Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication.

Translation and mRNA decay constitute key players in the post-transcriptional control of gene expression. We examine the mechanisms by which the 5-untranslated region (UTR) of nonaberrant mRNAs acts to modulate both these processes in Saccharomyces cerevisiae. Two classes of functional relationship between ribosome-5-UTR interactions and mRNA decay are identifiable. In the first of these, elements in the main open reading frame (ORF) dictate how the decay process reacts to inhibitory structures in the 5-UTR. The same types of stability modulation can be elicited by transregulation of translation via inducible binding of the iron-regulatory protein to an iron-responsive element located 9 nucleotides from the 5 cap. A eukaryotic translational repressor can therefore modulate mRNA decay via the 5-UTR. In contrast, translational regulation mediated via changes in the activity of the capbinding eukaryotic translation initiation factor eIF-4E bypasses translation-dependent pathways of mRNA degradation. Thus modulation of mRNA stability via the 5-UTR depends on disruption of the scanning process, rather than changes in translational initiation efficiency per se. In the second class of pathway, an upstream ORF (uORF) functions as a powerful destabilizing element, inducing termination-dependent degradation that is apparently independent of any main ORF determinants but influenced by the efficiencies of ribosomal recognition of the uORF start and stop codons. This latter mechanism provides a regulatable means to modulate the stability of nonaberrant mRNAs via a UPFdependent pathway.The steady-state abundance of mRNA in the eukaryotic cell is determined by the relative rates of its transcription and degradation. mRNA decay rates are not uniform, but rather vary over at least a 100-fold range (1-6), thus influencing significantly the rates of expression of individual genes. Moreover, modulation of mRNA decay constitutes an important means of regulating gene expression (3, 7-10). The mechanism of mRNA degradation has accordingly been the subject of intensive research activity and has been found to be a complex process, following a number of pathways (2,(11)(12)(13).Any model of mRNA decay has to take into account that the same molecules that are turned over by the action of degradative enzymes also serve as templates for translation (see e.g. 14). Indeed, a number of investigations have indicated that translation influences the decay process. First, the rapidly degraded mRNAs of yeast MAT␣1 (15) and mammalian early response genes (16) contain translation-dependent destabilizing elements within their respective coding regions. Second, the presence of nonsense codons in the reading frames of at least some yeast mRNAs accelerates their degradation (17-19). In mammalian systems, nonsense codons destabilize nuclear, rather than cytoplasmic, mRNA (20,21). In yeast, the pathway of accelerated decay triggered by nonsense codons, referred to as nonsense-mediated decay, is dependent on trans-acting factors encoded by the UPF genes (22...

Cap proximity is a requirement to enable secondary structures and RNA-binding proteins to repress translational initiation via the 5-untranslated region (5-UTR) of mammalian mRNAs. We show that in Saccharomyces cerevisiae, unlike mammalian cells, the in vitro translational repressive effect of the mammalian iron regulatory protein 1 (IRP1) is independent of the site of its target in the 5-UTR, the iron-responsive element (IRE). In vitro studies demonstrate that the binding affinity of

4E-binding proteins (4E-BPs) are believed to have important regulatory functions in controlling the rate of translation initiation in mammalian cells. They do so by binding to the mRNA cap-binding protein, eIF4E, thereby inhibiting formation of the cap-binding complex, a process essential for cap-dependent translation initiation. We have reproduced the translation-repressive function of human 4E-BP1 in yeast and find its activity to be dependent on substitution of human eIF4E for its yeast counterpart. Translation initiation and growth are inhibited when human 4E-BP1 is expressed in a strain with the human eIF4E substitution, but not in an unmodified strain. We have compared the relative affinities of human 4E-BP1 for human and yeast eIF4E, both in vitro using an m 7 GTP cap-binding assay and in vivo using a yeast two-hybrid assay, and find that the affinity of human 4E-BP1 for human eIF4E is markedly greater than for yeast eIF4E. Thus yeast eIF4E lacks structural features required for binding to human 4E-BP1. These results therefore demonstrate that the features of eIF4E required for binding to 4E-BP1 are distinct from those required for cap-complex assembly.The initiation of translation requires recognition by small ribosomal subunits of the 5Ј end of mRNA. In eukaryotes, this is mediated by the assembly of a complex of proteins, known as eukaryotic initiation factor (eIF) 1 4F, which recognizes the mRNA 5Ј cap structure. Modulation of eIF4F assembly is an important mechanism by which the rate of translation initiation is regulated according to the general demand for protein synthesis. The two most conserved components of eIF4F, found in all eukaryotes, are eIF4E, which binds to the cap structure directly, and eIF4G, a multifunctional polypeptide that binds to eIF4E and to other essential translation initiation factors. The binding of eIF4E to eIF4G is crucial for cap-dependent translation initiation and is believed to be subject to an important regulatory mechanism in mammalian cells involving 4E-binding proteins (4E-BPs), the subject of this study (reviewed in Refs. 1-6). 4E-BPs constitute a family of small polypeptides, of which one, 4E-BP1, has been the most extensively studied. 4E-BP1 shares with eIF4G a binding site for eIF4E and competes with eIF4G for binding to eIF4E, thus inhibiting the assembly of eIF4F (7,8). The competition appears to be inversely phosphorylation-dependent. 4E-BP1 is known to be phosphorylated in response to stimuli that promote increases in the rate of translation (9), but only binds to eIF4E in its hypophosphorylated state (10, 11). Phosphorylation has been shown in vitro to cause dissociation of 4E-BP1 from eIF4E (10), thus relieving the competition for eIF4G binding (7). How important a regulatory mechanism is this in vivo? And, if regulation of translation initiation through 4E-BPs is important in mammals, to what extent has this regulatory mechanism been conserved throughout evolution? In order to start to answer these questions and to establish a system with which to dissec...

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