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The determinants of mRNA stability include specific cis-acting destabilizing sequences located within mRNA coding and noncoding regions. We have developed an approach for mapping coding-region instability sequences in unstable yeast mRNAs that exploits the link between mRNA translation and turnover and the dependence of nonsense-mediated mRNA decay on the activity of the UPF1 gene product. This approach, which involves the systematic insertion of in-frame translational termination codons into the coding sequence of a gene of interest in a upf1delta strain, differs significantly from conventional methods for mapping cis-acting elements in that it causes minimal perturbations to overall mRNA structure. Using the previously characterized MATalpha1 mRNA as a model, we have accurately localized its 65-nucleotide instability element (IE) within the protein coding region. Termination of translation 5' to this element stabilized the MATalpha1 mRNA two- to threefold relative to wild-type transcripts. Translation through the element was sufficient to restore an unstable decay phenotype, while internal termination resulted in different extents of mRNA stabilization dependent on the precise location of ribosome stalling. Detailed mutagenesis of the element's rare-codon/AU-rich sequence boundary revealed that the destabilizing activity of the MATalpha1 IE is observed when the terminal codon of the element's rare-codon interval is translated. This region of stability transition corresponds precisely to a MATalpha1 IE sequence previously shown to be complementary to 18S rRNA. Deletion of three nucleotides 3' to this sequence shifted the stability boundary one codon 5' to its wild-type location. Conversely, constructs containing an additional three nucleotides at this same location shifted the transition downstream by an equivalent sequence distance. Our results suggest a model in which the triggering of MATalpha1 mRNA destabilization results from establishment of an interaction between translating ribosomes and a downstream sequence element. Furthermore, our data provide direct molecular evidence for a relationship between mRNA turnover and mRNA translation.

Double-stranded Leishmania RNA virus 1-4 (LRV 1-4) has at least four open reading frames (ORFs). The two small ORFs located near its 5' terminus, ORF1 and ORFx, could encode 34- and 60-amino acid polypeptides, respectively. ORF2 encodes an 82-kDa major capsid protein, and ORF3 encodes a 98-kDa polypeptide which contains the consensus sequence for RNA-dependent RNA polymerases of plus-strand and double-stranded RNA viruses. The complete sequence of LRV 1-4 shows that ORF2 and ORF3 overlap by 71 nucleotides, and that ORF3 lacks a potential translation initiation site, suggesting that the viral polymerase may be synthesized as a 180-kDa fusion protein with the virus capsid. In this report, we present evidence for the synthesis of a fusion protein through a ribosomal frameshift. In vitro-translation experiments and immunostudies involving antiserum against the viral capsid protein demonstrated that the overlapping 71 nucleotides of ORF2 and ORF3 are contained in a region which promotes translational frameshifting. Computer analysis of the putative frameshift region revealed a potential pseudoknot structure located within the overlapping 71 nucleotide sequence.

Retroviruses employ translation frameshift for gene expression. Translation frameshift takes place at the slippery heptanucleotide sequence on mRNA. A downstream stem-loop structure (and a pseudoknot formed by the stem-loop) enhances the level of ribosomal frameshifting. Here, we describe that an upstream termination codon suppresses the ribosomal frameshifting, leading to reduction in the expression of downstream genes.

INFECTIOUS ELEMENTS OF SACCHAROMYCES CEREVISIAE 250 BIOLOGY OF THE YEAST dsRNA VIRUSES AND THE KILLER PHENOMENON 250 L-A VIRUS STRUCTURE: T 5 1 WITH 60 ASYMMETRIC Gag DIMERS 251 VIRAL REPLICATION CYCLES 251 L-A ENCODES Gag AND Gag-Pol 252 REPLICATION AND TRANSCRIPTION OF VIRAL RNA: IN VITRO SYSTEMS 254 TRANSLATION OF VIRAL mRNA 254 The SKI2,3,8 System Blocks the Translation of Non-Poly(A) mRNA 255 M1 Propagation Depends Critically on Free 60S Ribosomal Subunit Levels 256 Do SKI2, 3, and 8 Determine 60S Subunit Interaction with Poly(A)? 257 Gag Makes Decapitated Decoys To Distract the SKI1/XRN1 Exoribonuclease 257 Lethality of ski1 ski2 and ski1 ski3 double mutants 258 Gag-Pol Fusion Protein Formed by a 21 Ribosomal Frameshift: How and Why 258 Mechanism of 21 ribosomal frameshifting 258 How critical is the efficiency of frameshifting? 259 Chromosomal genes affecting the efficiency of frameshifting 259 Can 21 ribosomal frameshifting be used as a target of antiviral drugs? 259 POSTTRANSLATIONAL PROCESSING 259 MAK3 N-Acetyltransferase Modification of Gag Is Necessary for Assembly 259 Killer Preprotoxin Is Processed To Form Mature Toxin 260 KEX1 and KEX2 Processing Proteases and Mammalian Prohormone Processing 260 RNA PACKAGING: IN VITRO AND IN VIVO 260 Evidence for cis Packaging by L-A 260 Does Packaging Control Translation? 260 L-BC IS CLOSELY RELATED TO L-A 261 CONCLUSIONS AND PROSPECTS 261 REFERENCES 262

Members of the Paramyxoviridae family utilize a variety of different strategies to increase coding capacity within their P cistrons. Translation initiation at alternative 5'-proximal AUG codons is used by measles virus (MV) to express the virus-specific P and C proteins from overlapping reading frames on their mRNAs. Additional species of mRNAs are transcribed from the MV P cistron by the insertion of extra nontemplated G residues at a specific site within the P transcript. Addition of only a single nontemplated G residue results in the expression of the V protein, which contains a unique carboxyl terminus. We have used an Escherichia coli system to express MV P cistron-related mRNAs and proteins. We have found that ribosomal frameshifting on the MV P protein mRNA is capable of generating a previously unrecognized P cistron-encoded protein that we have designated R. Some ribosomes which have initiated translation of the P protein mRNA use the sequence TCC CCG AG (24 nucleotides upstream of the V protein stop codon) to slip into the -1 reading frame, thus translating the sequence as TC CCC GAG. The resulting R protein terminates five codons downstream of the frameshift site at the V protein stop codon. We have gone on to use a chloramphenicol acetyltransferase reporter system to demonstrate that this MV-specific sequence is capable of directing frameshifting during in vivo translation in eukaryotic cells. Analysis of immunoprecipitated proteins from MV-infected cells by two-dimensional gel electrophoresis allowed detection of a protein species consistent with R protein in MV-infected cells. Quantitation of this protein species allowed a rough estimation of frameshift frequency of approximately 1.8%. Significant stimulation of ribosomal frameshift frequency at this locus of the MV P mRNA was mediated by a downstream stimulator element which, although not yet fully defined, appeared to be neither a conventional stem-loop nor an RNA pseudoknot structure.

Introduction. Accurate maintenance of the translational reading frame by ribosomes is essential for the production of functional proteins and unsurprisingly, errors in frame maintenance have been estimated to occur at rates probably lower than 5 × 10-5 per codon (Kurland, 1992). However, an increasing number of examples have been documented where a purposeful shift in reading frame is programmed into the mRNA and serves an essential function (Gesteland et al., 1992; Atkins & Gesteland, 1995). Highly efficient ribosomal frameshifting is an example of such a programmed frameshift site. In response to certain signals in the mRNA, ribosomes are induced to move into the -1 reading frame (in a 5′ direction) at a specific point and continue translation in the new reading frame. The phenomenon was first described in 1985 as the way in which the Gag-Pol polyprotein of the retrovirus Rous sarcoma virus (RSV) is expressed from the overlapping gag and pol open reading frames (ORFs) (Jacks & Varmus, 1985) and to date, most examples of frameshifting come from virus systems.

The mechanism favoured for -1 frameshifting at typical retroviral sites is a pre-translocation simultaneous slippage model. An alternative post-translocation mechanism would also generate the same protein sequence across the frameshift site and therefore in this study the strategic placement of a stop codon has been used to distinguish between the two mechanisms. A 26 base pair frameshift sequence from the HIV-1 gag-pol overlap has been modified to include a stop codon immediately 3' to the heptanucleotide frameshift signal, where it often occurs naturally in retroviral recoding sites. Stop codons at the 3'-end of the heptanucleotide sequence decreased the frame-shifting efficiency on prokaryote ribosomes and the recording event was further depressed when the levels of the release factors in vivo were increased. In the presence of elevated levels of a defective release factor 2, frameshifting efficiency in vivo was increased in the constructs containing the stop codons recognized specifically by that release factor. These results are consistent with the last six nucleotides of the heptanucleotide slippery sequence occupying the ribosomal E and P sites, rather than the P and A sites, with the next codon occupying the A site and therefore with a post-translocation rather than a pre-translocation -1 slippage model.

A single-base-pair deletion in exon 7 of the human carbonic anhydrase II gene was found to be the molecular defect in a group of independently ascertained, clinically heterogeneous, Hispanic carbonic anhydrase II-deficient patients, all of whom had ancestors from the Caribbean islands. This mutation predicts a +1 frameshift at codon 227 and incorporation of 12 missense amino acids before an early stop codon at position 239 produces a 27-kDa truncated carbonic anhydrase II. Expression of the Hispanic mutant cDNA in bacteria produced predominantly the 27-kDa protein, which was inactive. However, a minor 29-kDa polypeptide species was also produced that had 10% the specific activity of the wild-type enzyme after affinity purification. Amino acid sequencing showed that the 29-kDa mutant protein was produced by two frameshift events: a +1 frameshift at codon 227 due to the single-base deletion and a -1 ribosomal frameshift at codon 237 that restored the original reading frame after 11 missense amino acids were incorporated. Antibody against the 11-amino acid frameshift peptide detected the 29-kDa mutant protein in lysates of transfected COS cells. These results indicate that ribosomal frameshift can partially rescue the human carbonic anhydrase II frameshift mutation and suggest a mechanism whereby a compensatory ribosomal frameshift can ameliorate the consequences of certain frameshift mutations. Whether individual differences in efficiency of ribosomal frameshift contribute to clinical heterogeneity in patients with such mutations deserves further study.

The pathogenicity of influenza A viruses is a multigenic trait, which is orchestrated by the global networks between eight viral genomic constituents and their cellular interacting partners. A recent report provided information on the finding of a new PA ribosomal frameshifting product, the PA-X protein, in the influenza A virus segment 3, and an endonuclease property was suggested for a possible role of the PA-X protein. In cultured cells, viral growth was not affected by the PA-X protein expression. However, the reduced pathogenicity of mice appeared to be closely associated with the PA-X protein expression. It was also revealed that the PA-X protein was able to modulate host gene expression. Considered together, the PA-X protein can be a cellular signaling modulator and subsequently control viral pathogenicity. By reviewing recent publications, we present new insights in the contribution of the PA-X protein to the cellular signaling network and the resultant viral pathogenicity.

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.

A ribosomal frameshift at the gag-pro junction of mouse mammary tumor virus (MMTV) gives rise to the protein p30. The protein consists of two domains, the zinc-finger-containing nucleocapsid (NC) protein portion with 95 residues and a C-terminal extension comprising 154 residues. The C-terminal domain shows similarity in sequence with the enzyme dUTPase from other sources. In this paper, we demonstrate that p30 is a functional dUTPase. Overproduction of the NC protein in Escherichia coli, using the native frameshift sequence at the gag stop codon, caused a detectable expression of dUTPase ascribed to a low frequency of readthrough. By a 1-base insertion, eliminating the gag stop codon and fusing the gag and pro reading frames, a plasmid, pET-3d-NCDU, directing overexpression of p30, was constructed. The overproduced protein, purified by phosphocellulose chromatography, shows both zinc-binding and dUTPase activity. Analytical gel filtration and sequence homology to other dUTPases suggest a trimeric assembly of p30 subunits. MMTV thus possesses two different forms of the nucleocapsid protein, the ordinary NC protein and the p30, having the NC protein connected to a domain of dUTPase.

We have analyzed in cell culture the sequence elements that control the level of ribosomal frameshifting in the human T-cell leukemia virus type II (HTLV-2) gag-pro junction. The slippery sequence of HTLV-2 is sufficient to dictate a basal level of frameshifting. This level is enhanced by its upstream sequence context and by the downstream stem-loop structure which is located at an optimal distance of 7 bases. Frameshifting in human immunodeficiency virus gag-pol is similar to that of HTLV-2 gag-pro. However, experiments using hybrid cassettes of HTLV-2 and human immunodeficiency virus type 1 frameshift elements show that while the slippery sequence of HTLV-2 is less efficient, the stem-loop structure is a more efficient enhancer.

The nucleotide (nt) sequence of the Vibrio cholerae acfD gene (encoding an accessory colonization factor) has been determined. The acfD gene encompasses 254 nt that are predicted to encode an 88-amino-acid (aa) protein. Additionally, an open reading frame of 184 aa, designated orfZ , was detected that overlaps the 3' end of acfD by 45 nt. Computer-assisted homology searches revealed that OrfZ possesses aa sequence similarity to the C terminus of the fliC product of Salmonella muenchen and S. rubislaw . Additionally, OrfZ shares limited aa sequence similarity to a region of the S. typhimurium fliA product. Interestingly, a V. cholerae acfD :: TnphoA mutant demonstrates reduced motility and an altered swarming phenotype in semi-solid media when compared to wild-type V. cholerae . The possibility that the reduced motility and altered morphology result from polar effects on orfZ expression is currently being investigated.

Sendai virus P/C mRNA, human erythrocyte membrane protein 4.1 mRNA and PDGF-B chain mRNA were used to test whether translation initiation from non-AUG codons in COS1 cells was mRNA species dependent. Site-directed mutants of the authentic translation start sites of these mRNAs to alternate start codons showed that while P/C mRNA is capable of initiating translation from non-AUG start sites the other two mRNAs are not. Our study shows that translation initiation from non-AUG codons is mRNA species dependent and suggests that higher order structure of an mRNA determines the non-AUG translation start site.

A frameshift event is necessary for expression of the products of the pol gene in a number of retroviruses, including human immunodeficiency virus type 1 (HIV-1). The basic signals necessary for frameshifting consist of a shifty sequence in which the ribosome slips and a downstream stimulatory structure which can be either a stem-loop or a pseudoknot. In HIV-1, much attention has been paid to the frameshift site itself, and only recently has the role of the downstream structure been examined. Here we used a luciferase-based experimental system to analyze in vivo the cis and trans factors potentially involved in controlling frameshifting efficiency at the gag-pol junction of HIV-1. We demonstrated that high-level frameshifting is dependent on the presence of a palindromic region located downstream of the site where the frameshift event takes place. Frameshifting efficiencies were found to be identical in mouse fibroblasts and the natural host cells of the virus, i.e., CD4+ human lymphoid cells. Furthermore, no increase in frameshifting was observed upon virus infection. Previous observations have shown that viral infection leads to specific alteration of tRNAs involved in translation of shifty sites (D. Hatfield, Y.-X. Feng, B.J. Lee, A. Rein, J.G. Levin, and S. Oroszlan, Virology 173:736-742, 1989). The results presented here strongly suggest that these modifications do not affect frameshifting efficiency.

A special site on the (+) strand of the L-A dsRNA virus induces about 2% of ribosomes translating the gag open reading frame to execute a -1 frameshift and thus produce the viral gag-pol fusion protein. Using constructs in which a -1 ribosomal frameshift at this site was necessary for expression of lacZ we isolated chromosomal mutants in which the efficiency of frameshifting was increased. These mutants comprise eight genes, named mof (maintenance of frame). The mof1-1, mof2-1, mof4-1, mof5-1 and mof6-1 strains cannot maintain M1 dsRNA at 30 degrees, but, paradoxically, do not lose L-A. The mof2-1, mof5-1 and mof6-1 strains are temperature sensitive for growth at 37 degrees, and all three show striking cell cycle phenotypes. The mof2-1 strains arrest with mother and daughter cells almost equal in size, mof5-1 arrests with multiple buds and mof6-1 arrests as single large unbudded cells. mof2-1 and mof5-1 strains are also Pet-. The mof mutations show differential effects on various frameshifting signals.

Two proteins, one of 31 kDa and one of 16 kDa, are encoded by a segment of the phage lambda tail gene region that contains two overlapping reading frames, neither of which is long enough to encode the larger protein. We show that the abundant 16-kDa protein (gpG) is encoded by the upstream open reading frame, gene G. The 31-kDa protein, gpG-T, is encoded jointly by gene G and the overlapping downstream T open reading frame. gpG-T is synthesized as the result of a translational frameshift that occurs when a ribosome translating the G gene slips back by one nucleotide at a position six codons from the C terminus of the gene and thereby bypasses the G termination codon to continue on in the T open reading frame. The resulting protein shares 135 residues of N-terminal amino acid sequence with gpG, followed by 144 amino acid residues of unique sequence. The frameshift event occurs with a frequency of approximately 4% at the sequence G GGA AAG, which encodes the dipeptide -Gly-Lys- in both the zero and -1 reading frames. The frameshift frequencies of point mutants in this "slippery sequence" argue that codon-anticodon interactions with both the glycyl and the lysyl-tRNA are important for frameshifting to occur. We find no clear evidence for a pausing mechanism to enhance frameshifting, as is seen in other well-characterized frameshifts. No simple secondary structure has been predicted for the region downstream from the slippery sequence, but this downstream sequence does contribute to the frameshifting rate. Our results together with those of Katsura and Kuhl show that the frameshift product, gpG-T, has an essential role in lambda tail assembly, acting prior to tail shaft assembly. The role of gpG in tail assembly is not known. We find that both gpG and the gpG-T are absent from mature virions.

Yeast controls propagation of the L-A dsRNA virus, and thus pathogenicity, by partially blocking translation of viral mRNA. L-A makes a Gag-Pol fusion protein by a-1 ribosomal frameshift, regulated by the host but critical for satellite RNA propagation. Discovery of the KEX proteases, by their requirement for killer toxin expression from a satellite dsRNA of L-A, led to the identification of mammalian prohormone processing proteases.

The open reading frame of the human T-cell leukemia virus type II pro gene is arranged at a -1 position relative to the gag gene. Synthesis of the Gag-Pro fusion polyprotein is facilitated by ribosomal frameshift into the reading frame of the pro gene. Cloning of a synthetic 41-bp oligonucleotide corresponding to the gag-pro junction within a heterologous gene (nef of human immunodeficiency virus type I) and mutation analysis revealed that two cis-acting signals, an adenosine residue stretch and a dyad symmetry sequence, flanking the UAA termination codon, are required for efficient ribosomal frameshifting between gag and pro. The stability of the stem-loop structure is crucial for frameshifting.

The expression of the pol gene of human immunodeficiency virus type 1 (HIV-1) occurs by a ribosomal frameshift between the gag and the pol genes. The Gag-Pol polyprotein is produced at levels of 5 to 10% of that of the Gag protein, and is incorporated into virions to provide the viral protease, reverse transcriptase, and integrase which are essential for replication. The mechanism(s) by which the Gag-Pol polyprotein are targeted to the HIV virion is unknown, although it is believed to be via an interaction with the Gag protein. To further explore the mechanism by which the Gag-Pol polyprotein is incorporated into virions, we have constructed a mutation which changes an aspartic acid in the protease active site to asparagine (pHXB2pro-); a four-amino-acid insertion into the protease gene (pHXB2Smal); and insertion of translational termination codons in the protease gene following the gag gene (pHXB55). Transfection of these proviral genomes into COS-1 cells resulted in intracellular expression of only Pr55gag, demonstrating the inactivation of the viral protease. The expression of Pr55gag was evident in cells transfected with pHXB2pro- during a short pulse and first 3 hr of chase period, whereas at later times the intracellular levels of Pr55gag were greatly reduced. In contrast, the intracellular Pr55gag expressed from transfection of pHXB2Smal or pHXB55 were evident even after 6- or 12-hr chase times. To ascertain the effects of the mutations on the assembly and release of viruslike particles, the supernatants from the transfected cells were analyzed for the presence of Pr55gag. The release of Pr55gag from cells transfected with pHXB2pro- occurred as early as 1 hr following chase period, and increased for up to 3 hr. In contrast, reduced levels of Pr55gag were detected in the medium from cells transfected with pHXB2Smal or pHXB55. Subcellular fractionation studies demonstrated that the Pr55gag expressed from transfection of pHXB2pro- was rapidly targeted to intracellular membranes, while the majority of the Pr55gag expressed from transfection of pHXB2Smal or pHXB55 was distributed evenly between the cytoplasm and membrane fractions. Finally, the released viruslike particles obtained from the transfection of proviral genome pHXB2pro- were stable to mild detergent treatment, whereas particles obtained from transfection of pHXB2Smal and pHXB55 were relatively unstable. These results demonstrate that subtle changes in the Gag-Pol polyprotein of HIV-1 can have significant effects on the assembly and physical stability of the released virus.

The putative RNA-dependent RNA polymerase of potato leafroll luteovirus (PLRV) is expressed by -1 ribosomal frameshifting in the region where the open reading frames (ORF) of proteins 2a and 2b overlap. The signal responsible for efficient frameshift is composed of the slippery site UUUAAAU followed by a sequence that has the potential to adopt two alternative folding patterns, either a structure involving a pseudoknot, or a simple stem-loop structure. To investigate the structure requirements for efficient frameshifting, mutants in the stem-loop or in the potential pseudoknot regions of a Polish isolate of PLRV (PLRV-P) have been analyzed. Mutations that are located in the second stem (S2) of the potential pseudoknot structure, but are located in unpaired regions of the alternative stem-loop structure, reduce frameshift efficiency. Deletion of the 3' end sequence of the alternative stem-loop structure does not reduce frameshift efficiency. Our results confirm that -1 frameshift in the overlap region depends on the slippery site and on the downstream positioned sequence, and propose that in PLRV-P a pseudoknot is required for efficient frameshifting. These results are in agreement with those recently published for the closely related beet western yellows luteovirus (BWYV).

We have tested the effect of increased ribosomal fidelity on a modified version of the programmed release factor 2 (RF2) translational frameshift. In the constructs tested, the original UGA codon at the site of the shift was replaced by either of two sense codons, UGG (tryptophan), which allows a frameshift of approximately 13%, or CUG (leucine), which allows a frameshift of only approximately 2%. We confirmed the results of Curran and Yarus [Curran, J. F. & Yarus, M. (1989) J. Mol. Biol. 209, 65-77] in a wild-type ribosomal host, including a reduction of the UGG shift following induction of tRNA(Trp) from a plasmid copy of the tRNA gene. But to our surprise, in a hyperaccurate streptomycin pseudo-dependent host, the UGG frameshift increased to more than 50%. When we added a tRNA(Trp) plasmid to these cells, induction of the tRNA(Trp) gene reduced the shift back to approximately 7%. Messenger RNA levels did not vary greatly under these different induced conditions. Other increased accuracy alleles also showed increased frameshifting with UGG at the frameshift site. All increased accuracy alleles led to slower translation rates, and there appeared to be a proportionality between the extent of reduction of synthesis for the in-frame reporter and the extent of UGG frameshift for the out-of-frame reporter. There were little effects of increased accuracy on the lower level CUG frameshift. However, over-production of the cognate tRNA(1Leu) dramatically reduced even this lower level of shift, despite the fact that tRNA(1Leu) is already the most abundant isoacceptor in Escherichia coli. These results can be rationalized by following the hypothesis of Curran and Yarus as follows: with wild-type ribosomes, limited availability of tRNA(Trp) (about 1% of total tRNA) facilitates a pause at the UGG codon (due to the vacant A site), allowing increased opportunity for ribosome realignment. Excess tRNA(Trp) reduces the time the A site is vacant and thus reduces the frameshift. The slower hyperaccurate ribosomes increase the pause time and thus increase the opportunity for shifting, a process again reversed by increasing the in-frame cognate tRNA(Trp). These data provide strong support for a model in which the extent of ribosome pause time at a programmed frameshift site is a major determinant in the efficiency of the frameshift and in which tRNA availability can be a major influence on this process.

Previous work characterized ribosomal frameshifting within the sequence C UUC AAG provoked by lysyl-tRNA limitation. The ribosome frameshift is one base to the left of the AAG lysine codon, as shown by dotted overlining above. We now show that the frequency of this leftward ribosome frameshift is strongly influenced by the identity of the bases two, three and four positions to the left of the actual frameshift site. The nature of these influences coincides exactly the possibilities of base-pairing between the sequence and the anticodon of the P-site peptidyl-tRNA when shifted one base to the left just upstream of the frameshift site. We conclude that a peptidyl shift in the P-site is intimately involved in leftward frameshifting in the adjacent A site when it codes for an aminoacyl-tRNA in short supply.

The genomic RNA of beet western yellows virus (BWYV) contains a potential translational frameshift signal in the overlap region of open reading frames ORF2 and ORF3. The signal, composed of a heptanucleotide slippery sequence and a downstream pseudoknot, is similar in appearance to those identified in retroviral RNAs. We have examined whether the proposed BWYV signal functions in frameshifting in three translational systems, i.c. in vitro in a reticulocyte lysate or a wheat germ extract and in vivo in E. coli. The efficiency of the signal in the eukaryotic system is low but significant, as it responds strongly to changes in either the slip sequence or the pseudoknot. In contrast, in E. coli there is hardly any response to the same changes. Replacing the slip sequence to the typical prokaryotic signal AAAAAAG yields more than 5% frameshift in E. coli. In this organism the frameshifting is highly sensitive to changes in the slip sequence but only slightly to disruption of the pseudoknot. The eukaryotic assay systems are barely sensitive to changes in either AAAAAAG or in the pseudoknot structure in this construct. We conclude that eukaryotic frameshift signals are not recognized by prokaryotes. On the other hand the typical prokaryotic slip sequence AAAAAAG does not lead to significant frameshifting in the eukaryote. In contrast to recent reports on the closely related potato leafroll virus (PLRV) we show that the frameshifting in BWYV is pseudoknot-dependent.

Previous experiments have shown that limitation for certain aminoacyl-tRNA species results in phenotypic suppression of a subset of frameshift mutant alleles, including members in both the (+) and (−) incorrect reading frames. Here, we demonstrate that such phenotypic suppression can occur through a ribosome reading frame shift at a hungry AAG codon calling for lysyl-tRNA in short supply. Direct amino acid sequence analysis of the product and DNA sequence manipulation of the gene demonstrate that the ribosome frameshift occurs through a movement of one base to the left, so as to decode the triplet overlapping the hungry codon from the left or 5′ side, followed by continued normal translation in the new, shifted reading frame.

The ruvC gene of Escherichia coli specifies a nuclease that resolves Holliday junction intermediates in genetic recombination (B. Connolly, C.A. Parsons, F.E. Benson, H.J. Dunderdale, G.J. Sharples, R.G. Lloyd, and S.C. West, Proc. Natl. Acad, Sci. USA 88:6063-6067, 1991). The gene was located between aspS and the ruvAB operon by DNA sequencing and deletion analysis of ruvC plasmids and was shown to encode a protein of 18,747 Da. Analysis of the DNA flanking ruvC indicated that the gene is transcribed independently of the LexA-regulated ruvAB operon and is not under direct SOS control. ruvC lies downstream of an open reading frame, orf-33, for a protein which migrates during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 33-kDa polypeptide. These two genes probably form an operon. However, expression of ruvC was found to be very poor relative to that of orf-33. A double ribosomal frameshift between these genes is proposed as a possible reason for the low level of RuvC. Two further open reading frames of unknown function were identified, one on either side of the orf-33-ruvC operon.

The signals necessary for the translational frameshift in the gene 10 message of bacteriophage T7 include the previously identified frameshift site and the 3' non-coding region, over 200 bases downstream. The functional components of the frameshift site are identified in this study and show that the site most probably operates by the retroviral type two site mechanism. However, the base pairing requirements for the first tRNA are much more relaxed after the slip than is seen in other examples. The element at the 3' end of the gene, also necessary for frameshifting, is examined but only the extreme 5' side of the transcriptional terminator stem-loop structure in the 3' non-coding region seems to be required. No simple secondary structural model can explain the involvement of this sequence. The T7 frameshift site can be replaced with either a T3 site or a E. coli dnaX site. Both show higher levels of frameshifting than with the T7 site.

The genomic RNA of the coronavirus IBV contains an efficient ribosomal frameshift signal at the junction of the overlapping 1a and 1b open reading frames. The signal is comprised of two elements, a heptanucleotide "slip-site" and a downstream tertiary RNA structure in the form of an RNA pseudoknot. We have investigated the structure of the pseudoknot and its contribution to the frameshift process by analysing the frameshifting properties of a series of pseudoknot mutants. Our results show that the pseudoknot structure closely resembles that which can be predicted from current building rules, although base-pair formation at the region where the two pseudoknot stems are thought to stack co-axially is not a pre-requisite for efficient frameshifting. The stems, however, must be in close proximity to generate a functional structure. In general, the removal of a single base-pair contact in either stem is sufficient to reduce or abolish frameshifting. No primary sequence determinants in the stems or loops appear to be involved in the frameshift process; as long as the overall structure is maintained, frameshifting is highly efficient. Thus, small insertions into the pseudoknot loops and a deletion in loop 2 that reduced its length to the predicted functional minimum did not influence frameshifting. However, a large insertion (467 nucleotides) into loop 2 abolished frameshifting. A simple stem-loop structure with a base-paired stem of the same length and nucleotide composition as the stacked stems of the pseudoknot could not functionally replace the pseudoknot, suggesting that some particular conformational feature of the pseudoknot determines its ability to promote frameshifting.

Normal translation of the gene for E. coli release factor 2 (RF-2) is characterized by a +1 frameshift event that occurs with 30-50% efficiency. Frameshifting on synthetic RF-2 mRNA by eukaryotic ribosomes has also been observed, even though they lack the capability to interact with the frameshift signal in the same manner as prokaryotic ribosomes. We have mutagenized the sequence of the RF-2 gene to eliminate the need for a frameshift, thereby allowing frameshifting efficiency to be measured by direct comparison of RF-2 production from the mutant with production from the wild-type. Measurements using this approach confirm that frameshifting by rabbit reticulocyte lysate ribosomes occurs at the frameshift region, but with a limited efficiency of approximately 0.4%.

Two distinct mRNA transcripts were synthesized in Escherichia coli during expression of FSendA, an endoglucanase gene from Fibrobacter succinogenes AR1. Expression of FSendA required a ribosomal frameshift between open reading frame 1 (ORF1) and ORF2 to allow contiguous translation of a 453 amino acid protein (1). The primary transcript initiated upstream of ORF1 and the secondary transcript from within ORF1. Both transcripts terminated downstream of ORF2 and termination was essential for endoglucanase expression. Deletion of the primary transcript promoter region allowed read-through of the secondary transcript beyond the terminator region, indicating that a component of the intact FSendA gene allowed efficient transcription termination. The possibility of autogenous regulation by translation products is suggested.

The L-A double-stranded RNA (dsRNA) virus of Saccharomyces cerevisiae has two open reading frames (ORFs). ORF1 encodes the 80-kDa major coat protein (gag). ORF2, which is expressed only as a 180-kDa fusion protein with ORF1, encodes a single-stranded RNA-binding domain and has the consensus sequence for RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses (pol). We show that the 180-kDa protein is formed by -1 ribosomal frame-shifting by a mechanism indistinguishable from that of retro-viruses. Analysis of the "slippery site" suggests that a low probability of unpairing of the aminoacyl-tRNA from the 0-frame codon at the ribosomal A site reduces the efficiency of frameshifting more than the reluctance of a given tRNA to have its wobble base mispaired. Frameshifting of L-A requires a pseudoknot structure just downstream of the shift site. The efficiency of the L-A frameshift site is 1.8%, similar to the observed molar ratio in viral particles of the 180-kDa fusion protein to the major coat protein.

The basic mechanisms of mRNA translation are ubiquitous among all organisms, in that the accurate decoding of triplet codon sequences programs serial amide linkages of amino acid residues by ribosomal complexes. In general, the fidelity of this process is dependent on both accurate recognition of mRNA codons by aminoacyl tRNAs and maintenance of the corresponding open reading frame. However, in a growing number of cases, deviations from this triplet codon rule are observed, indicating that the information content of an mRNA to encode protein may extend beyond its primary structure. These cases, collectively referred to as translational recoding (for review, see Gesteland and Atkins 1996), present exceptions to the venerable genetic code and are typically subdivided among three primary mechanisms: (1) frameshifting, in which translating ribosomes are induced to slide one nucleotide forward or backward at a distinct point in the transcript, with protein synthesis then continuing in the +1 or 11 reading frame, respectively; (2) alternative codon usage, where stop codons are not interpreted as sites of translational termination but, rather, encode an amino acid residue; and (3) translational bypassing, where the translational machinery traverses a gap in the mRNA coding sequence yet yields a single polypeptide chain. Programmed 11 ribosomal frameshift signals are among the most extensively characterized of these translational recoding phenomena (for review, see Brierly 1995; Dinman 1995; Farabaugh 1996). Although current examples are largely limited to viral systems, some bacterial 11 frameshifting events have also been documented (Blinkowa and Walker 1990; Tsuchihashi and Brown 1992; Chandler and Fayet 1993; Engelberg-Kulka and Schoulaker-Schwarz 1994). At present, no examples of eukaryotic mRNAs exhibiting 11 frameshift activity have been reported. However, a number of viruses infecting eukaryotic cells utilize programmed 11 ribosomal frameshifts, demonstrating that the cis elements involved in the frameshifting process are operational in eukaryotes. In viral systems, the efficiency of frameshifting is an essential determinant of the stoichiometry of synthesized viral protein products, which must be rigidly maintained for efficient propagation of the virus (Brierly 1995 and references therein; Dinman and Wickner 1995). In work presented in this issue, (Hammell et al. 1999), a bioinformatic approach was used to screen prokaryotic and eukaryotic DNA sequence databases for potential 11 frameshift signals. Because of the complexity of 11 frameshift sites and the sequence variability observed among these elements in nature, a multicomponent search algorithm was required to identify candidate sites from bulk database entries. This was made possible in part by the plethora of information available describing structural features of frameshift signals, much of which is described in Hammell et al. (1999) and elsewhere (Brierly 1995; Chen et al. 1996; Marczinke et al. 1998). This strategy represents a marked contrast to many examples of database searching, which typically involve a single motif or are limited to primary structure parameters (Fickett 1996; Altschul et al. 1997; Aravind and Landsman 1998; O’Neill 1998). Database searches using this compound algorithm for 11 frameshift sites yielded a host of potential signals in all databases tested, including eukaryotic sequences, with frequencies significantly higher than found in random sequence populations. In a number of cases, frameshift signals were conserved in homologous mRNAs from different species. A convincing argument for the validity of these searches was provided by the functional demonstration of 11 frameshifting activity for two selected signals (from Saccharomyces cerevisiae RAS1 and human CCR5 mRNAs) in a recombinant assay system. Furthermore, in four cases, potential 11 frameshift signals colocalized to sites of mutation linked to heritable diseases in humans, raising the possibility that 11 frameshift activity may be a physiologically relevant component of regulated gene expression in humans. Modifications of the parameters employed in this search algorithm may yet reveal additional candidate frameshift signals, as the potential for 38-RNA pseudoknot formation was a constraint placed upon their selection in this study. An RNA pseudoknot is not requisite for 11 frameshifting at the gag–pol overlap of HIV-1 (Jacks et al. 1988), for example, which only contains a weak 38-stem–loop structure in vivo (Parkin et al. 1992). The identification of functional 11 frameshift signals in chromosomally encoded eukaryotic mRNAs raises several interesting questions for further investigation. First, it will be important to determine if ribosomal frameshifting occurs in vivo in the context of these transcripts. If so, what is the function of the frameshift event? A ribosomal frameshift may serve to generate alternate protein isoforms, with common aminoCorresponding author. E-MAIL gbrewer@wfubmc.edu; FAX (336) 7169928. Insight/Outlook

Folded structures in mRNAs can stimulate reprogramming of ribosomes to make one protein from two different reading frames. The first crystal structure of a frameshift stimulatory RNA pseudoknot reveals remarkable features.

Coronaviruses have the potential to cause significant economic, agricultural and health problems. The severe acute respiratory syndrome (SARS) associated coronavirus outbreak in late 2002, early 2003 called attention to the potential damage that coronaviruses could cause in the human population. The ensuing research has enlightened many to the molecular biology of coronaviruses. A programmed -1 ribosomal frameshift is required by coronaviruses for the production of the RNA dependent RNA polymerase which in turn is essential for viral replication. The frameshifting signal encoded in the viral genome has additional features that are not essential for frameshifting. Elucidation of the differences between coronavirus frameshift signals and signals from other viruses may help our understanding of these features. Here we summarize current knowledge and add additional insight regarding the function of the programmed -1 ribosomal frameshift signal in the coronavirus lifecycle.

A novel hepatitis B viral (HBV) protein of 35-40 kDa, characterized by antibodies and proposed as an X-C fusion protein, was previously described in core particles isolated from HBV-, WHV-, and GSHV-infected livers. The X and C genes are two adjacent genes in all mammalian hepadnaviruses but are not contiguous in WHV and GSHV. After examination of the X and preC/C junction sequences of 10 HBV, 4 WHV, and 1 GSHV, we found that the ORF of preC can be extended 7 more sense codons upstream so that X overlaps with the preC/C gene in all sequences. The number of overlapping base pairs (bp) is varied: 46 bp in HBV, 19 bp in WHV, and 10 bp in GSHV. In this region a conserved A-track was found to be followed by a pair of inverted repeats, suggesting that a ribosomal frameshift may occur for X-C fusion protein production. To assess this possibility, we have used an in vitro transcription and translation coupling system to identify X-C protein production. Two recombinant SP6 plasmids were used. One contained a full length of the X and preC/C gene of wild-type HBV-DNA and the other fused the X-preC/C gene by inserting a 10-bp HindIII linker at the junction of the X-preC/C region. No X-C fusion protein was detected from the wild-type plasmid. In contrast a large amount of X-C fusion protein was produced from the linker-inserted clone. It appears, therefore, that the X-C fusion protein is unlikely to be produced via the mechanism of ribosomal frameshifting.

In the human hepatitis B virus (HBV) genome, the 5' end of the polymerase coding sequence overlaps with the 3' end of the core protein coding sequence. Recent results obtained from genetic studies have suggested that translation of HBV polymerase initiates from the first ATG codon of the polymerase reading frame and is not a result of frameshift translation from the core protein reading frame, as in the case of retroviruses. By using in vitro-synthesized SP6 RNA transcripts, we now demonstrate that HBV core protein-specific mRNA can direct the synthesis of polymerase from the internal polymerase ATG codon in rabbit reticulocyte lysates and Xenopus oocytes. A related message with an additional 60 nucleotides at the 5' end (pre-core protein mRNA) was not as efficient as the core protein mRNA for translation of polymerase. Furthermore, translation of polymerase from the core protein mRNA was not inhibited by the cap analog m7GpppG. This result, together with the results described above, indicates that translation of HBV polymerase occurs in a novel, cap-independent manner.

As the details of the interactions amongst tRNA, elongation factors, ribosomes and mRNA during the elongation phase of protein synthesis come into increasing focus, several new avenues for translational control have emerged. The recent advent of the high-level ribosomal frameshift site has opened up one such avenue for alternative events during the elongation and termination phases. These extreme cases of sequence-dependent variation in reading frame step size have also exposed a new lead into unsuspected interactions that occur during decoding and translocation. Analysis of ribosomal frameshifting is thus informative both from the standpoint of novel genetic control mechanisms, as well as illuminating new, and complex, macromolecular interactions occurring in protein synthesis.

Nucleotide sequencing studies on the genomic RNA of infectious bronchitis virus (IBV) has revealed the presence, at its 5′ end of two very large open reading frames (ORFS) termed F1 and F2 (Boursnell et al., 1987). The 5′ proximal ORF, Fl has the capacity to encode a polypeptide of approximately 440K, while the distal ORF, F2, which overlaps Fl by about 40 nucleotides (ntds) in a different (-1) reading frame, can encode a 300K polypeptide. Our previous work has indicated that both these open reading frames can be expressed as a single polypeptide from genomic mRNA by ribosomal frame-shifting within the overlap region (Brierley et al., 1987, 1989). This conclusion was based on experiments which showed that sequences from the F1–F2 junction region, when introduced into a heterologous mRNA could direct efficient ribosomal frame-shifting during translation in vitro and in vivo. Thus the primary translation products of IBV genomic RNA in the infected cell consists of a 440K product, corresponding to the Fl ORF, and an additional 740K ‘read-through’ product representing fusion of the F1 and F2 ORFs (Figure 1).

The three major structural genes of human immunodeficiency virus type 1 (HIV-1) are arranged in the viral genome in the order of 5’ gag-pol-env 3’. The gag gene encodes four group specific antigens, MA(p17), CA(p24), NC(p7) and p6, while the pol gene codes for protease, reverse transcriptase/ribonuclease H (RT/RH, 66 kDa) and integrase (IN, 32 kDa) (Chassagne et al.., 1986; Henderson et al., 1988; Kramer et al., 1986; Lightfoot et al., 1986; Lillehoj et al., 1988). The primary translational product of the gag gene is a 55 kDa polyprotein (pr55gag) (Kalyanarman et al., 1984; Sarngadharan et al., 1985), and that of the pol gene is a gag-pol fusion protein 160 kDa in size (pr160gag-Pol) achieved by ribosomal frameshift near the 3’ end of the gag gene (Gendelman et al., 1987; Jacks et al., 1988). They are processed to the individual viral proteins by the protease encoded by the pol gene after the enzyme cleaves itself from the pr160gag-Pol precursor by autocatalysis (Farmerie et al., 1987; Graves et al., 1988). There are at least seven protease cleavage sites in the gag and pol polyprotein precursors (Darke et al., 1988), as depicted in Figure 1. The numbering of amino acid residues at the cleavage sites is based on the amino acid sequence predicted from the nucleotide sequence of clone HXB2 of HIV-1 isolate, HTLV HIB (Ratner et al., 1985).

Ferredoxin I (Fed-1) mRNA abundance is modulated by an internal light regulatory element that includes sequences both 5' and 3' of the translational initiation site. To test the hypothesis that the light response mediated by this element might be coupled to translation, we transformed tobacco plants with gene constructs blocked in translational initiation or elongation. Here, we report that such mutations abolish the light response in vivo. A nonsense mutation could be rescued by restoring the open reading frame with a different sequence, even when the new codon caused an amino acid substitution. Our data establish that the light response requires a translatable reading frame and thus provide strong circumstantial evidence for post-transcriptional modulation of Fed-1 mRNA levels. The Fed-1 system is presently the only higher plant example of a developmentally regulated change in mRNA abundance that requires translation of the affected mRNA.

We have inserted the sequence 5'-AAG-GAGGU-3', which is complementary to the 3' terminus of Escherichia coli 16S rRNA, in a reading frame and analyzed its effect on the accuracy and overall rate of translation in vivo. Translation over the sequence yields a 50% ribosomal frameshift if the reading phase is A-AGG-AGG-U. The other two possible frames do not give shifts. The introduction of a UAA stop codon before (UAA-AGG-AGG-U) but not after (A-AGG-AGG-UAA) the AGG codons abolishes the frameshift. The change in the reading phase occurs exclusively to the +1 direction. Efficient frameshifting is also induced by the sequence A-AGA-AGA-U. The arginine codons AGG and AGA are read by minor tRNA. Suppression of frameshifting takes place when a gene for minor tRNA(Arg) is introduced on a multicopy plasmid. We suggest that frameshifting during translation of the A-AGG-AGG-U sequence is due to the erroneous decoding of the tandem AGG codons and arises by depletion of tRNA(Arg). The complementarity of tandem AGG codons to the 3' terminus of 16S rRNA is a coincidence and apparently not related to the shift. Replacing the AGG-AGG sequence by the optimal arginine codons CGU-CGU does not increase the overall rate of translation.

Missense and nonsense suppressor tRNAs, selected for their ability to read a new triplet codon, were observed to suppress one or more frameshift mutations in trpA of Escherichia coli. Two of the suppressible frameshift mutants, trpA8 and trpA46AspPR3, were cloned, sequenced, and found to be of the +1 type, resulting from the insertion of four nucleotides and one nucleotide, respectively. Twenty-two suppressor tRNAs were examined, 20 derived from one of the 3 glycine isoacceptor species, one from lysT, and one from trpT. The sequences of all but four of the mutant tRNAs are known, and two of those four were converted to suppressor tRNAs that were subsequently sequenced. Consideration of the coding specificities and anticodon sequences of the suppressor tRNAs does not suggest a unitary mechanism of frameshift suppression. Rather, the results indicate that different suppressors may shift frame according to different mechanisms. Examination of the suppression windows of the suppressible frameshift mutations indicates that some of the suppressors may work at cognate codons, either in the 0 frame or in the +1 frame, and others may act at noncognate codons (in either frame) by some as-yet-unspecified mechanism. Whatever the mechanisms, it is clear that some +1 frameshifting can occur at non-monotonous sequences. A striking example of a frame shifting missense suppressor is a mutant lysine tRNA that differs from wild-type lysine tRNA by only a single base in the amino acid acceptor stem, a C to U70 transition that results in a G·U base pair. It is suggested that when this mutant lysine tRNA reads its cognate codon, AAA, the presence of the G·U base pair sometimes leads either to a conformational change in the tRNA or to an altered interaction with some component of the translation machinery involved in translocation, resulting in a shift of reading frame. In general, the results indicate that translocation is not simply a function of anticodon loop size, that different frameshifting mechanisms may operate with different tRNAs, and that conformational features, some far removed from the anticodon region, are involved in maintaining fidelity in translocation.

Double-stranded DNA derived from influenza B virus genome RNA segment 8, which codes for the NS1 and NS2 proteins, was constructed by hybridization of full-length cDNA copies of RNA segment 8 and of the NS1 mRNA. This DNA was cloned in plasmid pBR322 and sequenced. The NS1 mRNA (approximately 1,080 viral nucleotides) contains nonviral nucleotides at its 5' end and is capable of coding for a protein of 281 amino acids. Sequencing of the NS2 mRNA has shown that it contains an interrupted sequence of 655 nucleotides and is most likely synthesized by a splicing mechanism. The first approximately 75 virus-specific nucleotides at the 5' end of the NS2 mRNA are the same as are found at the 5' -end of the NS1 mRNA. This region contains the initiation codon for protein synthesis and coding information for 10 amino acids common to the two proteins. The approximately 350-nucleotide body region of the NS2 mRNA can be translated in the +1 reading frame, and the sequence indicates that the NS1 and NS2 protein-coding regions overlap by 52 amino acids translated from different reading frames. Thus, between the influenza A and B viruses, the organization of the NS1 and NS2 mRNAs and the sizes of the NS2 mRNA and protein are conserved despite the larger size of the influenza B virus RNA segment, NS1 mRNA, and NS1 protein.

A genetic approach has been used to establish the molecular basis of 4-base codon recognition by frameshift suppressor tRNA containing an extra nucleotide in the anticodon. We have isolated all possible base substitution mutations at the position 4 (N) in the 3'-CCCN-5' anticodon of a Saccharomyces cerevisiae frameshift suppressor glycine tRNA encoded by the SUF16 gene. Base substitutions at +1 frameshift sites in the his4 gene have also been obtained such that all possible 4-base 5'-GGGN-3' codons have been identified. By testing for suppression in different strains that collectively represent all 16 possible combinations of position 4 nucleotides, we show that frameshift suppression does not require position 4 base pairing. Nonetheless, position 4 interactions influence the efficiency of suppression. Our results suggest a model in which 4-base translocation of mRNA on the ribosome is directed primarily by the number of nucleotides in the anticodon loop, whereas the resulting efficiency of suppression is dependent on the nature of position 4 nucleotides.