Special peptidyl-tRNA molecules can promote translational frameshifting without slippage.

Recently we described an unusual programmed +1 frameshift event in yeast retrotransposon Ty3. Frameshifting depends on the presence of peptidyl-tRNA(AlaCGC) on the GCG codon in the ribosomal P site and on a translational pause stimulated by the slowly decoded AGU codon. Frameshifting occurs on the sequence GCG-AGU-U by out-of-frame binding of a valyl-tRNA to GUU without slippage of peptidyl-tRNA(AlaCGC). This mechanism challenges the conventional understanding that frameshift efficiency must correlate with the ability of mRNA-bound tRNA to slip between cognate or near-cognate codons. Though frameshifting does not require slippery tRNAs, it does require special peptidyl-tRNAs. We show that overproducing a second isoacceptor whose anticodon had been changed to CGC eliminated frameshifting; peptidyl-tRNA(AlaCGC) must have a special capacity to induce +1 frameshifting in the adjacent ribosomal A site. In order to identify other special peptidyl-tRNAs, we tested the ability of each of the other 63 codons to replace GCG in the P site. We found no correlation between the ability to stimulate +1 frameshifting and the ability of the cognate tRNA to slip on the mRNA--several codons predicted to slip efficiently do not stimulate frameshifting, while several predicted not to slip do stimulate frameshifting. By inducing a severe translational pause, we identified eight tRNAs capable of inducing measurable +1 frameshifting, only four of which are predicted to slip on the mRNA. We conclude that in Saccharomyces cerevisiae, special peptidyl-tRNAs can induce frameshifting dependent on some characteristic(s) other than the ability to slip on the mRNA.     

Pulling the ribosome out of frame by +1 at a programmed frameshift site by cognate binding of aminoacyl-tRNA.

Programmed translational frameshifts efficiently alter a translational reading frame by shifting the reading frame during translation. A +1 frameshift has two simultaneous requirements: a translational pause which occurs when either an inefficiently recognized sense or termination codon occupies the A site, and the presence of a special peptidyl-tRNA occupying the P site during the pause. The special nature of the peptidyl-tRNA reflects its ability to slip +1 on the mRNA or to facilitate binding of an incoming aminoacyl-tRNA out of frame in the A site. This second mechanism suggested that in some cases the first +1 frame tRNA could have an active role in frameshifting. We found that overproducing this tRNA can drive frameshifting, surprisingly regardless of whether frameshifting occurs by peptidyl-tRNA slippage or out-of-frame binding of aminoacyl-tRNA. This finding suggests that in both cases, the shift in reading frame occurs coincident with formation of a cognate codon-anticodon interaction in the shifted frame.     

Predicting ribosomal frameshifting efficiency

Many retroviruses use -1 ribosomal frameshifting as part of the mechanism in translational control of viral protein synthesis. Quantitative prediction of the efficiency of -1 frameshifting is crucial for understanding the viral gene expression. Here we investigate the free energy landscape for a minimal -1 programmed ribosomal frameshifting machinery, including the codon-anticodon base pairs at the slippery site, the downstream messenger RNA structure and the spacer between the slippery site and the downstream structure. The free energy landscape analysis leads to a quantitative relationship between the frameshifting efficiency and the tension force generated during the movement of codon-anticodon complexes, which may occur in the A/T to A/A accommodation process or the translocation process. The analysis shows no consistent correlation between frameshifting efficiency and global stability of the downstream mRNA structure.     

Nonsense suppressor and antisuppressor mutations at the 1409-1491 base pair in the decoding region of Escherichia coli 16S rRNA.

Using a genetic selection for suppressors of a UGA nonsense mutation in trpA, we have isolated a G to A transition mutation at position 1491 in the decoding region of 16S rRNA. This suppressor displayed no codon specificity, suppressing UGA, UAG and UAA nonsense mutations and +1 and -1 frameshift mutations in lacZ. Subsequent examination of a series of mutations at G1491 and its base-pairing partner C1409 revealed various effects on nonsense suppression and frameshifting. Mutations that prevented Watson-Crick base pairing between these residues were observed to increase misreading and frameshifting. However, double mutations that retained pairing potential produced an antisuppressor or hyperaccurate phenotype. Previous studies of antibiotic resistance mutations and antibiotic and tRNA footprints have placed G1491 and C1409 near the site of codon-anticodon pairing. The results of this study demonstrate that the nature of the interaction of these two residues influences the fidelity of tRNA selection.     

Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit

During translation, some +1 frameshift mRNA sites are decoded by frameshift suppressor tRNAs that contain an extra base in their anticodon loops. Similarly engineered tRNAs have been used to insert nonnatural amino acids into proteins. Here, we report crystal structures of two anticodon stem-loops (ASLs) from tRNAs known to facilitate +1 frameshifting bound to the 30S ribosomal subunit with their cognate mRNAs. ASL(CCCG) and ASL(ACCC) (5'-3' nomenclature) form unpredicted anticodon-codon interactions where the anticodon base 34 at the wobble position contacts either the fourth codon base or the third and fourth codon bases. In addition, we report the structure of ASL(ACGA) bound to the 30S ribosomal subunit with its cognate mRNA. The tRNA containing this ASL was previously shown to be unable to facilitate +1 frameshifting in competition with normal tRNAs (Hohsaka et al. 2001), and interestingly, it displays a normal anticodon-codon interaction. These structures show that the expanded anticodon loop of +1 frameshift promoting tRNAs are flexible enough to adopt conformations that allow three bases of the anticodon to span four bases of the mRNA. Therefore it appears that normal triplet pairing is not an absolute constraint of the decoding center.     

Genome Expansion by tRNA +1 Frameshifting at Quadruplet Codons

Inducing tRNA +1 frameshifting to read a quadruplet codon has the potential to incorporate a non-canonical amino acid (ncAA) into the polypeptide chain. While this strategy is attractive for genome expansion in biotechnology and bioengineering endeavors, improving the yield is hampered by a lack of understanding of where the shift can occur in an elongation cycle of protein synthesis. Lacking a clear answer to this question, current efforts have focused on designing +1-frameshifting tRNAs with an extra nucleotide inserted to the anticodon loop for pairing with a quadruplet codon in the aminoacyl-tRNA binding (A) site of the ribosome. However, the designed and evolved +1-frameshifting tRNAs vary broadly in achieving successful genome expansion. Here we summarize recent work on +1-frameshifting tRNAs. We suggest that, rather than engineering the quadruplet anticodon-codon pairing scheme at the ribosome A site, efforts should be made to engineer the pairing scheme at steps after the A site, including the step of the subsequent translocation and the step that stabilizes the pairing scheme in the +1-frame in the peptidyl-tRNA binding (P) site.     

Prokaryotic-style frameshifting in a plant translation system: conservation of an unusual single-tRNA slippage event

Ribosomal frameshifting signals are found in mobile genetic elements, viruses and cellular genes of prokaryotes and eukaryotes. Typically they comprise a slippery sequence, X XXY YYZ, where the frameshift occurs, and a stimulatory mRNA element. Here we studied the influence of host translational environment and the identity of slippery sequence-decoding tRNAs on the frameshift mechanism. By expressing candidate signals in Escherichia coli, and in wheatgerm extracts depleted of endogenous tRNAs and supplemented with prokaryotic or eukaryotic tRNA populations, we show that when decoding AAG in the ribosomal A-site, E.coli tRNA(Lys) promotes a highly unusual single-tRNA slippage event in both prokaryotic and eukaryotic ribosomes. This event does not appear to require slippage of the adjacent P-site tRNA, although its identity is influential. Conversely, asparaginyl-tRNA promoted a dual slippage event in either system. Thus, the tRNAs themselves are the main determinants in the selection of single- or dual-tRNA slippage mechanisms. We also show for the first time that prokaryotic tRNA(Asn) is not inherently 'unslippery' and induces efficient frameshifting when in the context of a eukaryotic translation system.     

Programmed translational -1 frameshifting on hexanucleotide motifs and the wobble properties of tRNAs

Programmed -1 ribosomal frameshifting, involving tRNA re-pairing from an AAG codon to an AAA codon, has been reported to occur at the sequences CGA AAG and CAA AAG. In this study, using the recoding region of insertion sequence IS3, we have investigated the influence on frameshifting in Escherichia coli of the first codon of this type of motif by changing it to all other NNA codons. Two classes of NNA codons were distinguished, depending on whether they favor or limit frameshifting. Their degree of shiftiness is correlated with wobble propensity, and base 34 modification, of their decoding tRNAs. A more flexible anticodon loop very likely makes the tRNAs with extended wobble more prone to liberate the third codon base, A, for re-pairing of tRNALys in the -1 frame.     

Mutation of a highly conserved base in the yeast mitochondrial 21S rRNA restricts ribosomal frameshifting

A mutation shown to cause resistance to chloramphenicol inSaccharomyces cerevisiae was mapped to the central loop in domain V of the yeast mitochondrial 21S rRNA. The mutant 21S rRNA has a base pair exchange from U₂₆₇₇ (corresponding to U₂₅₀₄ inEscherichia coli) to C₂₆₇₇, which significantly reduces rightward frameshifting at a UU UUU UCC A site in a + 1 U mutant. There is evidence to suggest that this reduction also applies to leftward frameshifting at the same site in a – 1 U mutant. The mutation did not increase the rate of misreading of a number of mitochondrial missense, nonsense or frameshift (of both signs) mutations, and did not adversely affect the synthesis of wild-type mitochondrial gene products. It is suggested here that ribosomes bearing either the C₂₆₇₇ mutation or its wild-type allele may behave identically during normal decoding and only differ at sites where a ribosomal stall, by permitting non-standard decoding, differentially affects the normal interaction of tRNAs with the chloramphenicol resistant domain V. Chloramphenicol-resistant mutations mapping at two other sites in domain V are described. These mutations had no effect on frameshifting.     

Analysis of tetra- and hepta-nucleotides motifs promoting -1 ribosomal frameshifting in Escherichia coli

Programmed ribosomal -1 frameshifting is a non-standard decoding process occurring when ribosomes encounter a signal embedded in the mRNA of certain eukaryotic and prokaryotic genes. This signal has a mandatory component, the frameshift motif: it is either a Z_ZZN tetramer or a X_XXZ_ZZN heptamer (where ZZZ and XXX are three identical nucleotides) allowing cognate or near-cognate repairing to the -1 frame of the A site or A and P sites tRNAs. Depending on the signal, the frameshifting frequency can vary over a wide range, from less than 1% to more than 50%. The present study combines experimental and bioinformatics approaches to carry out (i) a systematic analysis of the frameshift propensity of all possible motifs (16 Z_ZZN tetramers and 64 X_XXZ_ZZN heptamers) in Escherichia coli and (ii) the identification of genes potentially using this mode of expression amongst 36 Enterobacteriaceae genomes. While motif efficiency varies widely, a major distinctive rule of bacterial -1 frameshifting is that the most efficient motifs are those allowing cognate re-pairing of the A site tRNA from ZZN to ZZZ. The outcome of the genomic search is a set of 69 gene clusters, 59 of which constitute new candidates for functional utilization of -1 frameshifting.     

Anticodons, frameshifts, and hidden periodicities in tRNA sequences

Fourier analysis of the short-range periodicities for the complete set of sequences coding for tRNA genes in genome of Bacillus subtilis proves that periodicities with periods p = 2, 3, 4, and 6 sites are the inherent properties of tRNAs. The related periodicities should be understood in a broad statistical sense and their identifying needs the elaborate statistical methods. To improve the statistics, the analysis of significant periodicities was performed for the binary R-Y, S-W, and K-M sequences. Generally, such short-range periodicities are produced via biased positioning of particular nucleotides rather than via the tandem multiplication and subsequent modifications of repeats, though the latter mechanism may also be realized. Quasi-coherently piercing long segments of tRNA, the short-range periodicities create the effective long-range structural coupling between the acceptor stem and the anticodon loop and may participate in the mechanisms of molecular recognition. The periodicities with p = 2 and 4 provide the natural ground for the translation with spontaneous or programmed frameshifting and are present in tRNAs decoding the most frameshift-prone codons. The observation of short-range periodicities suggests that the mechanisms of amino-acylation of tRNAs and codon-anticodon pairing are not independent. Their study may also provide the important information related to the origin and evolution of the genetic code.     

Recognition and positioning of mRNA in the ribosome by tRNAs with expanded anticodons

Mutant tRNAs containing an extra nucleotide in the anticodon loop are known to suppress +1 frameshift mutations, but in no case has the molecular mechanism been clarified. It has been proposed that the expanded anticodon pairs with a complementary mRNA sequence (the frameshift sequence) in the A site, and this quadruplet "codon-anticodon" helix is translocated to the P site to restore the correct reading frame. Here, we analyze the ability of tRNA analogs containing expanded anticodons to recognize and position mRNA in ribosomal complexes in vitro. In all cases tested, 8 nt anticodon loops position the 3' three-quarters of the frameshift sequence in the P site, indicating that the 5' bases of the expanded anticodon (nucleotides 33.5, 34, and 35) pair with mRNA in the P site. We also provide evidence that four base-pairs can form between the P-site tRNA and mRNA, and the fourth base-pair involves nucleotide 36 of the tRNA and lies toward (or in) the 30 S E site. In the A site, tRNA analogs with the expanded anticodon ACCG are able to recognize either CGG or GGU. These data imply a flexibility of the expanded anticodon in the A site. Recognition of the 5' three-quarters of the frameshift sequence in the A site and subsequent translocation of the expanded anticodon to the P site results in movement of mRNA by four nucleotides, explaining how these tRNAs can change the mRNA register in the ribosome to restore the correct reading frame.     

Evolution of +1 Programmed Frameshifting Signals and Frameshift-Regulating tRNAs in the Order Saccharomycetales

Programmed translational frameshifting is a ubiquitous but rare mechanism of gene expression in which mRNA sequences cause the translational machinery to shift reading frames with extreme efficiency, up to at least 50%. The mRNA sequences responsible are deceptively simple; the sequence CUU-AGG-C causes about 40% frameshifting when inserted into an mRNA in the yeast Saccharomyces cerevisiae. The high efficiency of this site depends on a set of S. cerevisiae tRNA isoacceptors that perturb the mechanism of translation to cause the programmed translational error. The simplicity of the system might suggest that it could evolve frequently and perhaps be lost as easily. We have investigated the history of programmed +1 frameshifting in fungi. We find that frameshifting has persisted in two structural genes in budding yeasts, ABP140 and EST3 for about 150 million years. Further, the tRNAs that stimulate the event are equally old. Species that diverged from the lineage earlier both do not employ frameshifting and have a different complement of tRNAs predicted to be inimical to frameshifting. The stability of the coevolution of protein coding genes and tRNAs suggests that frameshifting has been selected for during the divergence of these species. [Reviewing Editor: Dr. Niles Lehman]     

Eight new mtDNA sequences of glass sponges reveal an extensive usage of + 1 frameshifting in mitochondrial translation

Three previously studied mitochondrial genomes of glass sponges (phylum Porifera, class Hexactinellida) contained single nucleotide insertions in protein coding genes inferred as sites of + 1 translational frameshifting. To investigate the distribution and evolution of these sites and to help elucidate the mechanism of frameshifting, we determined eight new complete or nearly complete mtDNA sequences from glass sponges and examined individual mitochondrial genes from three others. We found nine new instances of single nucleotide insertions in these sequences and analyzed them both comparatively and phylogenetically. The base insertions appear to have been gained and lost repeatedly in hexactinellid mt protein genes, suggesting no functional significance for the frameshifting sites. A high degree of sequence conservation, the presence of unusual tRNAs, and a distinct pattern of codon usage suggest the “out-of-frame pairing” model of translational frameshifting. Additionally, we provide evidence that relaxed selection pressure on glass sponge mtDNA – possibly a result of their low growth rates and deep-water lifestyle – has allowed frameshift insertions to be tolerated for hundreds of millions of years. Our study provides the first example of a phylogenetically diverse and extensive usage of translational frameshifting in animal mitochondrial coding sequences.     

An extended signal involved in eukaryotic -1 frameshifting operates through modification of the E site tRNA

By using a sensitive search program based on hidden Markov models (HMM), we identified 74 viruses carrying frameshift sites among 1500 fully sequenced virus genomes. These viruses are clustered in specific families or genera. Sequence analysis of the frameshift sites identified here, along with previously characterized sites, identified a strong bias toward the two nucleotides 5' of the shifty heptamer signal. Functional analysis in the yeast Saccharomyces cerevisiae demonstrated that high frameshifting efficiency is correlated with the presence of a Psi39 modification in the tRNA present in the E site of the ribosome at the time of frameshifting. These results demonstrate that an extended signal is involved in eukaryotic frameshifting and suggest additional interactions between tRNAs and the ribosome during decoding.     

rRNA-mRNA base pairing stimulates a programmed -1 ribosomal frameshift.

Base pairing between the 3' end of 16S rRNA and mRNA is shown to be important for the programmed -1 frameshifting utilized in decoding the Escherichia coli dnaX gene. This pairing is the same as the Shine-Dalgarno pairing used by prokaryotic ribosomes in selection of translation initiators, but for frameshifting the interaction occurs within elongating ribosomes. For dnaX -1 frameshifting, the 3' base of the Shine-Dalgarno sequence is 10 nucleotides 5' of the shift site. Previously, Shine-Dalgarno rRNA-mRNA pairing was shown to stimulate the +1 frameshifting necessary for decoding the release factor 2 gene. However, in the release factor 2 gene, the Shine-Dalgarno sequence is located 3 nucleotides 5' of the shift site. When the Shine-Dalgarno sequence is moved to the same position relative to the dnaX shift site, it is inhibitory rather than stimulatory. Shine-Dalgarno interactions by elongating ribosomes are likely to be used in stimulating -1 frameshifting in the decoding of a variety of genes.     

Destabilization of codon-anticodon interaction in the ribosomal exit site

The affinities of the exit (E) site of poly(U) or poly(A)-programmed Escherichia coli ribosomes for the respective cognate tRNA and a number of non-cognate tRNAs were determined by equilibrium titrations. Among the non-cognate tRNAs, the binding constants vary up to about tenfold (10(6) to 10(7) M-1 at 20 mM-Mg2+) or 50-fold (10 mM-Mg2+), indicating that codon-independent binding is modulated to a considerable extent by structural elements of the tRNA molecules other than the anticodon. Codon-anticodon interaction stabilizes tRNA binding in the E site approximately fourfold (20 mM-Mg2+) or 20-fold (10 mM-Mg2+), corresponding to delta G degree values of -3 and -7 kJ/mol (0.7 and 1.7 kcal/mol), respectively. Thus, the energetic contribution of codon-anticodon interaction to tRNA binding in the E site appears rather small, particularly in comparison to the large effects on the binding in A and P sites and to the binding of complementary oligonucleotides or of tRNAs with complementary anticodons. This result argues against a role of the E site-bound tRNA in the fixation of the mRNA on the ribosome. In contrast, we propose that the role of the E site is to facilitate the release of the discharged tRNA during translocation by providing an intermediate, labile binding site for the tRNA leaving the P site. The lowering of both affinity and stability of tRNA binding accompanying the transfer of the tRNA from the P site to the E site is predominantly due to the labilization of the codon-anticodon interaction.     

A twisted tRNA intermediate sets the threshold for decoding

Putting together consistent cryo-EM structure, transient kinetic and mutant tRNA suppressor data, it appears that a deformed or waggling aminoacyl-tRNA is the critical transitional structure examined by the ribosome during decoding at the A site. The unusual conformation may be required for effective proofreading of the codon-anticodon complex.     

Adjacent codon-anticodon interactions of both tRNAs present at the ribosomal A and P or P and E sites

A labeled tRNA present at the A, P or E site can be partially chased from the ribosome, a cognate nonlabeled tRNA as chasing substrate being 3-12-times more efficient than non-cognate tRNA at a molar ratio tRNA: 70 S = 10:1. These findings indicate that a tRNA bound to a programmed ribosome undergoes codon-anticodon interaction at all three sites (A, P and E site). Furthermore, both labeled tRNA present on the ribosome can be chased more effectively with cognate than with non-cognate substrate at the same time. This finding provides strong evidence that both tRNAs present on the ribosome exhibit simultaneous codon-anticodon interaction. This is valid for both the pretranslocational state (Ac[3H]Lys-tRNALys in the A and [14C]tRNALys in the P site) as well as the posttranslocational state (Ac[3H]Lys-tRNALys in the P and [14C]tRNALys in the E site).     

Maintaining the ribosomal reading frame: the influence of the E site during translational regulation of release factor 2

Maintenance of the translation reading frame is one of the most remarkable achievements of the ribosome while decoding the information of an mRNA. Loss of the reading frame through spontaneous frameshifting occurs with a frequency of one in 30,000 amino acid incorporations. However, at many recoding sites, the mechanism that controls reading frame maintenance is switched off. One such example is the programmed +1 frameshift site of the prfB gene encoding the termination factor RF2, in which slippage into the forward frame by one nucleotide can attain an efficiency of approximately 100%, namely, four orders of magnitude higher than normally observed. Here, using the RF2 frameshift window, we demonstrate that premature release of the E site tRNA from the ribosome is coupled with high-level frameshifting. Consistently, in a minimal system, the presence of the E site tRNA prevents the +1 frameshift event, illustrating the importance of the E site for reading-frame maintenance.