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Originally published In Press as doi:10.1074/jbc.M703391200 on July 9, 2007

J. Biol. Chem., Vol. 282, Issue 36, 26026-26034, September 7, 2007
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Role of a tRNA Base Modification and Its Precursors in Frameshifting in Eukaryotes*

William F. Waas1, Zhanna Druzina1, Melanie Hanan, and Paul Schimmel2

From the Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, April 24, 2007 , and in revised form, June 11, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Little is known about the role of specific base modifications of transfer RNAs. Wyosine bases are tRNAPhe-specific modifications that are distinguished by differentiated, lateral side chains and base methylations appended to the core ring structure of a universally conserved G37, adjacent to the anticodon of Phe tRNAs. Based on previous data, we hypothesized that this modification was needed for –1 frameshifting. Using a reporter system incorporating a SCV-LA yeast virus slippery site for detecting –1 frameshifts in vivo, yeast strains were created that enabled chemical-genetic dissection of the role of different functional groups of wyebutosine that are added in a three-step post-transcriptional set of reactions. With this system, hypomodification increased Phe-specific frameshifting, with incremental changes in frameshift efficiency after specific intermediates in the progression of wyebutosine synthesis. These data combined with investigations of wild-type and hypomodified tRNA binding to ribosomes suggest that frameshift efficiency is kinetically and not thermodynamically controlled. The progressive nature of frameshift efficiency with the stage of modification is consistent with a stepwise evolution and tuning of frameshift potential. The stepwise tuning of frameshift efficiency could explain why tRNAPhe in some eukaryotes is not fully modified but, rather, hypomodified to capture a specific frameshift potential.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Little is known about the biological role of specific base modifications of transfer RNAs. These modifications are found universally with some bacteria investing nearly 1% of their genome into tRNA modification genes (1). Yet most functions of tRNA that can be tested in vitro show little dependence on these modifications. Wyosine bases are eukaryote- and Archaea-specific modifications, characterized by a fluorescent, tricyclic 1H-imidazo[1,2-{alpha}]purine core structure. These guanosine modifications occur strictly in phenylalanine-specific tRNA (tRNAPhe) at position 37, on the 3'-side of the anticodon. Given the remarkable distribution of wyosine bases in eukaryotic Phe-specific tRNAs and in the light of our previous genetic studies of wyosine base biosynthesis (2), we set out to investigate its potential functional significance.

Wyosine bases from different organisms are distinguished by differentiated, lateral side chains and base methylations that are appended to the core ring structure (3–8). Wyebutosine (yW),3 a wyosine-family member of yeast tRNAPhe, is one of the most extensively hypermodified bases (see Fig. 1A). In Saccharomyces cerevisiae, yW is posttranscriptionally synthesized from a genetically encoded guanine in a multienzyme process whose details are not fully understood (9, 10) (see Fig. 1B). In vitro studies showed that loss of the modification has little effect on the interactions between tRNAPhe and phenylalanyl-tRNA synthetase or its ability to perform in in vitro protein synthesis (11, 12). The hydrophobic nature of wyebutosine contributes to base-stacking interactions with neighboring bases (A36 and A38) and restricts conformational flexibility of the anticodon loop (13–17). Removal of yW from tRNAPhe resulted in local changes of the anticodon and was thought to destabilize codonanticodon interactions in the ribosomal P and A sites (18–21). These observations seem relevant to in vitro studies that link yW to the preservation of the reading frame at phenylalanine codons and to the influence of yW on –1 ribosomal-programmed frameshifting (22, 23). Thus, change of the position 37 modification of tRNAPhe from wyebutosine-37 to 1-methyl-G37 resulted in a 3-fold increase of –1 programmed ribosomal frameshifting in rabbit reticulocyte lysates.

Many viruses (e.g. retroviruses and totiviruses) rely on programmed –1 frameshifting to allow translation of multiple proteins or protein variants from a single promoter (24–27). For such organisms the efficiency of frameshifting is critical because it determines the ratio of structural and catalytic proteins and, therefore, controls viral propagation. For example, a small (2-fold) change in –1 frameshifting frequency completely eradicated the SCV-LA virus from S. cerevisiae (28). Given the perspective of this previous work, we set out to explore the potential role of the wyebutosine base modification in –1 frameshifting. Because yW is synthesized through a multistep pathway, our interest was drawn to the question of how the potential for frameshifting might also be built up step-by-step. Thus, the significance of yW modification substructures of yeast tRNAPhe to –1 frameshifting, its frequency, and its specificity were investigated by structural ablation using a series of characterized yeast knock-out strains that are blocked at specific, successive steps of wyebutosine synthesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
tRNA Strains—Wild-type and deletion strains ({Delta}YPL207w and {Delta}YML005w) of S. cerevisiae were purchased from Open Biosystems. All three strains were of the MAT{alpha} leu2{Delta}0 met15{Delta}0 ura3{Delta}0 genotype. Genes {Delta}YPL207w and {Delta}YML005w were renamed as TYW1 and TYW2, respectively (9). The YPL207w and {Delta}YML005w open reading frames were substituted with a KanR cassette (Table 1).


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  		TABLE 1
Isogenic S. cerevisiae strains producing tRNAPhe with specific structural variants of G37

The common genetic background of the strains was MAT{alpha} leu2{Delta}0 met15{Delta}0 ura3{Delta}0.

 
Each deletion strain is blocked at separate, successive steps of wyebutosine biosynthesis (see Fig. 1B), and therefore, it produces tRNAPhe pools that are homogeneously under-modified at position 37. Composition analysis of tRNAPhe from S. cerevisiae {Delta}TYW1 strain by tandem liquid chromatography–mass spectroscopy revealed the presence of the first wyebutosine precursor, 1-methylguanosine (m1G, see Fig. 1), at position 37 of tRNAPhe (2). An analogous study of {Delta}TYW2 ({Delta}YML005w) strain demonstrated accumulation of imG14 precursor in tRNAPhe (29).

Cell growth of each deletion and wild-type strains was measured in rich YPD medium (2% peptone, 1% yeast extract and 2% glucose). Neither strain showed a detectable defect in rate or yield relative to the parent wild-type strain that might hinder consequent in vivo studies.

–1 PRF Reporter Constructs—Dual-luciferase reporter construct pJD511 containing ADH1 promoter, lucR, an ~200 base sequence from SCV-LA virus, lucF and CYC1 terminator was a generous gift of J. D. Dinman (University of Maryland) (30). The slippery sequence variants (pSCV-I–pSCV-VI) of the SCV-LA-derived bicistronic reporter were produced by site-directed mutagenesis (QuikChange) of pJD511 plasmid. 0-frame control plasmid contained polylinker sequence only (no frame-shifting sequence) and firefly luciferase is in the same reading frame as Renilla luciferase, producing both proteins. Plasmid sequences were confirmed by automated sequencing.

Plasmids were first introduced into S. cerevisiae by chemical transformation using standard protocols. Transformants were isolated on media lacking leucine (0.067% Leu(–) DO supplement (BD Biosciences), 0.67% nitrogenous base (Sigma), 2% glucose); pSCV restored leucine biosynthesis.

Dual-luciferase Assays—Yeast strains (wt, {Delta}TYW1, {Delta}TYW2) harboring reporter constructs were grown in Leu(–) dropout media (2 ml) for 7 h at 30 °C after a 1:5 dilution of overnight culture. Cells were harvested by centrifugation, washed with 500 µl of ice-cold 1x phosphate-buffered saline (containing protease inhibitor mixture, Roche Applied Science), centrifuged again for 10 min at 15700 x g, and resuspended in 100 µl of the same buffer. Cells were lysed with 100 µl of glass beads (Sigma, 425–600 µm, prewashed in phosphate-buffered saline) in a Vortex mixer at 4 °C 5 times 2 min each. Lysates were clarified by centrifuging for 10 min, and total protein concentration was determined by Bradford method. Preparation routinely produced 0.1–1.0 mg/ml protein.

Activities of expressed Renilla and Firefly luciferases were determined in a luminometer (MicroLumant LB 96P) with 20 µl of cell lysate using the Dual-Luciferase® assay system reagents (Promega E1910) following manufacturer's protocol. Each strain was assayed in seven replicates. Raw luminescence data were converted to a lucF/lucR ratio for each sample. This ratio was then normalized to the strain-specific 0-frame control and multiplied by 100% to obtain frameshift efficiencies for each recoding signal. By virtue of this manipulation, frameshifting is 100% for control plasmids, although strain variation is not large.

tRNA Preparation and Purification—Bulk tRNA containing tRNAPhe structural variants was isolated from yeast strains (wt, {Delta}TYW1, {Delta}TYW2). Bulk tRNA purification and fractionation and purification of aminoacylated Phe-tRNAPhe structural variants were as described in Waas et al. (2). Briefly, purified bulk tRNA containing wt or hypomodified tRNAPhe was fractionated by reverse-phase HPLC (Vydac C4 semi-preparative HPLC column, Deerfield, IL). Fractions enriched for tRNAPhe were aminoacylated with [3H]phenylalanine using yeast phenylalanyl-tRNA synthetase. Aminoacylated [3H]Phe-tRNAPhe variants were further purified by reverse-phase HPLC as described (2). Because the retention times of Phe-tRNAPhe and deacylated tRNAPhe are grossly different, the resulting Phe-tRNAPhe pool was free of deacylated tRNA contamination. Using this procedure we obtained Phe-tRNAPhe structural variants with amino acid acceptances that were higher than 1800 pmol/A260 (2). The uniformity of purified tRNAPhe structural variants was additionally confirmed by matrix-assisted laser desorption ionization mass spectrometry (2, 29).

All three structural variant were aminoacylated simultaneously with the same batch of [3H]Phe, and the charged tRNAs were purified under identical conditions. We demonstrated that no less than 95% of detected [3H]Phe was linked to tRNA. Specific activities of the three [3H]Phe-tRNAPhe variants were uniform and equal to 1.32 x 104 cpm/pmol. The procedure for N-acetylation of Phe-tRNAPhe to prepare P-site substrate NAc-Phe-tRNAPhe was performed similar to previously described (31).

Ribosome Purification—80 S ribosomes were purified from commercially available S. cerevisiae strain (INVSc1, Invitrogen) as described in Algire et al. (32) with the following modification. Yeast cells, resuspended in 30 ml of low salt buffer (100 mM potassium acetate, 20 mM Hepes-KOH, pH 7.6, 2.5 mM magnesium acetate, 1 mg/ml heparin, 1 mini-EDTA free protease inhibitor tablet (Roche Applied Science)), were lysed with 425–600-µm glass beads using Bead-Beater (Biospec) in 3 pulses of 3 min each at 4 °C. Cells were spun for 30 min at 50,000 x g, and the supernatant was collected and centrifuged again. KCl was added to the supernatant to the final concentration of 500 mM and centrifuged again for 4 h at 50,000 x g. Ribosome pellets were resuspended in 6 ml of high salt buffer (500 mM KCl, 100 mM potassium acetate, 20 mM Hepes-KOH, pH 7.6, 2.5 mM magnesium acetate, 1 mg/ml heparin, 2 mM dithiothreitol). Puromycin and GTP were added to the final concentration of 1 mM each, and the mixture was incubated on ice for 45 min. Ribosome mixture was layered on top of 3 ml of sucrose cushion (500 mM KCl, 100 mM potassium acetate, 20 mM Hepes-KOH, pH 7.6, 2.5 mM magnesium acetate, 2 mM dithiothreitol, 1 M sucrose) and centrifuged overnight at 152,000 x g at 4 °C. Pellets were resuspended in ribosome storage buffer (20 mM Hepes-KOH, pH 7.6, 10 mM MgCl2, 1 mM dithiothreitol, 250 mM sucrose, 0.1 mM EDTA), separated into aliquots, frozen in liquid nitrogen, and stored in –80 °C. The concentration of 80 S ribosomes was calculated based on the assumption that 1 A260 = 20 pmol (80 S extinction coefficient at 260 nM is 5 x 107 cm–1 M–1) (32, 33).

Acid Treatment of [3H]AcPhe-tRNAPhe—To remove wyebutosine modification of position 37 of tRNAPhe, wt [3H]Phe-tRNAPhe and [3H]Phe-tRNAPhe (m1G-37) were treated with mild acid as previously described (12). Briefly, aminoacylated tRNA was incubated in 100 mM ammonium formate, pH 2.9, for 3 h at 37°C. tRNA was exchanged into 5 mM ammonium acetate, pH 4.5, and concentrated using Microcon YM10 filters (Millipore).

Millipore Filter Binding Assay: Saturation Experiments with [14C]NAcPhe-tRNAPhe—0.8 µM 80 S was incubated with 0.8 mg/ml poly(U) RNA (Sigma) in binding buffer (30 mM Hepes, pH 7.5, 50 mM NH4Cl, 10 mM MgCl2, 2 mM spermidine, 0.05 mM spermine, 5 mM beta-mercaptoethanol) for 20 min at 37 °C followed by 20 min of incubation on ice. 2.5-µl aliquots were taken into separate tubes, and 7.5 µl of an increased concentration [14C]NAc-Phe-tRNAPhe was added (to reach molar ratios of 0.5–10 tRNA to 80 S). The incubation was continued for 20 min at 37 °C. Tubes were transferred onto ice, and tRNA binding was determined by a filter -binding assay. Each sample was diluted to 100 µl with binding buffer and immediately applied onto a presoaked 0.45-µm nitrocellulose filter (Millipore); filters were washed 3 times with 500–750 µl of binding buffer containing 0.2 mg/ml total yeast RNA (to decrease nonspecific binding of tRNA to filters). Values were corrected for control samples lacking ribosomes, which were typically 1–4% of applied labeled tRNA. Filters were air-dried, placed into a scintillation vial, treated with 100 µl of 0.1 M NaOH to release Phe of tRNA (to reduce quenching), and counted in a scintillation counter using EcoLite scintillant (ICN).

A-site tRNA Binding—80 S yeast ribosomes were preincubated with poly(U) mRNA in 140–150 µl of binding buffer for 20 min at 37 °C. 10–20 µl of P-site substrate N-AcPhe-tRNAPhe in the same buffer was added, giving a final concentration of 60 nM 80 S, 0.4 mg/ml poly(U), 75 nM N-AcPhe-tRNAPhe. The incubation was continued for 20 min at 37 °C and 20 min on ice. For the alternative A-site complex (see "Results"), 0.1 µM mRNA with sequence AUG UUU ACG AUU AUU AUU AUU (synthesized by IDT technologies) and 0.1 µM Escherichia coli tRNAMetf as P-site substrate were used (purchased from Chemical Block, Russia). After incubation, 5-µl aliquots of P-site tRNA-80 S complex were transferred into separate tubes and mixed with 5 µl of increasing concentration of [3H]Phe-tRNAPhe to titrate ribosomal A-site. The complexes were incubated an additional 20 min at 37 °C, transferred to ice, and filtered as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experimental Strategy—In the yeast S. cerevisiae, the hypermodified wyebutosine is synthesized through post-transcriptional, sequential action of at least five enzymes, designated as TRM5p and TYW1p to TYW4p (Fig. 1B) (9). The disruption of TYW1 results in production of m1G37, a hypomodified tRNAPhe lacking both yW side chains and the core tricyclic ring structure (it is specifically m1G37 tRNAPhe that has been identified in certain mammalian tumor cell lines (11, 34)). After conversion of G37 to m1G by TRM5p, the next enzyme, TYW1p, converts m1G to 9H-imidazo[1,2-{alpha}]purin-9-one, 3,4-dihydro-6-methyl-3-beta-D-ribofuranosyl (4-demethylwyosine, imG14). The actions of TYW2p, -3p, and -4p then alter imG14 to yW through reactions whose details are not fully understood. Thus, a {Delta}TYW2 strain produces tRNAPhe with a tricyclic, archaeal-like (wyosine-family bases at position 37 of tRNAPhe are also seen in Archaea) form of wyosine that lacks the typical side chains of yW, i.e. 4-demethylwyosine-37 (imG14-37). Our goal was to work with yeast strains in which one of the genes needed for yW synthesis was knocked out, so that a specific intermediate, like m1G37 or imG14-37, would build up. We could then test that tRNA, with a specific hypomodification at position 37, for its activity in programmed ribosomal frameshifting.

For these investigations isogenic yeast strains were selected that were proven to accumulate the specific hypomodified tRNAPhe (Table 1). Taking advantage of previous work and strain constructions, the common genetic background for the "wild-type" and deletion strains was MAT {alpha} leu2{Delta}0 met15{Delta} 0 ura3{Delta} 0 (2). Similar strains encoding the {Delta}TYW1 and, separately, the {Delta}TYW2 alleles, were viable and, as demonstrated previously by mass spectrometry, produced tRNAPhe with m1G and imG14, respectively, at position 37 (2, 29). A strain carrying a {Delta}TRM5 allele was also considered, but its viability was marginal. More importantly, the m1G37 methyltransferase is not pathway-specific (a {Delta}TRM5-bearing strain encodes other hypomodified tRNAs (for example, Arg-, Leu-, and Pro-specific tRNAs) in addition to hypomodified tRNAPhe (35)). Disruption of the TYW1 and TYW2 gene products, which are pathway-specific, thus provides an important simplification for interpretation of any data generated in vivo. With all these considerations in mind, three different species of tRNAPhe (wild-type, m1G, and imG14) were used for investigations of frameshifting in vivo.

Experimental Rationale for Use of Yeast Double-stranded RNA Virus as Part of the Reporter System—Our reporter constructs were developed to mimic viral frameshifting in which "–1" (or –1 PRF) is most common. In viruses, programmed –1 ribosomal frameshifting redirects translating ribosomes into a new reading frame, thus bypassing an in-frame stop codon and continuing synthesis of an extended protein. As a consequence, frameshifting allows for expression of two polypeptide chains from a single mRNA at a ratio specified by the primary sequence of the viral gene as well as by the predisposition of the host for frameshifting.


Figure 1
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  		FIGURE 1.
A, secondary structure of tRNAPhe from S. cerevisiae with modified nucleotides (yW at position 37 is boxed). B, biosynthetic pathway of yW in S. cerevisiae tRNAPhe (9).

 
We noted that many laboratory strains of S. cerevisiae carry a double-stranded RNA virus known as SCV-LA. The viral genome codes for a capsid polypeptide (cap) and cap-RNA polymerase fusion (cap-pol) that is produced as a result of –1 PRF (36–38). The signal for frameshifting is provided by an ~200-base sequence between cap and pol genes. Contained within this sequence are three elements that are each required for –1 PRF, a heptameric slippery site, a linker region, and a secondary structure element (36, 39). In SCV-LA virus, similarly to other viruses (including retroviruses), efficiency of –1 PRF is crucial because it determines the ratio of structural and enzymatic proteins. Growing evidence suggests that modulation of frameshift efficiency can reduce viral infectivity and have a dramatic effect on virus propagation and viability (25, 40, 41).

For our experiments the functional –1 PRF unit from SCV-LA virus was transplanted into a bicistronic LucF/LucR reporter system (see below) (Fig. 2). In yeast that harbors SCV-LA, a specific UUA-reading tRNALeu slips one nucleotide from UUA to insert leucine at a UUU (codes for Phe) codon. This construction allowed the inherent frameshifting frequency to be determined in detail (30). When placed in the wild-type or {Delta}TYW1 and {Delta}TYW2 genetic backgrounds, the reporter system enabled us to analyze in vivo relationship between the discrete (but substantial) modification state of tRNAPhe and –1 programmed ribosomal frameshifting efficiency in a codon-specific manner. The dependence of frameshifting on the slippery sequence context could also be studied by incorporating several variants of the SCV-LA heptameric frameshift element into our test system. For these purposes, we changed the "pre-slippage" UUA codon to either UUU or UUC (codons for Phe) so as to specifically position tRNAPhe in the ribosomal A-site (Fig. 2).

Combining Yeast RNA Virus Construct with a Dual Luciferase Reporter System—To have a direct visual readout, we used a dual luciferase reporter system developed by Dinman and co-workers (30, 42). The construct specifies synthesis of two reporters that are driven by an ADH1 promoter. The two reporters are Renilla luciferase (encoded by lucR) and firefly luciferase (encoded by lucF). As shown in Fig. 2, viral coding sequences for the cap and pol proteins were replaced, respectively, with the coding sequences for lucR and lucF. Thus, the SCV-LA sequence that promotes frameshifting was placed between the coding sequences for the two luciferases. This arrangement promoted constitutive expression of lucR, whereas the production of lucF depended on frameshifting at the SCV-LA intervening sequence. Therefore, a –1 frameshift produced a protein harboring both Firefly and Renilla luciferases connected by a small peptide linker.


Figure 2
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  		FIGURE 2.
Schematic of –1 PRF reporter construct. Dual luciferase reporters were designed to mimic viral frameshifting (bottom panel). The frameshifting signal encoded between LucR and LucF is provided by an ~200-nt sequence from SCV-LA virus and contains a heptameric slippery sequence, a linker, and a secondary structure element. The slippery sequence codons are shown in 0-frame (Pre-slippage) and in –1 frame (Post-slippage) with corresponding A-site codons underlined (top panel). CAP, capsid polypeptide; POL, polymerase RNA.

 
A construction encoding a 0-frame control was also assembled. A control plasmid was used to measure the translation level of an in-frame firefly luciferase coding sequence relative to that of the Renilla luciferase. The luminescence from lucR and lucF was measured sequentially in the same experiment with two different molecular substrates, with a quench step after the first measurement (thus, in these experiments luminescence from lucR served as a convenient internal control). The frequency of programmed ribosomal frameshifting was then determined by dividing the lucF/lucR luminescence ratio from cells carrying the frameshift reporter vector by the same ratio from the cells carrying the 0-frame control plasmid.

The major advantage of this system is the internally controlled nature of the constructs. Normalization allowed for comparison of frameshifting rates in different cell types (strains) and minimized the potential for artifacts due to inherent variability of protein synthesis rate and differences in mRNA abundance/stability. Because LucF acts as an internal control, there was no need to depend on error-prone protein quantification or standardization procedures for individual samples (30).


Figure 3
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  		FIGURE 3.
Frameshifting efficiencies in cells bearing the pSCV-I and pSCV-II constructs (%). wt and deletion yeast strains producing tRNAPhe with Wyebutosine (light gray), imG14 (dark gray), or 1-methyl-G (black) modifications at position 37 and harboring the indicated reporter plasmids (slippery sequences are indicated for each reporter) were prepared as described under "Materials and Methods." Frameshift efficiencies were determined by dividing the ratio of the firefly to Renilla luciferase activities from each sample to the ratio obtained from the 0-frame control strain (both luciferases are in-frame and constitutively expressed).

 
Modification Status of G37 of tRNAPhe Correlates with –1 PRF at Phenylalanine-specific A-site Codons—Six different variations of the heptameric slippery site were created (Fig. 2). These were designated as pSCV-I–VI. We reasoned that because wyebutosine is a tRNAPhe-specific modification, frameshifting would respond specifically to codons (UUU and UUC) for phenylalanine. To demonstrate this point, we used constructs pSCV-I and pSCV-II containing G GGU UUA and G GGU UUU heptameric sequences, respectively. Because UUU codes for phenylalanine, frameshifting at the slippery sequence of pSCV-II was expected to respond to yW hypomodification. In contrast, the pSCV-I A-site sequence codes for leucine and should not be sensitive to the modification status of tRNAPhe or deletion of the corresponding biosynthetic gene.

With all three strains of S. cerevisiae, wild-type, {Delta}TYW1, and {Delta}TYW2, –1 frameshifting was observed, and its frequency was substantially increased by hypomodification at position 37. In particular, –1 frameshifting was at a frequency of 35% (compared with the 0-frame control) for the hypomodified m1G37-containing tRNAPhe (Fig. 3). This level of frameshifting was almost twice that for wild-type tRNAPhe. For the next intermediate, the more modified imG14-37 tRNAPhe, the frameshift frequency was between that of wild type and m1G37 tRNAPhe. However, with the UUA codon of pSCV-I replacing the UUU codon of pSCV-II, little frameshifting was observed, and no sensitivity to the status of tRNAPhe modification could be seen. These results support the idea that the modification status of tRNAPhe affects –1 frameshifting at the slippery sequence and does so in a way that is specific to the A-site codon for Phe. The data also demonstrate the incremental nature of the effects of guanosine-37 modification states.


Figure 4
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  		FIGURE 4.
Frameshifting efficiencies of pSCV-II–pSCV-IV constructs. A, pre-slippage A-site-dependent effects on frameshift efficiencies. B, replot of data in A as normalized frameshifting efficiencies, which show large relative increase in efficiency with ablation of yW side chains; –1 PRF efficiency of each sample was normalized to –1 PRF efficiency of the wild type yeast strain.

 
Interplay between the G37 Modification State and the Slippery Sequence of mRNA—The codon-anticodon interaction is a critical event for translation and for ribosomal recoding by frameshifting. Previous work showed that –1 PRF correlates with the theoretical strength between A- and P-site interactions of tRNAs with the mRNAs to which they are bound (43). To consider the role of G37 modifications in establishing these interactions, we designed experiments where strains harboring tRNAPhe G37 variants were challenged with reporter constructs coding for permutations of the slippery sequence (pSCV-II–pSCV-VI). Because tRNAPhe has only a single isoacceptor with a GAA anticodon, only UUU and UUC A-site codons need be considered. We made two P-site sequence variants (GGU (wild type) and GAU) in pairwise combinations with each of the UUU and UUC codons. Thus, for example, differences in –1 PRF frequencies were compared using pSCV-II versus -III (or pSCV-IV versus -V) constructs that contain alterations of the A-site mRNA sequence. In addition, we made a sixth construct, pSCV-VI, that had a GUU codon at the P site and a UUU codon at the A-site. This construct gave us a third P-site variation to test in conjunction with the UUU A-site.

The frequency of –1 PRF was markedly reduced by changing U to C at the terminal position of the slippery sequence (Fig. 4A). This reduction was seen with both the GGU and the GAU P-site triplet that immediately precedes the Phe codon. This result is consistent with the previous observation of Brierley et al. (43) with the infectious bronchitis virus sequence in a rabbit reticulocyte lysate cell-free translation system, where frameshifting was more likely in cases where the codon-anticodon interaction in the pre-slip ribosomal A-site is weaker. The simplest explanation for this observation is that the specific interaction between the A-site Phe codon of the mRNA and the tRNAPhe GAA-anticodon sequence is operationally weaker with UUU than with UUC.

Interestingly, the frameshifting efficiencies remained largely unaffected when the P-site codon was changed from GGU to GAU (compare SCV-II versus SCV-IV and SCV-III versus SCV-V), indicating that the requirement for specific nucleotides in the P-site sequence are less stringent (Fig. 4B). Similarly, Brierley et al. (43) concluded that only minimal mRNA-tRNA pairing in the P site might be required for efficient frameshifting.

Surprisingly, for the slippery sequence G GUU UUU (SCV-VI contains a third P-site variation codon), sensitivity to hypomodification is lost, although –1 PRF frequency is robust. At present we cannot fully explain this observation. However, Brierley et al. (43) have demonstrated reduced sensitivity of frameshifting events to the absence/presence of downstream secondary elements in the context of extended uridine stretches at the slippery sequence (where position 7 is also uridine). Similarly, Carlson et al. (23) observed no influence by yW37 on –1 PRF at U UUU UU(U/C) sequences. It is plausible that, by substituting U at position 3 for pSCV-VI (versus G for pSCV-II or A for pSCV-IV) and thereby increasing Un content in the slippery sequence, sensitivity to modification at position 37 is attenuated.

Most importantly, the putative strength of the codon-anticodon interaction did not change the relative order of the efficiency of frameshifting that correlates with the state of modification. That is, the greater the state of modification, the lower the frequency of frameshifting. This relationship can be seen more clearly by assigning an arbitrary value of 1 to the frameshift efficiency seen with wild-type tRNAPhe and replotting the data (Fig. 4B). In this format it is easy to see that deletion of the wyebutosine side chain, as occurs with the {Delta}TYW2 genetic background, increased the –1 PRF frequency by 150–200% relative to the isogenic wild-type yeast strain. Further ablation of the structure (from a tricyclic core of 4-demethylwyosine to a purine of m1G, {Delta}TYW1) produced an even more significant increase (>200% relative to the wild-type yeast strain). Thus, hypomodification increased the frequency of –1 ribosomal frameshifting. These incremental changes in frameshift efficiency, therefore, correlate inversely with the increasing structural complexity of G37 modifications of A-site-bound tRNAPhe that are associated with specific intermediates in the progression of the wyebutosine biosynthetic pathway.

Binding of tRNAPhe Variants to Eukaryotic Ribosomes—The correlation in vivo between –1 frameshifting and the apparent strength of tRNA interactions at the ribosomal A site was further investigated in vitro. This correlation could be due to an inherent stronger equilibrium binding of the hypomodified tRNAs to the A site. From an experimental standpoint, equilibrium studies were most accessible and were pursued. For this purpose, we performed equilibrium binding studies of Phe-tRNAPhe to yeast 80 S ribosomes using two different mRNA systems.

Limited data are available on the functional role of yW-37 modification in tRNA binding to the ribosomal A site. Thus, Katunin and co-workers (18, 20) demonstrated that the presence of wyebutosine base increased the strength of binding of aminoacylated tRNAPhe to the A site of E. coli 70 S ribosomes 10-fold. In their work, binding of a fully modified yeast tRNAPhe was compared with the binding of acid-treated tRNAPhe, specifically depurinated at the position 37. Such treatment removes wyebutosine from wild-type tRNA without breaking the RNA chain, thereby producing tRNAPhe depurinated at position 37 (12). Because frameshifting is a subtle, conformationally sensitive event, we worried that removal of a wyebutosine base could have a significant effect on the conformation of anticodon loop that in turn would result in destabilization of codon-anticodon interactions and tRNA binding to the ribosomes. This concern not withstanding, we first decided to determine whether acid-directed ablation of yW-37 had the same effect in an in vitro yeast as in the E. coli system.

We carried out acid treatment of wild-type tRNAPhe (yW-37) and purified yeast ribosomes that accepted up to 1.5 molecules of N-AcPhe-tRNAPhe (per 80 S particle in the presence of poly(U) mRNA). Because N-AcPhe-tRNAPhe is able to bind to both P and A sites, we concluded that 75% of our preparation of yeast ribosomes was active for tRNA binding. Then, to determine the affinity of Phe-tRNAPhe for the A site, conditions had to be found where the P site was fully occupied, whereas the A site was vacant. For E. coli ribosomes, the P site is occupied first and the half-life of N-AcPhe-tRNAPhe dissociation is ~2h (provided there is no tRNA exchange between P and A ribosomal sites during the experimental procedures) (44) (interference from the third, E-site, binding locus is unlikely because that site is specific for deacylated tRNA, and in any case, binding to that site is obscured by the filter binding technique that we used (45–47)). Thus, by use of ribosomes whose P site is preoccupied with a peptidyl-tRNA analog, the titration of the A site with aminoacyl-tRNAPhe reduces to a single-site model and allows for a determination of the Kd for the A-site complex.

Yeast 80 S ribosomes were first incubated with a 1.25-fold excess of N-AcPhe-tRNAPhe in the presence of poly(U). This procedure quantitatively filled the P site. Increasing concentrations of aminoacylated of [3H]Phe-tRNAPhe were then added to the P-site-filled ribosomes. Ribosomal complexes were subjected to nitrocellulose filtration. Similar to E. coli ribosomes, the A-site binding of wild-type Phe-tRNAPhe to yeast 80 S particles decreased at least 5-fold after acid-dependent depurination of yW-37 (Table 2) (as a control, the binding affinity of acid-treated tRNAPhe (m1G-37) remained unchanged (within the error of experiment) as expected, because m1G-containing tRNA is not susceptible to acid depurination). Because acid ablation actually removes the entire base and not just a pendant group and because we demonstrated that frameshifting at Phe codons is sensitive to modification changes far more subtle than acid ablation of yW-37 from the tRNA anticodon loop, we decided to proceed with studies of binding affinities of hypomodified Phe-tRNAPhe (m1G and imG14) to the A site of the yeast 80 S particle.


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  		TABLE 2
Dissociation constants Kd (nM) of binding of 3H-Phe-tRNAPhe variants to the A site of yeast 80 S ribosomes at pH 7.5, 10 mM MgCl2

Binding was measured using Millipore filter method as described under "Materials and Methods." ND, not determined.

 
Using the approach described above, we identified a Kd for yeast wild-type Phe-tRNAPhe (yW-37) binding to the A site as 154 ± 50 nM (Table 2) (this value is similar to the reported dissociation constants of 20–100 nM for yeast Phe-tRNAPhe binding to the A site of E. coli 70 S ribosomes (18, 44).) Interestingly, binding of structural analogs of Phe-tRNAPhe containing the hypomodified nucleotide in position 37, tRNA-m1G, or tRNA-imG14 demonstrated somewhat stronger binding to the ribosomal A site, with dissociation constants of 49 ± 27 and 46 ± 21 nM, respectively. This difference in binding of wild-type and hypo-modified tRNA, whereas small (3-fold), was reproducible and had a p value <0.006.

To avoid possible ambiguity coming from the degenerate nature of the poly (U)-programmed system and, in addition, the possibility of a P-site binding contribution to the A-site Kd value, similar binding studies were performed with 80 S ribosomes programmed with an mRNA coding for tRNAMetf and tRNAPhe at the first two codons (AUG UUU). In this system the ribosomal P site was occupied by deacylated E. coli tRNAMetf, whereas the A site was titrated by structural variants of Phe-tRNAPhe, as described above. Essentially the same results were obtained for this system. A-site binding of fully modified Phe-tRNAPhe (Kd = 96 ± 9 nM) was 2.5–3-fold weaker than that of the hypo-modified tRNAPhe isoacceptors (Kd = 41 ± 6 or 30 ± 9 nM for imG-14 or m1G, respectively) (Table 2). Thus, acid ablation of the entire base produced a result opposite to that observed with the more subtle, natural changes produced by genetics.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major motivation for this work was to find at least one rationale for the strong selective pressure that has designated phenylalanine-specific tRNAs in eukaryotes for wyosine-family base modification on the 3'-side of the anticodon. Generally, base modifications at any position in a tRNA do not produce large effects, at least with respect to the available assays related to proteins synthesis. For choosing to do in vivo studies of frameshifting in S. cerevisiae, we were particularly struck by the realization that only the smallest change in frameshift efficiency (e.g. 2-fold, see above) could result in the difference between viability and non-viability in specific circumstances. We, therefore, surmised that, if the effects of wyosine base modifications on frameshifting were small but at least measurable, then the small effects could in themselves be sufficient to account for the widespread distribution of wyosine modifications in eukaryotes and Archaea. The data presented in Figs. 3 and 4 establish clearly that yW-related modifications at position 37 alter –1 frameshifting in vivo by a magnitude that is well outside experimental error. Thus, the effects of yW-family modifications on –1 frameshifting may in themselves offer the explanation for the widespread distribution of these modifications in eukaryotes and Archaea.

Our data also show that –1 frameshifting itself is dependent on the degree of modification of each intermediate state in the multistep biosynthetic pathway. We were able to study three progressive states of modification, m1G, imG14, and yW (wild type). Frameshifting was greatest with the least modified m1G intermediate and then dropped progressively as additional elaborations to the core base were added. Because the entire multistep pathway most likely developed in a progressive, step-by-step way, our results suggest that the differences in frameshift efficiency of each substructure could itself provide the selective force for the pathway to evolve progressively. In addition, the appearance in some organisms of mature tRNAPhe that are hypomodified with m1G or imG14 (4–6, 48, 49), for example, could be a result of a need for a finely tuned tRNAPhe-dependent frameshift potential in these organisms.

The general mechanism of –1 PRF is thought to reflect ribosome pausing by cis-acting elements of the mRNA that induce a –1 shift. Based on in vitro studies, frameshifting at a particular site has been proposed to be determined at least in part by the strength of interaction of the tRNA anticodon with the A-site codon before tRNA slippage (43). According to this hypothesis, if the codon-anticodon interaction in the ribosomal A site before tRNA slippage is relatively weak, then slippage is more likely to occur. Indeed, we observed in vivo a decrease in frameshifting efficiency upon switching the A-site codon triplet from UUU to the stronger pairing UUC (Fig. 4A). But we cannot determine whether, from the in vivo analysis, the effects of a codon difference on frameshift efficiency are under kinetic (transition state) or thermodynamic control. The in vivo data in general do not address whether the effects of the modification state on frameshift efficiency are from kinetic or thermodynamic parameters. This question is of some interest because a potentially related phenomena, translational fidelity, is generally thought to depend primarily on kinetic rather than thermodynamic discrimination (50).

To understand better the thermodynamic side, we used poly(U) as the mRNA for studying in vitro the A-site binding of three different tRNAPhe modified at position 37. In these studies we found a consistently stronger binding of m1G and imG14-modified substructures compared with the wild-type, fully modified structure (Table 2). Also, the m1G and imG14-modified substructures are more efficient in frameshifting. If thermodynamics determined the efficiency of frameshifting, then we would expect tighter A-site binding to yield less frameshifting (because the bound tRNA would have more trouble "slipping" ahead). This lack of correlation of binding in vitro with frameshifting in vivo is consistent with the idea that the results in vivo are determined more by kinetic considerations. Indeed, whereas m1G37-tRNAPhe is more active than imG14-37 tRNAPhe in frameshifting (Figs. 3 and 4), no significant difference could be seen between the two tRNAs in A-site mRNA binding in vitro (Table 2). Thus, if frameshifting is viewed as a specific example of a perturbation of translational fidelity, then our data can be harmonized with the idea that fidelity is kinetically determined.

Although our experiments show clearly that wyosine base modifications can modulate frameshifting, we do not know whether frameshifting per se is the driving force in evolution for the strong preservation of these modifications or whether frameshifting is secondary to a more fundamental role for these modifications. Because of the strong role of frameshifting in determining the viability of specific infectious viruses in eukaryotes, the selective pressure is obvious in these circumstances (virus-infected cells). Less clear is the role for frameshifting and position 37 modifications of tRNAPhe in non-virus-infected cells. However, it is becoming increasingly apparent that PRF is not limited to virus-infected cells but is more widespread and is likely employed by many organisms. Thus, frameshifting is used for expression of the E. coli (and certain other bacteria) dnaX gene, encoding two subunits of DNA polymerase III, i.e. {tau} and {gamma} (51). Frameshifting occurs with 50% efficiency at an A AAA AAG slippery site, which encodes for A- and P-site tRNALys containing the hypermodified nucleotide t6A at position 37. Recently, computational analyses revealed that functional –1 PRF signals are widespread in the S. cerevisiae genome. Confirming the analyses, selected tested putative –1 PRF signals promoted efficient frameshifting in vivo (52). Interestingly, the majority of –1 PRF events would direct translating ribosomes to premature stop codons, suggesting that in yeast PRF could be used to post-transcriptionally regulate gene expression through nonsense-mediated decay (52).


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM23562, a grant from the National Foundation for Cancer Research, and a Ruth L. Kirschstein National Research Service Award from the National Institutes of Health (to W. F. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8970; Fax: 858-784-8990; E-mail: schimmel{at}scripps.edu.

3 The abbreviations used are: yW, wyebutosine; m1G, N1-methylguanosine; imG14, 4-demethylwyosine; wt, wild type; PRF, programmed ribosomal frameshifting; HPLC, high performance liquid chromatography. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jonathan Dinman and Dr. Ewan Plant for providing –1 PRF reporter constructs and Dr. Jason Harger for helpful discussions and encouragement. We are grateful to the laboratory of Dr. Gerald Edelman for providing access to the MicroLumant luminometer.



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