Specific effects of ribosome-tethered molecular chaperones on programmed -1 ribosomal frameshifting

Abstract

The ribosome-associated molecular chaperone complexes RAC (Ssz1p/Zuo1p) and Ssb1p/Ssb2p expose a link between protein folding and translation. Disruption of the conserved nascent peptide-associated complex results in cell growth and translation fidelity defects. To better understand the consequences of deletion of either RAC or Ssb1p/2p, experiments relating to cell growth and programmed ribosomal frameshifting (PRF) were assayed. Genetic analyses revealed that deletion of Ssb1p/Ssb2p or of Ssz1p/Zuo1p resulted in specific inhibition of -1 PRF and defects in Killer virus maintenance, while no effects were observed on +1 PRF. These factors may provide a new set of targets to exploit against viruses that use -1 PRF. Quantitative measurements of growth profiles of isogenic wild-type and mutant cells showed that translational inhibitors exacerbate underlying growth defects in these mutants. Previous studies have identified -1 PRF signals in yeast chromosomal genes and have demonstrated an inverse relationship between -1 PRF efficiency and mRNA stability. Analysis of published DNA microarray experiments reveals conditions under which Ssb1, Ssb2, Ssz1, and Zuo1 transcript levels are regulated independently of those of genes encoding ribosomal proteins. Thus, the findings presented here suggest that these trans-acting factors could be used by cells to posttranscriptionally regulate gene expression through -1 PRF.

FIG. 1.

Dual-luciferase reporters for in vivo determination of PRF efficiencies in yeast. In all reporters, transcription is initiated from the yeast ADH1 promoter and terminated at sequence derived from the CYC1 3′ untranslated region. The Renilla and firefly luciferase genes are cloned in tandem and are separated by a multiple cloning site (MCS). In pYDL-control, the Renilla and firefly luciferase genes are in frame with one another and produce a fusion of the two proteins. In pYDL-Ty1 and pYDL-LA, the Ty1 +1 and L-A −1 PRF signals are inserted in the MCS and the firefly luciferase gene is in the +1 or −1 reading frame, respectively, relative to the Renilla luciferase gene. pYDL-LA0 is an alternative 0-frame control reporter in which a cytosine residue was inserted upstream of the L-A −1 PRF signal to inactivate frameshifting while also bringing the firefly and Renilla luciferase genes into frame with one another. PRF efficiencies were calculated by dividing the ratio of firefly to Renilla luciferase generated from cells harboring the −1 or +1 test vector by the ratio of firefly to Renilla luciferase generated from cells harboring either of the 0-frame control plasmids.

FIG. 2.

Deletion of the Rac-Ssb1/2 chaperone complex results in killer virus maintenance defects. The endogenous yeast L-A and M₁ killer viruses were introduced into isogenic [rho⁰] wild-type and mutant strains by cytoduction at the indicated temperatures. A. Killer virus maintenance phenotypes. Cytoductants were replica plated onto a lawn of diploid, Killer⁻ 5X47 indicator cells and incubated at 20°C for 3 days. Killer activity is scored by the zone of growth inhibition around colonies. B. Analysis to determine the presence of viral double-stranded RNAs. Total nucleic acids were extracted from isogenic cells, subjected to RNase A treatment in 500 mM NaCl, and separated through a 1% Tris-acetate-EDTA-agarose gel. Genomic DNA (gDNA) and the L-A and M₁ double-stranded RNAs are indicated.

FIG. 3.

Growth curves of isogenic cells in the presence and absence of translational inhibitors. Cultures were inoculated from saturated overnight cultures into prewarmed 500-μl wells with or without the indicated drugs to an OD₅₉₅ of 0.05. OD₅₉₅ measurements were taken every 17 min for 28 h and recorded automatically using a Synergy HT microplate reader. Doubling times in the presence of anisomycin (A) or sparsomycin (B) during log-phase growth were calculated for increasing drug concentrations. Inset graphs indicate the fold changes in doubling times of the mutants in the presence of increasing drug concentrations normalized to that of wild-type cells under the same conditions.

FIG. 4.

DMS protection experiments: representative autoradiograms probing the vicinity of the peptidyltransferase center using primer 25-6. In vivo probing of intact cells and in vitro probing using puromycin-treated purified ribosomes are indicated. Sequencing reactions indicated by CTAG are located to the left of each set. W, wild type; R−, ssz1Δ zuo1Δ mutant; and S−, ssb1Δ ssb2Δ mutant. Untreated samples are denoted by dashes, and DMS treatment in the in vivo assays is denoted by a wedge (80 mM or 160 mM DMS) and in the in vitro assays by + symbols. Representative bases of yeast 25S rRNA are numbered.

FIG. 5.

Modeling the effects of the Rac⁻ and ssb1Δ ssb2Δ mutants on ribosome structure and function. The upper illustrations depict cartoons of translating ribosomes in wild-type and mutant cells. The lower illustrations focus on events in the peptidyltransferase center at the molecular scale. Color-coded features include the nascent peptide (green), the 3′ ends of the aa- and peptidyl-tRNAs, the A- and P-loops (purple and blue, respectively), and the peptidyltransferase center (PTC) (red). A. Wild-type ribosomes. Incoming aa-tRNAs are accommodated in the A-site of the peptidyltransferase center along the path indicated by the dotted purple line. Peptidyl transfer between peptidyl- and aa-tRNAs then occurs, and nascent peptide continues to be extruded through the exit tunnel. B. Rac⁻ and ssb1Δ ssb2Δ mutants. Impaired chaperoning causes nascent peptides to back up into the exit tunnel, mispositioning the peptidyl-tRNA 3′ end and thus inhibiting accommodation of the aa-tRNA in the A-site (blocked dotted red line).