Specific Effects of Ribosome-Tethered Molecular Chaperones on Programmed -1 Ribosomal Frameshifting

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 complexresults in cell growth and translation fidelity defects. Tobetter understand the consequences of deletion of either RACor Ssb1p/2p, experiments relating to cell growth and programmedribosomal frameshifting (PRF) were assayed. Genetic analysesrevealed that deletion of Ssb1p/Ssb2p or of Ssz1p/Zuo1p resultedin specific inhibition of –1 PRF and defects in Killervirus maintenance, while no effects were observed on +1 PRF.These factors may provide a new set of targets to exploit againstviruses that use –1 PRF. Quantitative measurements ofgrowth profiles of isogenic wild-type and mutant cells showedthat translational inhibitors exacerbate underlying growth defectsin these mutants. Previous studies have identified –1PRF signals in yeast chromosomal genes and have demonstratedan inverse relationship between –1 PRF efficiency andmRNA stability. Analysis of published DNA microarray experimentsreveals conditions under which Ssb1, Ssb2, Ssz1, and Zuo1 transcriptlevels are regulated independently of those of genes encodingribosomal proteins. Thus, the findings presented here suggestthat these trans-acting factors could be used by cells to posttranscriptionallyregulate gene expression through –1 PRF.

INTRODUCTION

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Introduction

Programmed ribosomal frameshifting (PRF) is a posttranscriptionalregulatory mechanism in which elongating ribosomes are directedto shift the reading frame in response to specific cis-actingsignals in mRNAs. Many viruses, including human immunodeficiencyvirus type 1, use PRF to optimize the stoichiometric ratiosbetween their structural and enzymatic proteins (5, 11). Changesin PRF efficiency alter those ratios, inhibiting virion morphogenesis(9). In particular, retroviruses appear to be very susceptibleto such changes (4, 21, 33, 37). Thus, elucidation of the molecularmechanisms underlying PRF can aid in the rational design ofantiviral therapeutics (reviewed in reference 8). Though firstdiscovered in viruses, it is also becoming apparent that programmed–1 ribosomal frameshifting (–1 PRF) is also usedto regulate expression of cellular genes (reviewed in reference34). Given the widespread use of PRF, it is important to understandthe molecular mechanisms underlying PRF and to identify cellularfactors that might be used to regulate these processes.

Ribosomes can be directed to shift by one base in either the5' or 3' direction depending on the cis-acting signal. Programmed–1 ribosomal frameshifting is the result of a net shiftof the translational reading frame by 1 base in the 5' direction.The shift is typically directed by a tripartite cis-acting mRNAelement composed of a (from 5' to 3') heptameric slippery site,a spacer, and a thermodynamically stable structure, typicallyan mRNA pseudoknot (reviewed in reference 5). It is generallyaccepted that –1 PRF requires a change in the forwardkinetics of elongation, e.g., that the stable secondary structureforces elongating ribosomes to pause with their A- and P-sitetRNAs paired with bases 2 through 7 of the slippery site. Thenature of the slippery sites tends to be such that slippageof tRNAs by 1 base in the 5' direction results in codon-anticodonpairing in the nonwobble positions.

Yeast cells commonly harbor a symbiotic double-stranded RNAvirus called Killer (reviewed in reference 54). Killer is composedof the L-A helper virus, which uses –1 PRF to producethe correct ratio of structural (Gag) to enzymatic (Gag-Pol)proteins and a satellite called M₁, which encodes a secretedtoxin and relies on L-A as a source of viral particles and replicasefunction. The Killer⁺ phenotype of cells harboring L-A and M₁is due to the action of the secreted toxin, which kills nearbyuninfected cells (a proteolytic intermediate of the protoxinconfers immunity to infected cells). While changes in –1PRF tend to have profound effects on the propagation of M₁,L-A is much more resistant to these effects. Though the reasonsfor this remain unclear, one explanation could be that the L-Aplus strand, which is the substrate for packaging into nascentviral particles, is also the mRNA from which the viral proteinsare translated. The potentially high local concentrations ofviral proteins may allow the L-A mRNA plus strand to packageitself in cis, perhaps buffering the effects of changes in theratios of Gag to Gag-Pol. Nonetheless, the extreme sensitivityof M₁ (which must be packaged in trans) to changes in –1PRF efficiency makes the yeast Killer virus an excellent modelsystem for studies of –1 PRF.

Programmed +1 ribosomal frameshifting (+1 PRF) is the resultof a net shift of the translational reading frame by 1 basein the 3' direction. In Saccharomyces cerevisiae, the Ty1 retrotransposableelement heptameric sequence CUU AGG C directs a +1 PRF eventthat results in synthesis of a TYA-TYB fusion protein (reviewedin reference 11). This mechanism is driven by a ribosomal pauseat the "hungry" AGG-Arg codon which corresponds to a very lowabundance Arg-tRNA^CCU. The low abundance of the cognate A-sitetRNA is thought to cause a translational pause during whichthe P-site Leu-tRNA^UAG, which typically decodes the CUU codonin the 0-frame P-site, slips into the UUA codon in the overlapping+1 frame. This creates a new A-site codon, GGC, a commonly usedGly codon that is decoded by an abundant cognate tRNA. The establishmentof this codon-anticodon pair establishes the +1 frameshift.Changes in Ty1 +1 efficiency also have profound negative effectson Ty1 propagation.

A recurring motif in the history of modern molecular biologyis that basic molecular mechanisms are first identified in viralsystems. It is becoming apparent that PRF is widely used toregulate the expression of cellular as well as viral genes.Programmed +1 frameshifting is widely used to regulate expressionof ornithine decarboxylase antizyme (reviewed in reference 23),and in yeast, two other genes are known to use +1 PRF (2, 26).In mice, –1 PRF is required for translation of the 3'portions of the edr mRNA (27, 46), and in humans the paraneoplasticMa3 gene also uses a –1 frameshift (55).

Computational approaches have also been used to identify –1PRF signals in genome databases. A recent search of the yeastgenome for fusion genes produced by a –1 PRF event employinga two-step approach identified 189 candidate genes (3). Of 58mRNAs tested, almost half expressed full-length mRNAs encompassingboth open reading frames, and sequences derived from 11 of thesepromoted highly efficient –1 PRF. An earlier study designedto identify sequence and structure motifs resembling viral –1PRF signals identified significant numbers of putative –1PRF signals in many genomes, found evolutionarily conserved–1 PRF signals in homologous genes, identified known humandisease alleles that colocalized with putative –1 PRFsignals, and demonstrated efficient –1 PRF promoted bysequences in two yeast genes in vivo (16).

Notably, nearly all of the –1 PRF signals were predictedto direct elongating ribosomes into premature termination events,generating the hypothesis that –1 PRF could be used totarget mRNAs for rapid degradation via the nonsense-mediatedmRNA decay pathway. Proof-of-principle experiments using a viral–1 PRF signal inserted in the "genomic organization" intoa cellular reporter mRNA confirmed this notion (41). Additionalexperiments demonstrating an inverse relationship between –1PRF efficiency and mRNA half-lives suggested that regulationof –1 PRF could be employed to posttranscriptionally regulategene expression. More recent investigations along these lineshave identified a significant number of functional –1PRF signals that can act as mRNA destabilizing elements (J.D. Dinman, unpublished). Thus, trans-acting factors that affect–1 PRF efficiency could potentially regulate gene expression.

Genetic, biochemical, pharmacological, and molecular modelingstudies suggest that –1 PRF occurs either during or afteraccommodation of aminoacyl-tRNA (aa-tRNA) into the ribosomalA-site and prior to peptidyltransfer (reviewed in reference19). Alterations in the kinetics of aa- or peptidyl-tRNA bindingor in peptidyltransfer can affect the frequency of –1PRF (29-32, 39). In contrast, the Ty1 +1 PRF occurs while theribosomal A-site is unoccupied, i.e., after translocation butprior to delivery of aa-tRNA to the A-site. Since the –1and +1 PRF mechanisms occur at different stages of the elongationcycle, they can be used as foils in mechanistic studies of translationelongation (19).

A search of the literature suggested the ribosome-associatedchaperone complex as candidate trans-acting factors that mightbe used to regulate PRF. During the process of protein synthesis,unfolded peptides are inevitably produced. Cells have evolveda ribosome-associated chaperone complex to ensure that nascentpeptides are correctly folded and cleared from the ribosomepeptide exit tunnel (reviewed in reference 44). In yeast, thefunctional chaperone is a triad composed of a functional interactionbetween the Zuo1p/Hsp40 J-protein and Ssb1p/Ssb2p, coupled withthe Ssz1p Hsp70 homolog (14), an arrangement that is universallyconserved from bacteria to humans (20). The Ssz1p-Zuo1p-Ssb1/2pchaperone system is not present in bacteria. Bacteria have triggerfactor instead. Ribosomes isolated from mammalian cells possessa zuotin homolog called MPP11 (20). MPP11 forms a complex witha specific Hsp70 homolog (Hsp70L1), and the combination of MPP11and Hsp70L1 (mammalian RAC) can complement the lack of RAC inyeast (36). However, the issue of whether or not Ssb1/2p isconserved from yeast to humans remains unclear.

The current study was motivated by the observation that deletionof this complex has been shown to have functional consequences.Specifically, ssb1 ${Delta}$ ssb2 ${Delta}$ and ssz1 ${Delta}$ zuo1 ${Delta}$ cells grow slowly, arecold sensitive, are hypersensitive to high osmolarity and toaminoglycoside antibiotics (13, 35, 56), and promote increasedrates of nonsense and missense suppression under certain conditionsin yeast (42). Here, data are presented showing that –1PRF is specifically repressed in ssb1 ${Delta}$ ssb2 ${Delta}$ and ssz1 ${Delta}$ zuo1 ${Delta}$ cellsand that these cells are hypersensitive to two translationalinhibitors that were previously shown to affect this process.The implications of these findings with respect to their relevanceto posttranscriptional regulation of gene expression are discussed.

MATERIALS AND METHODS

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Materials and Methods

Yeast strains, media, and genetic methods. The S. cerevisiae strains used in this study are presented inTable 1. All experiments were carried out at 30°C unlessotherwise indicated. The ssz1 ${Delta}$ zuo1 ${Delta}$ (Rac^–) and ssb1 ${Delta}$ ssb2 ${Delta}$ strains were provided by Sabine Rospert. Escherichia coli DH5 ${alpha}$ was used to amplify plasmids, and transformations were performedby using standard calcium chloride methods (45). Yeast strainswere transformed by using the alkali cation method (22), andyeast media were prepared as previously described (10). TheL-A and M₁ viruses were transferred to [rho⁰] cells by cytoplasmicmixing (cytoduction) using a kar1-1 donor strain, and killerassays were performed as described previously (10). Total nucleicacids were isolated from the cytoductants as previously described(12) and treated with RNase A under high-salt conditions (500mM NaCl) to degrade single-stranded RNA. Nucleic acids wereextracted, separated through a 1% agarose-Tris-acetate-EDTAgel, and visualized with ethidium bromide.

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TABLE 1. Yeast strains used in this study

Plasmids and programmed ribosomal frameshifting assays. Reporter plasmids used to quantitatively monitor PRF in yeastcells are shown in Fig. 1. pYDL-control, pYDL-LA, and pYDL-Ty1have been described previously (18). These are dual-luciferasereporter plasmids based on the pRS series of yeast shuttle vectors(6, 47). From 5' to 3', each reporter contains the ADH1 promoter,a translational start site, the Renilla luciferase gene, a multiplecloning site, the firefly luciferase gene, and the CYC1 transcriptiontermination signal. In the 0-frame control pYDL-control, thefirefly luciferase gene is in the 0 frame with respect to theRenilla gene. In pYDL-LA, the firefly luciferase gene is inthe –1 frame respective to the Renilla gene and is 3'of the L-A viral –1 PRF signal. In pYDL-Ty1 the fireflyluciferase gene is in the +1 frame relative to the Renilla luciferasegene and is 3' of the Ty1 +1 PRF signal. Thus, in pYDL-LA andpYDL-Ty1, firefly luciferase is expressed only consequent toa –1 or +1 PRF event, respectively.

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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.

Another 0-frame control reporter was made to control for potentialeffects of the L-A mRNA pseudoknot on ribosome processivity.Oligonucleotide site-directed mutagenesis was performed to inserta cytosine 5' of the L-A –1 PRF signal in pYDL-LA. Theresulting construct, pYDL-LA0, maintains the sequence and mRNApseudoknot structure of pYDL-LA, but the –1 PRF signalis inactivated by placing the slippery site out of frame withrespect to the incoming ribosomes. The single base insertionmoves the firefly luciferase gene into the same reading frameas the Renilla luciferase gene, making the resulting constructa 0-frame control for pYDL-LA.

Luciferase assays were performed as described previously (18).Frameshifting data were analyzed using the statistical methoddescribed previously (24) to ensure that all data sets werenormally distributed and passed the 95% confidence levels.

In vivo and in vitro DMS modification and RNA sequencing. rRNAs were modified in vivo using dimethyl sulfate (DMS) aspreviously described (28). Briefly, logarithmically growingwild-type and mutant cultures were collected and treated witheither 80 mM or 160 mM dimethyl sulfate (molecular biology grade;Sigma, St. Louis, MO). Both untreated and stop controls wereincluded. Treatment with DMS proceeded for 3 min, ß-mercaptoethanolwas added to a final concentration of 0.7 M, and samples werethen treated with ice-cold water-saturated isoamyl alcohol.Cells were washed with water and collected by centrifugation,and nucleic acids were extracted with acid phenol and treatedwith DNase, and RNAs were precipitated with ethanol.

For in vitro DMS protection studies, ribosomes were purifiedfrom logarithmically growing cells by glass bead lysis and purifiedby ultracentrifugation as previously described (32). Purifiedribosomes were treated with 1 mM puromycin and 1 mM GTP for30 minutes at 37°C to remove aa-tRNAs and elongation factors.Treated ribosomes were then incubated with DMS as previouslydescribed (25). RNAs isolated from DMS-treated and untreatedcells and ribosomes were annealed with ³²P-end-labeled primers(Table 2) by incubation at 70°C followed by slow coolingand holding for 5 min to 40°C followed by immediate transferto ice. These oligonucleotides were designed to provide fullcoverage of the A-site finger (helix 38), the peptidyltransferasecenter, the A- and P-loops, and the sarcin/ricin loop. Primerextension and RNA sequencing were performed using avian myeloblastosisvirus reverse transcriptase and nucleoside triphosphates (Roche,Mannheim, Germany) at 42°C for 30 min. Products were separatedthrough 10% urea-acrylamide gels followed by visualization ona Kodak phosphorimager.

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TABLE 2. Oligonucleotides used for primer extension

Growth curves and drug sensitivity assays. Growth and drug sensitivity assays were performed in duplicatewith constant high-intensity shaking at 30°C. Optical density(OD) measurements were recorded automatically using a SynergyHT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT)at 595 nm and sampled every 17 min for 28 h. Cultures were inoculatedfrom saturated overnight cultures to a starting OD₅₉₅ of 0.05into prewarmed 500-µl wells with or without drugs. Anisomycinand sparsomycin were obtained from Sigma Aldrich (St. Louis,MO).

Data analysis was done as previously described (51, 52). Opticaldensity data were smoothed and log transformed. Estimation oflag phase was determined by calculating the slope over eighttime points, approximately 2.5 h. The intercept of the highestcalculated slope was designated the lag phase. Doubling timeswere determined by calculating the slope between every thirddata point on the growth curve. The lowest five of the sevenhighest slopes were averaged, giving the mean doubling time(DT). Doubling time was calculated as (log₁₀ 2)/mean DT_. Stationary-phaseOD was calculated by first determining the standard deviationof the final six OD measurements. A standard deviation of <2%of the final OD was used to designate the culture as being instationary phase. The OD change is determined by subtractingthe initial OD from the final OD.

RESULTS

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Results

Specific repression of –1 but not +1 PRF. A previous study demonstrated that mutants of the RAC-Ssb1p/2pchaperone complex exhibited increased rates of both nonsenseand missense suppression in the presence of paromomycin (42).Based on these observations, we assayed the effects of thesemutants on a different aspect of translational fidelity, PRF.Isogenic wild-type, ssz1 ${Delta}$ zuo1 ${Delta}$ (Rac^–), and ssb1 ${Delta}$ ssb2 ${Delta}$ cellswere transformed with the reporter plasmids shown in Fig. 1and PRF efficiencies were determined as previously described(18, 24). The mutants had little effect on +1 PRF (1.1- and0.9-fold difference with respect to the wild type, respectively).In contrast, translational fidelity as monitored by –1PRF was actually enhanced, i.e., deletion of either portionof the Rac-Ssb1/2 chaperone complex inhibited –1 PRF toapproximately half of the wild-type level (Table 3). Given theprevious findings of decreased translational fidelity by thesemutants, the results of these analyses were not anticipated.

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TABLE 3. Inactivation of the RAC-Ssb1/2 chaperone complex specifically inhibits –1 PRF^a

It is possible that a defect in ribosome processivity, e.g.,decreased ability to resolve and transit through the mRNA pseudoknotin the –1 PRF signal, could result in an apparent butfalse inhibition of –1 PRF. pYDL-LA0 was designed andutilized to address this possibility. This 0-frame control reporteris identical to pYDL-LA except for the addition of one cytosineresidue 5' of the L-A –1 PRF signal. This base insertioninactivates the –1 PRF signal by moving the slippery siteout of frame with regard to incoming ribosomes while retainingthe mRNA pseudoknot structure. Isogenic wild-type and mutantcells were transformed with pYDL-LA0 and pYDL-LA, and –1PRF efficiencies were determined. These results confirmed thatthe absence of the ribosome-tethered chaperone complex resultedin repression of –1 PRF (Table 3).

Killer virus maintenance defects. The efficiency of –1 PRF determines the ratio of structuralto enzymatic proteins available for virus particle assembly,and altering –1 PRF efficiency has a severe negative impacton the ability of yeast to propagate the L-A and M₁ Killer viruses(9). Thus, if inactivation of the ribosome-tethered chaperonetruly inhibited –1 PRF, then such cells would not be ableto propagate the Killer virus. The L-A helper and M₁ satelliteviruses were introduced into isogenic [rho⁰] wild-type and mutantcells by cytoduction, and cytoductants were then assayed forthe killer phenotype. While wild-type cells were Killer⁺, neitherthe ssz1 ${Delta}$ zuo1 ${Delta}$ nor the ssb1 ${Delta}$ ssb2 ${Delta}$ strain was able to maintainthe Killer phenotype (Fig. 2A).

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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.

To determine whether the Killer^– phenotype was due tovirus maintenance defects, as opposed to, e.g., defects in theprocessing or secretion of the killer toxin (reviewed in reference53), total nucleic acids were extracted from isogenic wild-typeand mutant cells, separated through an agarose-Tris-acetate-EDTAgel, and visualized by ethidium bromide staining. Compared tothose in wild-type cells, M₁ double-stranded RNA viral copynumbers were significantly decreased in the mutant cells; infact, trace amounts of M₁ double-stranded RNA could be visualizedin the mutants only by loading approximately five times moresample than extracts of wild-type cells (Fig. 2B). The inabilityof the RAC mutants to maintain M₁ provides independent confirmationthat they confer a defect in –1 PRF.

Quantitative growth defect analyses. The ssb1 ${Delta}$ ssb2 ${Delta}$ and Rac^– mutants are characterized by slowgrowth, cold sensitivity, hypersensitivity to high osmolarity,and hypersensitivity to aminoglycosides (13, 35, 56). To betterunderstand the functional significance of the ribosome-associatedchaperone complex, growth profiles of isogenic wild-type andmutant cells were quantitatively determined and compared. Assummarized in Fig. 3 and Table 4, almost twice as much timewas required for mutant cells to exit lag phase, and doublingtimes were similarly delayed in log phase. Mutant cells alsoreached stationary phase at significantly lower densities thandid their wild-type counterparts. These findings suggest thatinefficient utilization of energy resources consequent to defectiveprotein translation resulted in decreased rates of cell growthand premature medium depletion by the mutants.

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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.

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TABLE 4. Quantitative analyses of growth characteristics of cells lacking the RAC^– Ssb1/2 chaperone complex^a

Anisomycin and sparsomycin are small molecules that bind tothe peptidyltransferase center of ribosomes, interfering withnormal peptidyl transfer and tRNA binding (reviewed in reference38). Sensitivity to these drugs is indicative of changes withinthe functional center of the ribosomes that affect either peptidyltransfer or tRNA binding. Quantitative analyses of cell growthin the presence of these drugs were used to monitor the effectsof the RAC mutants on this region of the ribosome. While therate of increase in the doubling times in the mutants increased,the wild-type doubling time was essentially unaffected at thesame drug concentrations. This indicates that the mutants aresensitive to both anisomycin and sparsomycin, consistent witha defect in translation. (Fig. 3A and B).

DMS protection assays. Empirical studies and modeling of the mechanism of –1PRF suggested that defects in the ability to accommodate aa-tRNAsinto the ribosomal A-site should promote specific inhibitionof –1 PRF (reviewed in reference 19). Potentially, ifnascent peptides are not efficiently chaperoned upon exit fromthe ribosome, the ribosome exit tunnel could become congested,backing up into the peptidyltransferase center. This could resultin displacement of the 3' ends of peptidyl-tRNAs toward theA-site, which in turn might inhibit the ability of new aa-tRNAto properly accommodate the A-site. Alternatively, the absenceof the ribosome-associated chaperone complex could in some wayaffect ribosome assembly and hence its final structure. In eitherevent, changes in tRNA positioning or overall ribosome structurecould be reflected by changes in the accessibility of rRNA basesin this region to small chemical probes.

In vivo and in vitro DMS probings of rRNA were employed to testthis hypothesis. While in vitro probing can identify structuralchanges in the nontranslating ribosome population, in vivo analysisprovides a means to sample all of the ribosomes in all statesof translation (nontranslating ones as well), enabling a globalview of ribosome status. This can potentially allow detectionof innate structural differences as well as identifying elusivedifferences that may occur during specific stages of translationelongation.

Logarithmically growing cells were incubated with two differentconcentrations of DMS (or no-DMS controls), total RNAs wereextracted, and primer extension reactions using ³²P-labeledoligonucleotides sufficient to probe the entire peptidyltransferasecenter, the sarcin/ricin loop, and helix 38 were employed asdescribed in Materials and Methods. Puromycin-treated purifiedribosomes were also probed with DMS in vitro.

Figure 4 shows a representative autoradiogram of in vivo andin vitro DMS protection assays using primer 25-6. Although theseautoradiograms show DMS-specific changes in base protectionpatterns, there were no detectable changes between wild-typeand mutant ribosomes probed either in vivo or in vitro. Theapparent ssb1 ${Delta}$ ssb2 ${Delta}$ mutant-specific changes at positions 2932and 2935 in the in vitro assay are artifacts of this particularautoradiogram. The in vitro probing experiments demonstratethat lack of the ribosome-associated chaperone complex doesnot affect overall ribosome structure. As discussed below, however,the in vivo DMS protection assay may not be sensitive enoughto detect changes in protection patterns if a given defect affectsonly a small fraction of elongating ribosomes.

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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–, ssz1 ${Delta}$ zuo1 ${Delta}$ mutant; and S–, ssb1 ${Delta}$ ssb2 ${Delta}$ 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.

DISCUSSION

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Discussion

The ribosome-associated molecular chaperones interact with nascentpolypeptide chains, and it is hypothesized that they help themto fold properly and prevent their aggregation. The data presentedhere indicate that these chaperones are not only involved innascent polypeptide binding and protein folding, but also affectinnate ribosomal processes involved in translational fidelity.It has been suggested that nascent polypeptides can signal backwardsto the peptidyltransferase center (44). The lack of effectsof the mutants on +1 PRF, coupled with the inhibition of –1PRF, and stimulation of missense and nonsense suppression canbe used to infer the mechanistic consequences of such signalingon ribosome function.

As discussed in the integrated model, programmed ribosomal frameshiftingmust be considered within the context of the translation elongationcycle (19). In turn, the hybrid-states model of the translationelongation cycle has been described in terms of a series ofnine discrete steps (reviewed in reference 15). –1 PRFoccurs while the A- and P-sites of the large ribosomal subunitare occupied by tRNAs. Viewed within the context of the hybrid-statesmodel and taking other genetic and biochemical data into account,this event was narrowed down to a window occurring after deliveryof aa-tRNA to the A-site by elongation factor 1A (eEF1A) andprior to peptidyl transfer (19).

The 9-Å model, which examines the mechanism of –1PRF, further narrows this window, predicting that inhibitionof the accommodation step of the elongation cycle would decreasethe steady-state abundance of the substrate for –1 PRF,decreasing the frequency of this event (40). This predictionis supported by the observation that anisomycin, which inhibitsaccommodation by competing with the 3' end of the aa-tRNA forthe A-site of the peptidyltransferase center (17), specificallyinhibits –1 PRF (7, 24). Similarly, as accommodation isthe rate-limiting step of the elongation cycle (reviewed inreference 43), such defects would also be expected to resultin decreased fidelity of other events that rely on proper A-siteoccupancy states, whether it be the ability to discriminatebetween cognate and near-cognate aa-tRNAs (missense suppression)or between release factors and near- or noncognate aa-tRNAs(termination suppression). In contrast, since +1 PRF occurswhen the A-site of the small subunit is unoccupied (19), itshould not be affected by these mutants.

Figure 5 shows a set of cartoons describing how accommodationmay be inhibited by this class of mutants. In normal circumstances(Fig. 5A, lower panel), the 3' end of the acceptor tRNA movesfrom the A- to the P-site of the large subunit upon peptidyltransfer, occupying the A/P hybrid state, which is then followedby translocation, moving the peptidyl-tRNA to the P/P hybridstate. This in turn pushes the nascent peptide up into the exittunnel. The presumed role of the ribosome-associated chaperonecomplex is to act at the other end of the exit tunnel, helpingto ensure smooth passage of the nascent peptide out of the ribosome(Fig. 5A, upper panel).

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FIG. 5. Modeling the effects of the Rac^– and ssb1 ${Delta}$ ssb2 ${Delta}$ 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 ssb1 ${Delta}$ ssb2 ${Delta}$ 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).

Akin to the proposed activity of erythromycin, which binds tothe inside of the polypeptide exit tunnel, thus blocking peptidesynthesis (49), the absence of ribosome-tethered molecular chaperonescould result in peptide aggregation, blocking the polypeptideexit tunnel and creating problems inside the ribosome (Fig.5B, upper panel). If movement of the nascent peptide into thetunnel were inhibited, the 3' end of the acceptor tRNA wouldalso be prevented from fully occupying the P-site (Fig. 5B,lower panel). Such misplacement of the peptidyl-tRNA 3' endwould in turn inhibit the accommodation of the 3' end of theincoming aa-tRNAs into the A-site.

Although we had hoped that changes in the tRNA occupancy statusat the peptidyltransferase center might have been detectableas alterations in the accessibility of rRNA residues in thisarea, the in vivo DMS modification data were inconclusive. Thisis not surprising given the fact that only approximately 5%to 10% of translating ribosomes participate in the highly transient–1 PRF event. However, the absence of gross changes inrRNA modification patterns in purified ribosomes supports themodel discussed above by arguing against the notion that theobserved effects were indirectly due to gross changes in ribosomestructure.

The quantitative analyses of the effects of the mutants on cellgrowth point to the biological importance of the ribosome-associatedchaperone complex not only as cellular chaperones, but alsoas direct effectors of translation elongation. In a logarithmicallygrowing yeast cell, an overwhelming fraction of resources isdevoted to protein synthesis (reviewed in reference 50). Inthe growth conditions normally used in the laboratory, i.e.,unlimited nutritional resources, ideal temperatures, osmolarity,and aeration, the absence of this complex inhibits rates ofcell growth and division. However, conditions in the real worldare less than ideal, and the ability to posttranscriptionallyregulate the translational machinery can significantly extendvaluable resources and/or activate new biosynthetic pathways.Evidence that the RAC complex may be involved in such regulationcomes from DNA microarray experiments in which mRNAs encodingcomponents of the RAC complex are specifically downregulatedindependently of those encoding components of the translationalapparatus under conditions that affect chromatin remodelingand peroxisome assembly (1, 48). Thus, in addition to the obviousfunction of ensuring proper folding of nascent peptides, anotherintriguing possibility is that this complex may be used to regulateprotein translation by, e.g., regulating –1 PRF efficiencyor rates of nonsense and missense suppression. As such, it mayalso present an attractive target for drugs directed againstviruses that utilize these mechanisms.

ACKNOWLEDGMENTS

We are indebted to Sabine Rospert for generous sharing of yeaststrains. We also thank Ewan Plant and Jonathan Jacobs for criticalreview of the manuscript and Arturas Meskauskas for valuableconceptual discussions.

This work was supported by a grant to J.D.D. from the NationalInstitutes of Health (GM58859).

FOOTNOTES

* Corresponding author. Mailing address: Department of Cell Biology and Molecular Genetics, Microbiology Building, Room 2135, University of Maryland, College Park, MD 20742. Phone: (301) 405-0981. Fax: (301) 314-9489. E-mail: dinman{at}umd.edu.

REFERENCES

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