Genetic reversion of HIS4 initiator codon mutations in yeast has identified three unlinked genes, suil, sui2, and SUI3 (suppressors of initiator codon mutations), which when mutated confer the ability to initiate translation at HIS4 despite the absence of an AUG start codon. We have previously demonstrated that the SUI3 gene encodes the j3 subunit of the eukaryotic initiation factor 2 (eIF-2) and that mutations at a Zn(II) finger motif of SUI3 alter the start site selection process in yeast. In this report, molecular and biochemical characterizations show that the sui2 suppressor gene encodes the a subunit of eIF-2. The amino acid sequence of sui2 is 58% homologous to that encoded by the cDNA of the human eIF-2a. Mutations in the sui2 suppressor alleles occur in the amino-terminal portion of the protein and change amino acids that are identical at the same relative position in the yeast and human proteins. Protein sequence analysis shows that a sui2 mutant yeast strain allows initiation at a UUG codon in the absence of an AUG codon at HIS4. These data further suggest that eIF-2 is an important component of the preinitiation complex that mediates ribosomal recognition of a start codon during the scanning process.The eukaryotic translation initiation factor 2 (eIF-2) is composed of three nonidentical subunits: a, P3, and 'y(1). One role of eIF-2 established by biochemical studies (2) is that it functions during the early steps of translation initiation by forming a ternary complex with GTP and initiator tRNA. This complex then binds the small 40S ribosomal subunit, which in turn binds the 5' end of eukaryotic mRNA. According to the scanning model (3, 4), this preinitiation complex then scans the leader region until the first AUG codon is reached whereupon translation begins.Recent genetic studies in our laboratory have provided biological evidence that eIF-2 may also function in ribosomal selection of the initiator codon during t1e scanning process (5). By reverting his4-, his4-lacZ Saccharomyces cerevisiae initiator codon mutants (His-, white), three unlinked genes, suil, sui2, and SUI3, were identified that when mutated act in trans to restore both his4 and his4-lacZ expression [His', blue revertant colonies on synthetic dextrose minus histidine and 5-bromo-4-chloro-3-indolyl-,8-D-galactoside (X-Gal) In this report, we present the characterization of the sui2 suppressor gene. Our study shows that sui2 encodes the a subunit of eIF-2 and mutations in a also confer an alteration in start site selection by allowing initiation at a UUG codon in the mutant his4 message. As observed for the yeast and human P3 subunit of eIF-2, the yeast a subunit shows considerable identity to the amino acid sequence derived from a cDNA encoding the human a subunit of eIF-2 (7) and mutations in sui2 suppressor genes that restore his4 expression change amino acids that are identical at the same position in the human eIF-2a sequence. In light of similarities between the yeast and mammalian initiation processes (3,4,8,9), these ...
We have mutated various features of the 5' noncoding region of the HIS4 mRNA in light of established Saccharomyces cerevisiae and mammalian consensus translational initiator regions. Our analysis indicates that insertion mutations that introduce G+C-rich sequences in the leader, particularly those that result in stable stem-loop structures in the 5' noncoding region of the HIS4 message, severely affect translation initiation. Mutations that alter the length of the HIS4 leader from 115 to 39 nucleotides had no effect on expression, and sequence context changes both 5' and 3' to the HIS4 AUG start codon resulted in no more than a twofold decrease of expression. Changing the normal context at HIS4 5'-AAUAAUGG-3' to the optimal sequence context proposed for mammalian initiator regions 5'-CACCAUGG-3' did not result in stimulation of HIS4 expression. These studies, in conjunction with comparative and genetic studies in S. cerevisiae, support a general mechanism of initiation of protein synthesis as proposed by the ribosomal scanning model.Comparative analysis of 131 Saccharomyces cerevisiae genes (7) indicates that basic sequence and structural features associated with yeast translational initiator regions parallel those features associated with translational initiator regions from higher eucaryotic organisms that are proposed to initiate translation by a ribosomal scanning mechanism (16,17). Namely, the first AUG codon nearest the 5' end of the message serves as the start site of translation at 95% of yeast genes, 70% have leader regions with distances in the range of 20 to 80 nucleotides having an average length of 52 nucleotides, and sequence context both 5' and 3' to the AUG start codon is biased in base composition. However, yeast initiator regions do differ from mammalian initiator regions. Specifically, the base composition of yeast initiator regions is biased toward A nucleotides (46% A, 21% C, 21% U, and 12% G; from position -25 to -1 relative to the A of AUG) and sequences immediately 3' to the AUG reflect a nonrandom distribution of amino acids at the +2 amino acid position, as well as the preferred codon usage observed at yeast genes (7). These properties of yeast initiator regions are reflected in its consensus, 5'-YAUAAUGUCU-3', which contrasts the mammalian consensus, 5'-CCACCAUGG-3' (20), with the exception of a similar high bias for A nucleotides at the -3 position (75% and 79% for the yeast and mammalian consensus, respectively).The importance of the higher eucaryotic consensus to the mechanism of ribosomal recognition of an AUG start codon has been suggested by mutational studies (15,18,21,23,25). Alteration of this optimal sequence context at the rat preproinsulin gene can result in 20-fold differences in translational efficiency at the AUG start codon. As a result of inefficient initiation, the ribosome can bypass this region to initiate at a subsequent AUG (23). The significance of these effects would appear to relate to a functional role for sequence context in establishing different rates of in...
The SSL1 locus was identified as a trans-acting suppressor that restores HIS4 expression despite a stem-loop structure in the 5'-UTR. SSL1 encodes an essential protein of 52 kD with features characteristic of a protein with multiple zinc fingers. The mechanism of SSL1 suppression is not related to altering h/s4 transcription or removing the stem-loop sequence from the 5'-UTR; rather, 3-to 5-fold increases in His4 translational expression are observed indicating a post-transcriptional mechanism for SSL1 suppression. SSL1 suppressor mutants that are conditional for growth have altered polysome profiles at the restrictive temperature, and their cell-kee extracts are thermolabile in their ability to translate exogenously added mRNA. In addition, the mechanism of suppression appears to be specific for stem-loop structures placed near the 5' end of the message as opposed to a stem-loop located at a downstream position in the 5'-UTR. These observations suggest a role for this protein in promoting translation initiation presumably at the level of ribosomal binding to mRNA. Surprisingly, SSL1 suppressor mutations that are shown to confer an in vivo and in vitro defect in translation initiation also rendered yeast hypersensitive to UV irradiation. This latter phenotype was observed previously with a mutation in the SSL2 suppressor gene, which encodes the yeast homolog of the human gene ERCC-3, for which a defective form causes xeroderma pigmentosum. In light of the related effects of mutations in the SSL1 and SSL2 genes, the encoded proteins may functionally interact both to promote DNA repair and perform an essential function during translation initiation.
We have mutated various features of the 5' noncoding region of the HIS4 mRNA in light of established Saccharomyces cerevisiae and mammalian consensus translational initiator regions. Our analysis indicates that insertion mutations that introduce G + C-rich sequences in the leader, particularly those that result in stable stem-loop structures in the 5' noncoding region of the HIS4 message, severely affect translation initiation. Mutations that alter the length of the HIS4 leader from 115 to 39 nucleotides had no effect on expression, and sequence context changes both 5' and 3' to the HIS4 AUG start codon resulted in no more than a twofold decrease of expression. Changing the normal context at HIS4 5'-AAUAAUGG-3' to the optimal sequence context proposed for mammalian initiator regions 5'-CACCAUGG-3' did not result in stimulation of HIS4 expression. These studies, in conjunction with comparative and genetic studies in S. cerevisiae, support a general mechanism of initiation of protein synthesis as proposed by the ribosomal scanning model.
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