Theiler's murine encephalomyelitis virus contrasts with encephalomyocarditis and foot-and-mouth disease viruses in its functional utilization of the StopGo non-standard translation mechanism.

The picornaviruses' genome consists of a positive-sense ssRNA. Like many picornaviruses, cardioviruses synthesize two distinct polyprotein precursors from adjacent but non-overlapping genome segments. Both the [L-1ABCD-2A] and the [2BC-3ABCD] polyproteins are proteolytically processed to yield mature capsid and non-structural proteins, respectively. An unusual translational event, known as 'StopGo' or 'Stop-Carry on', is responsible for the release of the [L-1ABCD-2A] polyprotein from the ribosome and synthesis of the N-terminal amino acid of the [2BC-3ABCD] polyprotein. A common feature of these viruses is the presence of a highly conserved signature sequence for StopGo: -D(V/I)ExNPG(↓)P-, where -D(V/I)ExNPG are the last 7 aa of 2A, and the last P- is the first amino acid of 2B. Here, we report that, in contrast to encephalomyocarditis virus and foot-and-mouth disease virus, a functional StopGo does not appear to be essential for Theiler's murine encephalomyelitis virus viability when tested in vitro and in vivo.

The Drosophila gene for antizyme requires ribosomal frameshifting for expression and contains an intronic gene for snRNP Sm D3 on the opposite strand.

Previously, a Drosophila melanogaster sequence with high homology to the sequence for mammalian antizyme (ornithine decarboxylase antizyme) was reported. The present study shows that homology of this coding sequence to its mammalian antizyme counterpart also extends to a 5' open reading frame (ORF) which encodes the amino-terminal part of antizyme and overlaps the +1 frame (ORF2) that encodes the carboxy-terminal three-quarters of the protein. Ribosomes shift frame from the 5' ORF to ORF2 with an efficiency regulated by polyamines. At least in mammals, this is part of an autoregulatory circuit. The shift site and 23 of 25 of the flanking nucleotides which are likely important for efficient frameshifting are identical to their mammalian homologs. In the reverse orientation, within one of the introns of the Drosophila antizyme gene, the gene for snRNP Sm D3 is located. Previously, it was shown that two closely linked P-element transposon insertions caused the gutfeeling phenotype of embryonic lethality and aberrant neuronal and muscle cell differentiation. The present work shows that defects in either snRNP Sm D3 or antizyme, or both, are likely causes of the phenotype.

Antizyme expression: a subversion of triplet decoding, which is remarkably conserved by evolution, is a sensor for an autoregulatory circuit.

The efficiency of programmed ribosomal frameshifting in decoding antizyme mRNA is the sensor for an autoregulatory circuit that controls cellular polyamine levels in organisms ranging from the yeast Schizosaccharomyces pombe to Drosophila to mammals. Comparison of the frameshift sites and flanking stimulatory signals in many organisms now permits a reconstruction of the likely evolutionary path of the remarkably conserved mRNA sequences involved in the frameshifting.

Phylogenetic analysis of tmRNA genes within a bacterial subgroup reveals a specific structural signature.

Bacterial tmRNA mediates a trans-translation reaction, which permits the recycling of stalled ribosomes and probably also contributes to the regulated expression of a subset of genes. Its action results in the addition of a small number of C-terminal amino acids to protein whose synthesis had stalled and these constitute a proteolytic recognition tag for the degradation of these incompletely synthesized proteins. Previous work has identified pseudoknots and stem-loops that are widely conserved in divergent bacteria. In the present work an alignment of tmRNA gene sequences within 13 beta-proteobacteria reveals an additional sub-structure specific for this bacterial group. This sub-structure is in pseudoknot Pk2, and consists of one to two additional stem-loop(s) capped by stable GNRA tetraloop(s). Three-dimensional models of tmRNA pseudoknot 2 (Pk2) containing various topological versions of the additional sub-structure suggest that the sub-structures likely point away from the core of the RNA, containing both the tRNA and the mRNA domains. A putative tertiary interaction has also been identified.

Decoding of tandem quadruplets by adjacent tRNAs with eight-base anticodon loops.

To expand the genetic code for specification of multiple non-natural amino acids, unique codons for these novel amino acids are needed. As part of a study of the potential of quadruplets as codons, the decoding of tandem UAGA quadruplets by an engineered tRNA(Leu) with an eight-base anticodon loop, has been investigated. When GCC is the codon immediately 5' of the first UAGA quadruplet, and release factor 1 is partially inactivated, the tandem UAGAs specify two leucines with an overall efficiency of at least 10%. The presence of a purine at anticodon loop position 32 of the tRNA decoding the codon 5' to the first UAGA seems to influence translation of the following codon. Another finding is intraribosomal dissociation of anticodons from codons and their re-pairing to mRNA at overlapping or nearby codons. In one case where GCC is replaced by CGG, only a single Watson-Crick base pair can form upon re-pairing when decoding is resumed. This has implications for the mechanism of some cases of programmed frameshifting.

Identification of sequence motifs in oligonucleotides whose presence is correlated with antisense activity.

Design of antisense oligonucleotides targeting any mRNA can be much more efficient when several activity-enhancing motifs are included and activity-decreasing motifs are avoided. This conclusion was made after statistical analysis of data collected from >1000 experiments with phosphorothioate-modified oligonucleotides. Highly significant positive correlation between the presence of motifs CCAC, TCCC, ACTC, GCCA and CTCT in the oligonucleotide and its antisense efficiency was demonstrated. In addition, negative correlation was revealed for the motifs GGGG, ACTG, AAA and TAA. It was found that the likelihood of activity of an oligonucleotide against a desired mRNA target is sequence motif content dependent.

Thermodynamic criteria for high hit rate antisense oligonucleotide design.

Antisense oligonucleotides are used for therapeutic applications and in functional genomic studies. In practice, however, many of the oligonucleotides complementary to an mRNA have little or no antisense activity. Theoretical strategies to improve the 'hit rate' in antisense screens will reduce the cost of discovery and may lead to identification of antisense oligonucleotides with increased potency. Statistical analysis performed on data collected from more than 1000 experiments with phosphorothioate-modified oligonucleotides revealed that the oligo-probes, which form stable duplexes with RNA (DeltaG(o)37 < or = -30 kcal/mol) and have small self-interaction potential, are more frequently efficient than molecules that form less stable oligonucleotide-RNA hybrids or more stable self-structures. To achieve optimal statistical preference, the values for self-interaction should be (DeltaG(o)37) > or = -8 kcal/mol for inter-oligonucleotide pairing and (DeltaG(o)37) > or = -1.1 kcal/mol for intra-molecular pairing. Selection of oligonucleotides with these thermodynamic values in the analyzed experiments would have increased the 'hit rate' by as much as 6-fold.

RECODE: a database of frameshifting, bypassing and codon redefinition utilized for gene expression.

The RECODE database is a compilation of 'programmed' translational recoding events taken from the scientific literature and personal communications. The database deals with programmed ribosomal frameshifting, codon redefinition and translational bypass occurring in a variety of organisms. The entries for each event include the sequences of the corresponding genes, their encoded proteins for both the normal and alternate decoding, the types of the recoding events involved, trans-factors and cis-elements that influence recoding. The database is freely available at http://recode.genetics. utah.edu/.

Thermodynamic calculations and statistical correlations for oligo-probes design.

Optimization of probe design for array-based experiments requires improved predictability of oligonucleotide hybridization behavior. Currently, designing oligonucleotides capable of interacting efficiently and specifically with the relevant target is not a routine procedure. Multiple examples demonstrate that oligonucleotides targeting different regions of the same RNA differ in their hybridization ability. The present work shows how thermodynamic evaluations of oligo-target duplex or oligo self-structure stabilities can facilitate probe design. Statistical analysis of large sets of hybridization data reveals that thermodynamic evaluation of oligonucleotide properties can be used to avoid poor RNA binders. Thermodynamic criteria for the selection of 20 and 21mers, which, with high probability, interact efficiently and specifically with their targets, are suggested. The design of longer oligonucleotides can also be facilitated by the same calculations of DeltaG(o) (T) values for oligo-target duplex or oligo self-structure stabilities and similar selection schemes.

Eubacterial tmRNAs: everywhere except the alpha-proteobacteria?

Eubacterial tmRNAs mediate, at least in Escherichia coli, recycling of ribosomes stalled at the end of terminatorless mRNAs. A tmRNA-encoded peptide tag is added to abnormal protein products of truncated mRNAs. This tag is a specific signal for proteolysis of the aberrant protein. To obtain further sequence information, PCR was used to amplify more Eubacterial genes for tmRNA. Fifty-eight new tmDNA sequences including from members of nine additional phyla were determined. Remarkably, tmDNA sequences could be amplified from all species tested apart from those in the alpha-Proteobacteria, raising evolutionary implications.

The role of EF-Tu and other translation components in determining translocation step size.

The two EF-Tu encoding genes, tufA and tufB, of Salmonella typhimurium have been sequenced. Nearly all the differences from their Escherichia coli counterparts are third position changes which do not alter the encoded amino acids. Unexpectedly, most of the changes in one Salmonella tuf gene are paralleled by changes in the other tuf gene perhaps due to gene repair despite the distance separating the genes. Three mutants which cause mis-framing, have their substitutions at codon 375. Explanations for mutants which cause mis-framing are considered and the mechanism of normal reading frame maintenance discussed.

Structural probing and mutagenic analysis of the stem-loop required for Escherichia coli dnaX ribosomal frameshifting: programmed efficiency of 50%.

Three elements are crucial for the programmed frameshifting in translation of dnaX mRNA: a Shine-Dalgarno (SD)-like sequence, a double-shift site, and a 3' structure. The conformation of the mRNA containing these three elements was investigated using chemical and enzymatic probes. The probing data show that the structure is a specific stem-loop. The bottom half of the stem is more stable than the top half of the stem. The function of the stem-loop was further investigated by mutagenic analysis. Reducing the stability of the bottom half of the stem strongly effects frameshifting levels, whereas similar changes in the top half are not as effective. Stabilizing the top half of the stem gives increased frameshifting beyond the WT efficiency. The identity of the primary RNA sequence in the stem-loop is unimportant, provided that the overall structure is maintained. The calculated stabilities of the variant stem-loop structures correlate with frameshifting efficiency. The SD-interaction and the stem-loop element act independently to increase frameshifting in dnaX.

A functional mutant of tRNA(2Arg) with ten extra nucleotides in its TFC arm.

An unusual, spontaneously arising mutant of Escherichia coli tRNA(2Arg) has been deduced to have two extra nucleotides in its anticodon loop and a duplication of ten nucleotide residues in the TFC loop. This conclusion is based on its gene sequence, Northern blot analysis of isolated tRNA and the size of the in vitro-processed tRNA product. In vitro analyses showed that the mutant precursor is processed normally, albeit inefficiently, to a mature tRNA species 12 nucleotides longer than the wild-type. In addition, the mature tRNA functions as a frameshift suppressor in vivo. Several related mutants with more conservative changes within the gene sequence were similarly shown to be accurately processed, albeit with varying degrees of efficiency less than that of the wild-type. These results indicate that in spite of the high degree of evolutionary conservation of the tertiary structure of tRNAs, and the fact that no such naturally-occurring variant has been found, a greatly enlarged tRNA is capable of functioning in protein synthesis. The data also indicate that recognition sites for correct processing of precursor tRNAs may be unexpectedly tolerant of unusual context, and may depend on some specific features of the tertiary structure rather than the overall structure for accurate processing.

Seven, eight and nine-membered anticodon loop mutants of tRNA(2Arg) which cause +1 frameshifting. Tolerance of DHU arm and other secondary mutations.

The mutant tRNA(2Arg) encoded by the genetically-selected frameshift suppressor, sufT621, inserts arginine and causes a +1 reading-frame shift at the proline codon, CCG(U). There is an extra base, G36.1, in argV beta, one of the four identical genes for tRNA(2Arg) in the position between bases 36 and 37, corresponding to the 3' side of the anticodon. The new four-base anticodon, predicted from DNA sequencing to be 3' GGCA 5', is complementary to the four-base codon CCGU. Quadruplet translocation promoted by mutant argV does not require perfect complementarity between the codon and the anticodon since synthetic genes encoding derivatives of tRNA(2Arg) and tRNA(1Pro), with four-base anticodons complementary to three out of the four bases of CCGU, were also shown to be capable of frameshifting. Two other mutants of argV, inferred to have normal-size, seven-base anticodon loops, were also found to be capable of four-base-decoding demonstrating that quadruplet translocation promoted by mutant argV does not require an enlarged anticodon loop. Other alleles of argV, predicted to have nine bases in the anticodon loop, were also found to cause frameshifting. The DNA sequence of two of these showed in addition, either a deletion of G24, or a ten-base duplication in the region corresponding to the TFC arm. A general finding is that mutations in the DHU arm of tRNA(2Arg) are compatible with, and in one case necessary for, frameshifting.

Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions.

During translation of bacteriophage T4 gene 60 mRNA, ribosomes bypass 50 nucleotides with high efficiency. One of the mRNA signals for bypass is a stem-loop in the first part of the coding gap. When the length of this stem-loop is extended by 36 nucleotides, bypass is reduced to 0.35% of the wild-type level. Bypass is partially restored by a mutation in the C-terminal domain of Escherichia coli large ribosomal subunit protein L9. Previous work has shown that L9 is an elongated protein with an alpha-helix that connects and orients the N and C-terminal domains that both contain a predicted RNA binding site. We have determined two binding sites of L9 on 23 S rRNA. A 778 nucleotide RNA fragment encompassing domain V (nucleotides 1999 to 2776) of the 23 S rRNA is retained on filters by L9 and contains both sites. The N and C-terminal domains of L9 were shown to interact with nucleotides just 5' to nucleotide 2231 and 2179 of the 23 S rRNA, respectively, using the toeprint assay. These L9 binding sites on 23 S rRNA suggest that L9 functions as a brace across helix 76 to position helices 77 and 78 relative to the peptidyl transferase center. In this study, bypass on a mutant gene 60 mRNA has been used as an assay to probe the importance of particular L9 amino acids for function. Amino acid substitutions in the C-terminal domain are shown to partially restore bypass. These mutant L9 proteins have reduced binding to a 23 S rRNA fragment (nucleotides 1999 to 2274) containing domain V, to which L9 binds. They partially retain both the N and C-terminal domain interactions. On the other hand, substitutions of amino acids in the N-terminal domain, which greatly reduce RNA binding, do not restore bypass. The latter mutants have completely lost the N-terminal domain interaction. Addition of an amino acid to the alpha-helix also restores gene 60 bypass. RNA binding by this mutant is similar to that observed for the C-terminal domain mutants that partially restore bypass.

Deficiency of 1-methylguanosine in tRNA from Salmonella typhimurium induces frameshifting by quadruplet translocation.

The trmD gene encodes the tRNA(m1G37)methyltransferase, which methylates guanosine (G) to 1-methylguanosine (m1G) at position 37 of tRNAs that read CUN (leucine), CCN (proline), and CGG (arginine) codons. A mutant, trmD3, has previously been isolated, which at high temperature lacks m1G in tRNA, and this deficiency was correlated with a +1 frameshifting activity. In this study, the mechanism of this trmD3-induced frameshift involving mutant tRNA(Pro) and tRNA(Leu) species has been investigated. Potential frameshifting sites for proline tRNAs, CCC-N, were efficiently suppressed in the mutant strain. Hybrid beta-galactosidases encoded by plasmid constructs containing the sites CCC-U and CCC-A were subjected to amino-terminal sequencing. The protein sequences demonstrated that a quadruplet translocation had occurred and that a proline was inserted at these sites, suggesting that a tRNA(Pro) deficient in m1G is the frameshifting agent. Therefore, a mechanism involving a quadruplet codon-anticodon interaction is favoured for trmD3-dependent +1 frameshifting. Of the four potential sites for tRNA(Leu) (CCU-N), two, CCU-U and CCU-C, were significantly suppressed in the trmD3 mutant. Thus, species of tRNA(Leu) may also act as +1 frameshift suppressors. No -1 frameshifting activity was found with the trmD3 mutant.

The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons.

Expression of the RNA replicase domain of tobacco mosaic virus (TMV) and certain protein-coding regions in other plant viruses, is mediated by translational readthrough of a leaky UAG stop codon. It has been proposed that normal tobacco tyrosine tRNAs are able to read the UAG codon of TMV by non-conventional base-pairing but recent findings that stop codons can also be bypassed as a result of extended translocational shifts (tRNA hopping) have encouraged a re-examination. In light of the alternatives, we investigated the sequences flanking the leaky UAG codon using an in vivo assay in which bypass of the stop codon is coupled to the transient expression of beta-glucuronidase (GUS) reporter genes in tobacco protoplasts. Analysis of GUS constructions in which codons flanking the stop were altered allowed definition of the minimal sequence required for read through as UAG-CAA-UUA. The effects of all possible single-base mutations in the codons flanking the stop indicated that 3' contexts of the form CAR-YYA confer leakiness and that the 3' context permits read through of UAA and UGA stop codons as well as UAG. Our studies demonstrate a major role for the 3' context in the read through process and do not support a model in which teh UAG is bypassed exclusively as a result of anticodon-codon interactions. No evidence for tRNA hopping was obtained. The 3' context apparently represents a unique sequence element that affects translation termination.

Quadruplet codons: implications for code expansion and the specification of translation step size.

One of the requirements for engineering expansion of the genetic code is a unique codon which is available for specifying the new amino acid. The potential of the quadruplet UAGA in Escherichia coli to specify a single amino acid residue in the presence of a mutant tRNA(Leu) molecule containing the extra nucleotide, U, at position 33.5 of its anticodon loop has been examined. With this mRNA-tRNA combination and at least partial inactivation of release factor 1, the UAGA quadruplet specifies a leucine residue with an efficiency of 13 to 26 %. The decoding properties of tRNA(Leu) with U at position 33.5 of its eight-membered anticodon loop, and a counterpart with A at position 33.5, strongly suggest that in both cases their anticodon loop bases stack in alternative conformations. The identity of the codon immediately 5' of the UAGA quadruplet influences the efficiency of quadruplet translation via the properties of its cognate tRNA. When there is the potential for the anticodon of this tRNA to dissociate from pairing with its codon and to re-pair to mRNA at a nearby 3' closely matched codon, the efficiency of quadruplet translation at UAGA is reduced. Evidence is presented which suggests that when there is a purine base at position 32 of this 5' flanking tRNA, it influences decoding of the UAGA quadruplet.

Reported translational bypass in a trpR'-lacZ' fusion is accounted for by unusual initiation and +1 frameshifting.

I. Benhar and H. Engelberg-Kulka reported that a 55 nucleotide translational bypass occurs in decoding a fusion of the Escherichia coli tryptophan repressor, trpR, and lacZ genes. The start of the bypass occurred in the trpR gene and coding resumed in the lacZ gene. It was considered that bypassing likely occurred in expression of trpR itself to produce an additional 10 kDa product which may be biologically important. We report here that bypass is undetectable in the same and related trpR'-lacZ' fusions. The beta-galactosidase activity derived from the fusions is accounted for by unusual internal initiation and +1 frameshifting, both of which occur in the lacZ part of the fusion. The 10 kDa product reportedly encoded by the trpR gene was not detectable to a level of 1% of the full-length 12 kDa tryptophan repressor product, at least when expressed from a T7 promoter.

Structural studies of the RNA pseudoknot required for readthrough of the gag-termination codon of murine leukemia virus.

Retroviruses, such as murine leukemia virus (MuLV), whose gag and pol genes are in the same reading frame but separated by a UAG stop codon, require that 5-10 % of ribosomes decode the UAG as an amino acid and continue translation to synthesize the Gag-Pol fusion polyprotein. A specific pseudoknot located eight nucleotides 3' of the UAG is required for this redefinition of the UAG stop codon. The structural probing and mutagenic analyses presented here provide evidence that loop I of the pseudoknot is one nucleotide, stem II has seven base-pairs, and the nucleotides 3' of stem II are important for function. Stem II is more resistant to single-strand-specific probes than stem I. Sequences upstream of the UAG codon allow formation of two competing structures, a stem-loop and the pseudoknot.