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At least 19 recordsLinked to original sources
PubMed2026

AUA codon decoding by preferential use of tRNAIle(UAU) in Lactobacillus casei.

Modified nucleosides at the first (wobble) position of tRNA anticodons play critical roles in accurate decoding of the genetic code. In bacteria, the isoleucine AUA codon is typically decoded by tRNAIle(LAU), in which lysidine (L) at the wobble position of tRNAIle with a CAU anticodon ensures discrimination from the methionine AUG codon. However, some bacteria, such as Mycoplasma mobile, lack tRNAIle(LAU) and instead utilize tRNAIle(UAU). In this organism, the unmodified uridine at the wobble position is thought to enable specific decoding of AUA while avoiding AUG recognition. In our previous study, we identified a lactic acid bacterium in which both tRNAIle(LAU) and tRNAIle(UAU) coexist. Here, we show that tRNAIle(LAU) is scarcely aminoacylated in vivo, whereas tRNAIle(UAU) is efficiently aminoacylated. Notably, the presence of 4-thiouridine (s4U) at position 8 inhibits IleRS-dependent aminoacylation of tRNAIle(UAU) in vitro, suggesting a regulatory role of tRNA modification in this process. Moreover, tRNAIle(LAU) exhibits incomplete discrimination between AUA and AUG codons and binds to AUG in the ribosomal A-site binding assays. In contrast, tRNAIle(UAU) containing N 6-threonylcarbamoyladenosine (t6A) at position 37 showed a tendency toward improved discrimination between AUA and AUG codons and preferentially recognized AUA at the ribosomal A site. These results indicate that AUA decoding is predominantly mediated by preferential use of tRNAIle(UAU) rather than canonical tRNAIle(LAU), revealing an alternative mechanism of codon decoding based on differential utilization of tRNA isoacceptors, and providing an additional layer of translational control in bacteria.

RNA, Transfer, Ile↗
PubMed1992

Overproduction of a selenocysteine-containing polypeptide in Escherichia coli: the fdhF gene product.

The fdhF gene of Escherichia coli codes for the selenocysteine-including protein subunit of formate dehydrogenase H. The protein subunit consists of 715 amino acid residues containing a single selenocysteine residue at position 140 which is encoded by a UGA codon. The decoding of this opal termination codon occurs under anaerobic growth conditions by means of a specific tRNA, i.e. the selC gene product. The ability of E. coli cells to overproduce a selenopolypeptide was examined using the fdhF gene as a model system. Surprisingly, E. coli was able to synthesize the fdhF gene product at the level of approximately 12% of the total cellular protein. This was achieved by cloning fdhF in a multicopy plasmid together with a synthetic selC gene under the Ipp promoter. FdhF production was absolutely dependent upon the addition of selenium to the culture medium and was almost completely blocked in the presence of oxygen. The product was specifically labelled with 75Se, proving that it consisted of a selenoprotein. The product was purified to homogeneity and shown to exhibit the catalytic properties characteristic of formate dehydrogenase H.

Bacterial Proteins↗
PubMed1990

Cloning and expression of a mammalian peptide chain release factor with sequence similarity to tryptophanyl-tRNA synthetases.

The termination of protein synthesis is encoded by in-frame nonsense (stop) codons. Most organisms use three nonsense codons: UGA, UAG, and UAA. In contrast to sense codons, which are decoded by specific tRNAs, nonsense codons are decoded by proteins called release factors (RFs). Here we report the cloning of a mammalian RF cDNA by the use of monoclonal antibodies specific for rabbit RF. Functional studies showed that, when expressed in Escherichia coli, the protein encoded by this cDNA has in vitro biochemical characteristics similar to those of previously characterized mammalian RFs. DNA sequencing of this eukaryotic RF cDNA revealed a remarkable sequence similarity to bacterial and mitochondrial tryptophanyl-tRNA synthetases, with the greatest similarity confined to the synthetase active site, and no obvious similarity to bacterial RFs.

Amino Acid Sequence↗
PubMed1990

Features of the formate dehydrogenase mRNA necessary for decoding of the UGA codon as selenocysteine.

The fdhF gene encoding the 80-kDa selenopolypeptide subunit of formate dehydrogenase H from Escherichia coli contains an in-frame TGA codon at amino acid position 140, which encodes selenocysteine. We have analyzed how this UGA "sense codon" is discriminated from a UGA codon signaling polypeptide chain termination. Deletions were introduced from the 3' side into the fdhF gene and the truncated 5' segments were fused in-frame to the lacZ reporter gene. Efficient read-through of the UGA codon, as measured by beta-galactosidase activity and incorporation of selenium, was dependent on the presence of at least 40 bases of fdhF mRNA downstream of the UGA codon. There was excellent correlation between the results of the deletion studies and the existence of a putative stem-loop structure lying immediately downstream of the UGA in that deletions extending into the helix drastically reduced UGA translation. Similar secondary structures can be formed in the mRNAs coding for other selenoproteins. Selenocysteine insertion cartridges were synthesized that contained this hairpin structure and variable portions of the fdhF gene upstream of the UGA codon and inserted into the lacZ gene. Expression studies showed that upstream sequences were not required for selenocysteine insertion but that they may be involved in modulating the efficiency of read-through. Translation of the UGA codon was found to occur with high fidelity since it was refractory to ribosomal mutations affecting proofreading and to suppression by the sup-9 gene product.

Aldehyde Oxidoreductases↗
PubMed1987

Unusual genetic codes and a novel gene structure for tRNA(AGYSer) in starfish mitochondrial DNA.

The nucleotide sequence of a 3849-bp fragment of starfish mitochondrial genome was determined. The genes for NADH dehydrogenase subunits 3, 4, 5, and COIII, and three kinds of (tRNA(UCNSer), tRNA(His), and tRNA(AGYSer) were identified by comparing with the genes of other animal mitochondria so far elucidated. The gene arrangement of starfish mitochondrial genome was different from those of vertebrate and insect mitochondrial genomes. Comparison of the protein-encoding nucleotide sequences of starfish mitochondria with those of other animal mitochondria suggested a unique genetic code in starfish mitochondrial genome; both AGA and AGG (arginine in the universal code) code for serine, AUA (isoleucine in the universal code but methionine in most mitochondrial systems) for isoleucine, and AAA (lysine) for asparagine. It was also inferred that these AGA and AGG codons are decoded by serine tRNA(AGYSer) originally corresponding to AGC and AGU codons. This situation is similar to the case of Drosophila mitochondrial genome. Variations in the use of AGA and AGG codons were discussed on the basis of the evolution of animals and decoding capacity of various tRNA(AGYSer) species possessing different sizes of the dihydrouridine (D) arm.

Animals↗
PubMed1990

Recent advances in peptide chain termination.

Peptide chain termination occurs when a stop codon is decoded by a release factor. In Escherichia coli two codon-specific release factors (RF1 and RF2) direct the termination of protein synthesis, while in eukaryotes a single factor is required. The E. coli factors have been purified and their genes isolated. A combination of protein and DNA sequence data reveal that the RFs are structurally similar and that RF2 is encoded in two reading frames. Frame-shifting from one reading frame to the next occurs at a rate of 50%, is regulated by the RF2-specific stop codon UGA, and involves the direct interaction of the RF2 mRNA with the 3' end of the 16S rRNA. The RF genes are located in two separate operons, with the RF1 gene located at 26.7 min and the RF2 gene at 62.3 min on the chromosome map. Ribosomal binding studies place the RF-binding region at the interface between the ribosomal subunits. A possible mechanism of stop-codon recognition is reviewed.

Codon↗
PubMed2026

Molecular Determinants and Therapeutic Targeting of Stop Codon Readthrough in Eukaryotic Translation.

Accurate translation termination is essential for proteome integrity and in eukaryotes is primarily governed by the release factors eRF1 and eRF3, which ensure precise recognition of stop codons and efficient release of nascent polypeptides. However, proteome integrity is challenged by mutations that generate premature termination codons (PTCs), leading to truncated, nonfunctional proteins and degradation of the aberrant transcript via nonsense-mediated mRNA decay (NMD). Collectively, these events account for ∼1800 human genetic diseases. Translational readthrough, the process by which near-cognate tRNAs decode stop codons and allow ribosomes to continue elongation beyond the stop codon, represents a possibility to suppress PTCs and restore full-length protein synthesis. Initially discovered in viruses as a mechanism to expand coding capacity, readthrough is now recognized as a regulated feature of eukaryotic gene expression influenced by both cis-acting sequence elements and trans-acting factors. Recent evidence highlights the remarkable context dependence of readthrough, revealing variation across transcripts, tissues, and developmental stages. In this review, we examine the molecular determinants that define stop codon recognition and readthrough efficiency, with particular emphasis on nucleotide context. We further discuss the mechanisms and binding sites of small molecules that promote PTC readthrough, and summarize the clinical development landscape of readthrough-inducing compounds for the treatment of diseases caused by nonsense mutations.

Humans↗
PubMed1987

Physiological levels of normal tRNA(CAGGln) can effect partial suppression of amber mutations in the yeast Saccharomyces cerevisiae.

A number of ciliated protozoa are known to read the stop codons UAA and UAG as sense codons that specify glutamine during protein synthesis. In considering evolutionary mechanisms for this curious divergence from the standard genetic code, we propose the existence of progenitor tRNAs for glutamine that can weakly suppress UAA and UAG codons. It has been previously shown that multicopy plasmids that overexpress normal tRNA(CAAGln) and tRNA(CAGGln) genes from the yeast Saccharomyces cerevisiae can partially suppress a number of yeast ochre and amber mutations, respectively. In the present study we show that the tRNA(CAGGln) gene can also function as a weak amber suppressor when expressed in cells at physiological levels. This observation is consistent with a role of tRNA(CAGGln) as an evolutionary progenitor of tRNAs that strongly decode UAG codons.

Animals↗
PubMed1990

Codon recognition in polypeptide chain termination: site directed crosslinking of termination codon to Escherichia coli release factor 2.

An RNA synthesized in vitro was positioned on the Escherichia coli ribosome at the P site with tRNAala, and with a termination codon, UAA, as the next codon in the A site. Such a complex bound stoichiometric amounts of release factor 2 (RF-2); a corresponding RNA with UAC in place of UAA was not a template for the factor. An RNA containing 4-thio-UAA in place of the UAA supported binding of RF-2, and this has allowed site-directed crosslinking from the first position of the termination codon to answer two long standing questions about the termination of protein biosynthesis, the position of the termination codon and its proximity to the release factor during codon recognition. An RF-2.mRNA crosslinked product was detected, indicating the release factor and the termination codon are in close physical contact during the codon recognition event of termination. The 4-thio-U crosslinked also to the ribosome but only to the 30S subunit, and the proteins and the rRNA site concerned were identified. RF-2 decreased significantly the crosslinking to the ribosomal components, but no new crosslink sites were found. If the stop codon was deliberately displaced from the decoding site by one codon's length then a different pattern of crosslinking in particular to the rRNA resulted. These observations are consistent with a model of codon recognition by RF-2 at the decoding site, without a major shift in position of the codon.

Base Sequence↗
PubMed1991

UGA can be decoded as tryptophan at low efficiency in Bacillus subtilis.

Replacement of cat-86 codon 7 or 144 with the UGA codon permitted the gene to confer chloramphenicol resistance in wild-type Bacillus subtilis. UAA replacements of the same codons resulted in a chloramphenicol-sensitive phenotype in wild-type B. subtilis and a chloramphenicol-resistant phenotype in suppressor-positive strains. N-terminal sequencing showed that UGA at codon 7 was decoded as tryptophan in wild-type cells, at an efficiency of about 6%.

Bacillus subtilis↗
PubMed1978

The effect of specific structural modification on the biological activity of E. coli arginine tRNA.

Escherichia coli arginine tRNA1 has been modified at position s2C32 with iodoacetamide and a spin labelled derivative. The small effects on the charging ability of tRNA by the modifiications suggest that the synthetase does not bind to the tRNA in this region of the anticodon loop before the anticodon. A ternary complex of elongation factor Tu, GTP and the modified Arg-tRNA, can be formed allowing future studies of enzymatic binding to the ribosome. Using the triplet binding assay the native Arg-tRNA1 decodes all 4 codons beginning with CG. The modified Arg-tRNA1 has a restricted decoding but the decoding pattern is still unusual according to the Wobble Hypothesis.

Amino Acyl-tRNA Synthetases↗
PubMed2026

Pyridoxal-phosphate-dependent biosynthesis of aminovaleramide by AvaS in tRNA.

In eubacteria, decoding of isoleucine codon AUA requires a specialized tRNA (tRNAIle2) modified with lysidine (k2C) at the anticodon wobble position (C34), which switches decoding specificity from methionine (AUG) to isoleucine (AUA). Recently, aminovaleramide cytidine (ava2C) was discovered at the same tRNA position in several bacteria and plants and shown to support AUA decoding and Ile-specific aminoacylation. However, the enzyme catalyzing ava2C was unknown. Here, we report that tRNAIle-aminovaleramididine synthetase (AvaS) catalyzes ava2C biosynthesis in Pseudomonas aeruginosa PA14. AvaS converts k2C to ava2C through a pyridoxal-phosphate-dependent oxidative decarboxylation mechanism, supported by site-directed mutagenesis and in vitro enzymatic assays. Dual-reporter assays demonstrated that ava2C-modified tRNA exhibits lower AUA decoding efficiency than k2C-modified tRNA. Additionally, genome-wide screening revealed an unexpected link between ava2C levels and metabolic and stress response pathways influencing i6A/ms2i6A dynamics. Together, these findings define the molecular basis of ava2C biosynthesis and its broader cellular metabolic networks.

Journal Article↗
PubMed1985

Ribosomes are stalled during in vitro translation of alfalfa mosaic virus RNA 1.

In the presence of plant tRNAs the full-length translation product of alfalfa mosaic virus RNA 1 is produced in rabbit reticulocytes only at low mRNA concentration. At higher mRNA concentration translation is restricted to the 5' half of RNA 1. At high mRNA concentration the full-length product can be formed when additional plant tRNA and glutamine are supplied to the translation mixture. In contrast, in the presence of yeast or calf liver tRNA the translation pattern of alfalfa mosaic virus RNA 1 always results in the synthesis of the full-length product. Pulse-chase experiments in the presence of plant tRNAs show that the ribosomes pause at several positions in the 5' half of RNA 1. The pausing time is different at the different 'halting places'. Protein synthesis is resumed upon addition of glutamine, even when the addition is delayed for more than 3 h after the start of protein synthesis. Only one tRNA species, purified from wheat germ or tobacco, could promote full-length translation of RNA 1. This tRNA can be charged with glutamine. Analysis of the position of glutamine codons on RNA 1 shows a correlation between the positions of the CAA codons and the halting places of the ribosomes. The CAA codon (for any other codon) on its own cannot be responsible for the pausing of the ribosomes, since a variety of RNAs, known to contain all sense codons, are translated efficiently in rabbit reticulocyte lysates in the presence of plant tRNAs. Apparently other elements can restrict decoding of normal codons during protein chain elongation.

Animals↗
PubMed1986

Normal yeast tRNA(CAGGln) can suppress amber codons and is encoded by an essential gene.

We have isolated a gene that can encode yeast tRNA(CAGGln). When present on a multicopy plasmid, this gene suppresses the phenotype of a number of amber mutants, but has no effect on the ocher mutants tested. We therefore conclude that the anticodon CUG in tRNA(CAGGln) can decode the amber codon UAG by G-U mispairing, possibly by wobble base-pairing in the first codon position. This represents the second example we have observed in this laboratory of nonsense suppression in yeast by natural tRNA(Gln), involving G-U mispairing in the first codon position. Replacing the genomic copy of the cloned gene with a disrupted tRNA gene results in recessive lethality in heterozygous diploids and is lethal to haploid cells. This lethality can be rescued by transformation of cells with a single copy plasmid containing the tRNA(CAGGln) gene. Thus, the gene encoding tRNA(CAGGln) is apparently essential for viability in yeast, suggesting that it is normally present as a single copy gene.

Animals↗
PubMed1977

Rabbit liver tRNA1Val:I. Primary structure and unusual codon recognition.

The major valine acceptor tRNA1Val from rabbit liver was purified and its nucleotide sequence determined by in vitro [32P] - labeling with T4 phage induced polynucleotide kinase and finger-printing techniques. Its primary structure was found to be identical with the major valine tRNA from mouse myeloma cells. According to the wobble hypothesis this tRNA, which exclusively has an IAC anticodon, should decode the valine codons GUU, GUC and GUA only. However, this tRNA recognizes all four valine codons with a surprising preference for GUG. It is unknown whether this is due to the lack of A37 modification next to the 3' end of the anticodon IAC. The nature of the inosine-guanosine interaction remains to be clarified.

Animals↗
PubMed1987

Nucleotide sequences of three tRNA(Ser) from Drosophila melanogaster reading the six serine codons.

The nucleotide sequences of three serine tRNAs from Drosophila melanogaster, together capable of decoding the six serine codons, were determined. tRNA(Ser)2b has the anticodon GCU, tRNA(Ser)4 has CGA and tRNA(Ser)7 has IGA. tRNA(Ser)2b differs from the last two by about 25%. However, tRNA(Ser)4 and tRNA(Ser)7 are 96% homologous, differing only at the first position of the anticodon and two other sites. This unusual sequence relationship suggests, together with similar pairs in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, that eukaryotic tRNA(Ser)UCN may be undergoing concerted evolution.

Animals↗
PubMed2026

A Unified Mechanism of +1 Ribosomal Frameshifting.

Ribosomes decode 3-nucleotide codons and move in 1-codon increments to maintain the messenger RNA (mRNA) frame thereby accurately producing the encoded protein. In special cases, including viral genomes and regulatory cellular proteins, frameshifting occurs to expand the coding repertoire of an mRNA to make more than one protein. How these frameshifting events are induced and regulated is an active area of research. Here, we discuss recent progress in the understanding of +1 frameshifting (+1FS), during which the ribosome shifts by 1 mRNA nucleotide in the 3' direction. Structural and biochemical studies yielded insights into +1FS induced by mRNA slippery sequences and transfer RNA (tRNA) stem-loop expansion or modifications. tRNAs with an additional anticodon nucleotide are explored as a biotechnology tool for expanding the genetic code in an approach termed quadruplet decoding. We revisit the challenges of the quadruplet decoding model, discuss +1FS scenarios in bacteria and eukaryotes, and propose a unifying structural mechanism for +1FS.

Frameshifting, Ribosomal↗
PubMed1985

Partial suppression of an ochre mutation in Saccharomyces cerevisiae by multicopy plasmids containing a normal yeast tRNAGln gene.

We screened a yeast genomic library for recombinant DNA plasmids that complemented the ultraviolet (u.v.) sensitivity of a strain of Saccharomyces cerevisiae designated rad4-3 that is defective in excision repair of DNA. A multicopy plasmid (pNF4000) with a 9.4 X 10(3) base-pair yeast DNA insert partially complemented the u.v. sensitivity of rad4-3, but not of two other rad4 allelic mutants (rad4-2 and rad4-4), or of other u.v.-sensitive rad mutants. The yeast insert was analyzed by restriction mapping, DNA-DNA hybridization, DNA-tRNA hybridization and DNA sequencing. This analysis revealed the presence of a normal tRNAGln gene, a yeast sigma element situated 5' to the transfer RNA gene, a Ty element and a solo delta element. Deletion analysis of pNF4000 showed that the tRNAGln gene is required for partial complementation of the u.v. sensitivity of rad4-3. Furthermore, a multicopy plasmid containing a tRNAGln gene derived from a different region of the yeast genome also partially complemented the u.v. sensitivity of rad4-3. The rad4-3 mutation is suppressed following transformation with a plasmid containing the known ochre suppressor SUP11-o, indicating that it is an ochre mutation. We therefore conclude that when expressed in sufficient quantity, normal tRNAGln (which usually decodes the sense codon CAA) can weakly suppress the nonsense ochre codon UAA, and suggest that this represents an example of wobble occurring at the first rather than at the third position of the codon.

Base Sequence↗