We isolated a cloned DNA fragment containing PRTI, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae, by complementation of the temperature-sensitive prtl-l mutation. The entire PRT1 gene is contained within a 3.2-kilobase-pair segment of the cloned DNA in YEpl3 H1.2. Southern blot analysis demonstrated that PRTI is a single copy gene which is transcribed into a 2.3-kilobase RNA. We determined the direction of transcription and mapped the 5' and 3' ends of the gene.The initiation of protein biosynthesis is a complex process, involving numerous factors that must interact in a precisely ordered fashion. In the yeast Saccharomyces cerevisiae the isolation and characterization of temperaturesensitive mutants (23,24) has facilitated the study of the initiation of protein biosynthesis. ts187, containing the temperature-sensitive prtl-J allele, is one such mutant. The incorporation of radiolabeled amino acids into protein is greatly reduced in ts187 at the restrictive temperature (23), and an examination of the translational apparatus shows that polysomes shift to monosomes within minutes at the restrictive temperature. This result indicates that ts187 suffers a defect in initiation (36). In addition, in vitro translation systems prepared from ts187 cells grown at the restrictive temperature are inhibited in the formation of the 43S preinitiation complex (eIF2-Met tRNAf-GTP plus 40S ribosomal subunits) relative to in vitro systems prepared from wildtype cells or ts187 cells grown at the permissive temperature (17). Other aspects of protein biosynthesis such as methionine activation and methionylation of tRNAMet, formation of the ternary complex, mRNA binding, binding of the 60S subunit to form the 80S initiation complex, elongation, and termination are unaffected in in vitro translation systems prepared from heated ts187 cells (17). This evidence suggests that PRTJ codes for an initiation factor that is involved in the formation of the 43S preinitiation complex, probably a subunit of eIF-3 (33).Initiation factor eIF-3 has been purified from a number of systems including reticulocytes, wheat germ, liver and ascites (5, 38-40, 44, 45). It has a molecular weight between 500,000 and 700,000 and is composed of 7 to 10 polypeptides (33). Use of purified eIF-3 preparations has resulted in the assignment of several roles for this factor: a ribosome dissociation activity (4,38,40,47), stimulation of the formation of the 43S preinitiation complex (6,15,21,25,47), and mRNA binding (4,27,46,47).As a next step in the understanding of the process of the initiation of protein biosynthesis, we report the isolation and preliminary characterization of PRTI, a gene encoding one of the polypeptides of a eucaryotic initiation factor. MATERIALS AND METHODSStrains and growth conditions. S. cerevisiae MNB2-6D (a prtl ade leu2 tyri) was derived from a cross between ts187 (a * Corresponding author.gall adel ade2 ural his7 lys2 tyri) and LL20 (a leu2 his3). A364a (22) (a adel ade2 ural tyri his7 lys2 gall) an...
We isolated a cloned DNA fragment containing PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae, by complementation of the temperature-sensitive prtl-1 mutation. The entire PRT1 gene is contained within a 3.2-kilobase-pair segment of the cloned DNA in YEp13 H1.2. Southern blot analysis demonstrated that PRT1 is a single copy gene which is transcribed into a 2.3-kilobase RNA. We determined the direction of transcription and mapped the 5' and 3' ends of the gene.
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