A C-terminal deletion mutant of pokeweed antiviral protein inhibits programmed +1 ribosomal frameshifting and Ty1 retrotransposition without depurinating the sarcin/ricin loop of rRNA.

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes. Here, a series of PAP mutants were used to examine the relationship between depurination of the S/R loop and inhibition of +1 programmed ribosomal frameshifting (PRF) and to define PAP sequences critical for inhibition of +1 PRF and Ty1 retrotransposition in the yeast Saccharomyces cerevisiae. Using three different classes of mutants we present evidence that strong binding of a C-terminal PAP mutant (PAPc) to ribosomes is sufficient to inhibit +1 PRF and Ty1 retrotransposition in the absence of S/R loop depurination. PAPc did not affect the totivirus ScV-L-A and HIV-1-directed -1 PRF efficiencies or the ability of cells to maintain the M(1)-dependent killer phenotype, demonstrating the specificity of the effect of PAPc on +1 PRF.

Identification of putative programmed -1 ribosomal frameshift signals in large DNA databases.

The cis-acting elements that promote efficient ribosomal frameshifting in the -1 (5') direction have been well characterized in several viral systems. Results from many studies have convincingly demonstrated that the basic molecular mechanisms governing programmed -1 ribosomal frameshifting are almost identical from yeast to humans. We are interested in testing the hypothesis that programmed -1 ribosomal frameshifting can be used to control cellular gene expression. Toward this end, a computer program was designed to search large DNA databases for consensus -1 ribosomal frameshift signals. The results demonstrated that consensus programmed -1 ribosomal frameshift signals can be identified in a substantial number of chromosomally encoded mRNAs and that they occur with frequencies from two- to sixfold greater than random in all of the databases searched. A preliminary survey of the databases resulting from the computer searches found that consensus frameshift signals are present in at least 21 homologous genes from different species, 2 of which are nearly identical, suggesting evolutionary conservation of function. We show that four previously described missense alleles of genes that are linked to human diseases would disrupt putative programmed -1 ribosomal frameshift signals, suggesting that the frameshift signal may be involved in the normal expression of these genes. We also demonstrate that signals found in the yeast RAS1 and the human CCR5 genes were able to promote significant levels of programmed -1 ribosomal frameshifting. The significance of these frameshifting signals in controlling gene expression is not known, however.

Ribosomal protein L3 mutants alter translational fidelity and promote rapid loss of the yeast killer virus.

Programmed -1 ribosomal frameshifting is utilized by a number of RNA viruses as a means of ensuring the correct ratio of viral structural to enzymatic proteins available for viral particle assembly. Altering frameshifting efficiencies upsets this ratio, interfering with virus propagation. We have previously demonstrated that compounds that alter the kinetics of the peptidyl-transfer reaction affect programmed -1 ribosomal frameshift efficiencies and interfere with viral propagation in yeast. Here, the use of a genetic approach lends further support to the hypothesis that alterations affecting the ribosome's peptidyltransferase activity lead to changes in frameshifting efficiency and virus loss. Mutations in the RPL3 gene, which encodes a ribosomal protein located at the peptidyltransferase center, promote approximately three- to fourfold increases in programmed -1 ribosomal frameshift efficiencies and loss of the M1 killer virus of yeast. The mak8-1 allele of RPL3 contains two adjacent missense mutations which are predicted to structurally alter the Mak8-1p. Furthermore, a second allele that encodes the N-terminal 100 amino acids of L3 (called L3Delta) exerts a trans-dominant effect on programmed -1 ribosomal frameshifting and killer virus maintenance. Taken together, these results support the hypothesis that alterations in the peptidyltransferase center affect programmed -1 ribosomal frameshifting.