Translational suppressors and antisuppressors alter the efficiency of the Ty1 programmed translational frameshift.

Certain viruses, transposons, and cellular genes have evolved specific sequences that induce high levels of specific translational errors. Such "programmed misreading" can result in levels of frameshifting or nonsense codon readthrough that are up to 1,000-fold higher than normal. Here we determine how a number of mutations in yeast affect the programmed misreading used by the yeast Ty retrotransposons. These mutations have previously been shown to affect the general accuracy of translational termination. We find that among four nonsense suppressor ribosomal mutations tested, one (a ribosomal protein mutation) enhanced the efficiency of the Tyl frameshifting, another (an rRNA mutation) reduced frameshifting, and two others (another ribosomal protein mutation and another rRNA mutation) had no effect. Three antisuppressor rRNA mutations all reduced Tyl frameshifting; however the antisuppressor mutation in the ribosomal protein did not show any effect. Among nonribosomal mutations, the allosuppressor protein phosphatase mutation enhanced Tyl frameshifting, whereas the partially inactive prion form of the release factor eRF3 caused a slight decrease, if any effect. A mutant form of the other release factor, eRF1, also had no effect on frameshifting. Our data suggest that Ty frameshifting is under the control of the cellular translational machinery. Surprisingly we find that translational suppressors can affect Ty frameshifting in either direction, whereas antisuppressors have either no effect or cause a decrease.

Host genes that affect the target-site distribution of the yeast retrotransposon Ty1.

We report here a simple genetic system for investigating factors affecting Ty1 target-site preference within an RNAP II transcribed gene. The target in this system is a functional fusion of the regulatable MET3 promoter with the URA3 gene. We found that the simultaneous inactivation of Hir3 (a histone transcription regulator) and Cac3 (a subunit of the chromatin assembly factor I), which was previously shown by us to increase the Ty1 transposition rate, eliminated the normally observed bias for Ty1 elements to insert into the 5' vs. 3' regions of the MET3-URA3 and CAN1 genes. The double cac3 hir3 mutation also caused the production of a short transcript from the MET3-URA3 fusion under both repressed and derepressed conditions. In a hir3Delta single-mutant strain, the Ty1 target-site distribution into MET3-URA3 was altered only when transposition occurred while the MET3-URA3 fusion was actively transcribed. In contrast, transcription of the MET3-URA3 fusion did not alter the Ty1 target-site distribution in wild-type or other mutant strains. Deletion of RAD6 was shown to alter the Ty1 target-site preference in the MET3-URA3 fusion and the LYS2 gene. These data, together with previous studies of Ty1 integration positions at CAN1 and SUP4, indicate that the rad6 effect on Ty1 target-site selection is not gene specific.

Yeast Ty1 retrotransposition is stimulated by a synergistic interaction between mutations in chromatin assembly factor I and histone regulatory proteins.

A screen for host mutations which increase the rate of transposition of Ty1 and Ty2 into a chromosomal target was used to identify factors influencing retroelement transposition. The fortuitous presence of a mutation in the CAC3 gene in the strain in which this screen was undertaken enabled us to discover that double mutaions of cac3 and hir3, but neither of the two single mutations, caused a dramatic increase in the rate of retrotransposition. We further showed that this effect was not due to an increase in the overall level of Ty1 mRNA. Two subtle cac3 phenotypes, slight methyl methanesulfonate (MMS) sensitivity and reduction of telomeric silencing, were significantly enhanced in the cac3 hir3 double mutant. In addition, the growth rate of the double mutant was reduced. HIR3 belongs to a class of HIR genes that regulate the transcription of histones, while Cac3p, together with Cac1p and Cac2p, forms chromatin assembly factor I. Other combinations of mutations in cac and hir genes (cac3 hir1, cac3 hir2, and cac2 hir3) also increase Ty transposition and MMS sensitivity and reduce the growth rate. A model explaining the synergistic interaction between cac and hir mutations in terms of alterations in chromatin structure is proposed.

Farnesyl diphosphate synthetase. Molecular cloning, sequence, and expression of an essential gene from Saccharomyces cerevisiae.

Farnesyl diphosphate (FPP) synthetase is a key enzyme in isoprenoid biosynthesis which supplies C15 precursors for several classes of essential metabolites including sterols, dolichols, and ubiquinones. The structural gene for FPP synthetase was isolated on a 4.5-kilobase EcoRI genomic restriction fragment from the yeast Saccharomyces cerevisiae. The clone encodes a 40,483-dalton polypeptide of 342 amino acids with a high degree of similarity to the protein encoded by a putative rat liver clone of FPP synthetase (Clarke, C. F., Tanaka, R. D., Svenson, K., Wamsley, M., Fogelman, A. M., and Edwards, P. A. (1987) Mol. Cell Biol. 7, 3138-3146) and to an active site protein fragment from avian liver FPP synthetase (Brems, D. N., Bruenger, E., and Rilling, H. C. (1981) Biochemistry 20, 3711-3718). When cloned into the yeast shuttle vector YRp17, the 4.5-kilobase EcoRI fragment directed a 2-3-fold over-expression of FPP synthetase activity in transformed yeast cells. The levels of expression were independent of culture growth phase and orientation of the insert, indicative of a functional promoter in the clone. Disruption of the FPP synthetase gene from a diploid yeast strain, followed by dissection and analysis of tetrads, demonstrates that the gene is an essential, single copy number gene in yeast. The gene for FPP synthetase resides on chromosome XI as judged from Southern blots of separated yeast chromosomes.