prt1-26 mutation was shown to be an indirect consequence of a concerted arrest of mutant cells in the G 1 phase of the cell cycle (15). Thus, prt1 mutant alleles can allow sufficient protein synthesis for completion of an ongoing cell cycle (7) while blocking initiation of the next cell cycle and DNA replication.Prt1p has been characterized by analysis of the cloned PRT1 gene (24). In this report, we extend the characterization of Prt1p by showing that the open reading frame (ORF) of PRT1 does not accurately predict Prt1p and by identi...

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Section: Discussionsupporting

confidence: 82%

Section: Discussionsupporting

confidence: 81%

Mutational Analysis of the Prt1 Protein Subunit of Yeast Translation Initiation Factor 3

Evans

Rasmussen

Hanic-Joyce

et al. 1995

Molecular and Cellular Biology

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“…Analysis of the p110 cDNA Deduced Protein-Comparison of the predicted amino acid sequence of p110 subunit of mammalian eIF3 with protein sequences in the Swiss Prot Data Bank revealed that p110 has 32% identity and 56% similarity with the yeast S. cerevisiae protein Prt1p (25,26). The 82.7-kDa yeast Prt1p protein was shown to be a subunit of yeast eIF3 (22,23).…”

Section: Resultsmentioning

confidence: 99%

Biochemical Characterization of Mammalian Translation Initiation Factor 3 (eIF3)

Chaudhuri

Chakrabarti²,

Maitra

1997

Journal of Biological Chemistry

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Eukaryotic translation initiation factor 3 (eIF3), which plays an essential role in initiation of protein synthesis, was purified from rabbit reticulocyte lysates using an assay that specifically measures its ability to stimulate the binding of Met-tRNA f (as a Met-tRNA f ⅐eIF2⅐ GTP ternary complex) to 40 S ribosomal subunits. Purified eIF3 consisted of six major polypeptides of molecular masses 110, 67, 42, 40, 36, and 35 kDa but lacked the 170-kDa polypeptide reported to be a constituent of other eIF3 preparations. Characterization of purified eIF3 lacking the 170-kDa polypeptide showed that the eIF3-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex. eIF3, which was originally bound to the 40 S initiation complex, was released from the 40 S subunit during the subunit joining reaction. Additionally, chicken antibodies raised against rabbit reticulocyte eIF3 were used to immunochemically characterize eIF3 subunits and to isolate a 3.1-kilobase pair human cDNA that encodes the p110 subunit of mammalian eIF3. The derived amino acid sequence (calculated M r 95,214) shows that the p110 subunit is the mammalian homologue of Saccharomyces cerevisiae protein Prt1p, a subunit of yeast eIF3.

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“…The restriction map of the area 5' to SRA5 that is deleted from pW2 and pW3 (Fig. la) matches the restriction map of PRTJ (19). These data indicate that SRA5 is tightly linked to PRTJ on the right arm of chromosome XV, with the start codon of SRA5 approximately 0.6 kb downstream from the transcription termination site of PRT1.…”

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confidence: 51%

SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae.

Wilson¹,

Tatchell²

1988

Mol. Cell. Biol.

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sraS mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km, cyclic AMP (cAMP) phosphodiesterase activity. We have cloned SRA5 by complementation. It maps to the right arm of chromosome XV, tightly linked to PRT(, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase. Disruptions of SRAS allowed rasl ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP. sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP.The yeast Saccharomyces cerevisiae contains two genes, RAS] and RAS2, that are structurally and functionally homologous to the ras oncogenes of mammalian cells (9,10,17,24). RAS gene products bind guanine nucleotides and have intrinsic GTPase activity (for reviews, see references 1 and 13). Biochemical and genetic evidence indicates that, in S. cerevisiae, RAS gene products activate adenylate cyclase (5-7, 16, 17, 32 (4,6,11,30). SRA1 encodes the regulatory subunit of the cyclic AMP (cAMP)-dependent protein kinase, and SRA3 encodes one of the catalytic subunits (7). Dominant SRA4 mutations map to the cyri (adenylate cyclase) locus and result in high constitutive levels of RAS-independent adenylate cyclase activity (6). Mutations in SRA6 result in derepressed RAS] transcription (4), and mutations in SRA5 result in deficient low-Km cAMP phosphodiesterase activity (6).S. cerevisiae contains two known cAMP phosphodiesterases, a high-Km enzyme with broad specificity and a low-Km enzyme with narrow specificity (20,28). For this report, we cloned, sequenced, and mapped SRA5. We demonstrate by sequence homology that SRA5 encodes the low-Km cAMP phosphodiesterase of S. cerevisiae, and we describe some of the phenotypic consequences of its disruption.Cloning of ,fA5. The wild-type SRA5 gene was cloned by virtue of its ability to rescue a RAS' sraS-5 strain from dying upon nitrogen starvation in the presence of exogenous cAMP. Th; strain RW60-12C (RAS' sraS-5) was transformeO (2) ywith a yeast genomic library constructed in the ments all phenotypes of sra5-3 and sra5-5 previously described (6). Subcloning (21) and TnS mutagenesis (27) further delineated the location of the functional gene ( Fig. la and b).A URA3 gene was integrated at the pWl-insert chromosomal locus by transforming RW58 (ras2::LEU2 ura3) with pW4, an integrating plasmid containing URA3 and a functional portion of the pWl insert (Tables 1 and 2; Fig. 1). A stable Ura+ (Sra5S) transformant (RW158) was crossed with RW77 (ras2::LEU2 sraS-3 ura3); the segregation pattern of URA3 and sraS-3 was 27 parental ditype:0 nonparental ditype:0 tetratype. This linkage (less than 2 centimorgans) between the pWl insert locus and the sra5-3 locus indicates that the pWl insert contains SRA5 and not an extragenic suppressor thereof.Mapping of SRAS to chromosome XV. Preliminary assignment of SRA5 to chromosome VII or XV wa...

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Isolation and characterization of PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae.

Cited by 27 publications

References 48 publications

Mutational Analysis of the Prt1 Protein Subunit of Yeast Translation Initiation Factor 3

Mutational Analysis of the Prt1 Protein Subunit of Yeast Translation Initiation Factor 3

Biochemical Characterization of Mammalian Translation Initiation Factor 3 (eIF3)

SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae.

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