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PubMed1993

UGA codon context which spans three codons. Reversal by ms2i6A37 in tRNA, mutation in rpsD(S4) or streptomycin.

Mutant UGA codon contexts which previously have been identified at different positions in the lacI part of a fused lacIlacZ gene were characterized with respect to translational readthrough in another genetic surrounding at a constant location. Although readthrough levels are systematically higher in this new location the "tight/leaky" characteristics of these codon contexts are essentially fully determined by the two codons flanking the nonsense codon itself. Analysis of some UGA hybrid contexts shows that the contribution to the codon context character by the codon either at the 5'-side (CCA or AGC) or at the 3'-side (NGU) is independent of the nature of the codon at the other side of UGA if this codon is decoded by trpT(Su9) suppressor tRNA. In a trpT(Su9), miaA double mutant strain, which lacks the ms2i6A37 modification in this tRNA, suppression is decreased at all UGA contexts investigated. However, in one case the contribution to the codon context character by the determinant flanking at one side is negatively affected by the nature of the codon at the other side of UGA. Thus, the character of a nonsense codon context in this case results from both flanking codons acting in a co-operative manner with the tRNA reading the middle UGA codon. This negative context effect is counteracted by a rpsD12 (ribosomal protein S4) mutation or by a sublethal concentration of streptomycin in the growth medium. It is suggested that the ms2i6A37 base in trpT(Su9) suppressor tRNA increases the efficiency of this tRNA by protecting it from ribosomal proofreading which is induced by codon context.

Bacterial Proteins↗
PubMed1984

Suppressor sufJ: a novel type of tRNA mutant that induces translational frameshifting.

We describe the DNA sequence change responsible for the creation of a frameshift-suppressor gene in Salmonella typhimurium. The suppressor, sufJ, results from a base-pair insertion in the gene coding for a threonine transfer RNA (tRNA3Thr). Unlike previously studied frameshift suppressor mutations, the sufJ insertion does not fall within the sequence corresponding to the tRNA anticodon. The insertion (a G.C base pair) occurs within a run of three G.C base pairs in that region of the gene coding for one strand of the anticodon stem. In the secondary structure of the mature tRNA, the net result is that an extra, unpaired cytidine residue is pushed into the anticodon loop, thus increasing the size of the loop to eight nucleotides. These findings are discussed in connection with the peculiar "three-out-of-four" method of reading by the sufJ suppressor. A unifying model is presented accounting for the contrasting decoding behaviors of tRNAs with eight-nucleotide-long anticodon loops.

Alleles↗
PubMed1986

Antisuppressor mutations and sulfur-carrying nucleosides in transfer RNAs of Schizosaccharomyces pombe.

Antisuppressor mutations reduce the efficiency of nonsense suppressors. A mutation in the gene sin4 of Schizosaccharomyces pombe leads to loss of 5-(methoxycarbonylmethyl) thiouridine (mcm5s2U) from the first anticodon position of tRNAs. This resembles the phenotype of sin3 (Heyer, W. D., Thuriaux, P., Kohli, J., Ebert, P., Kersten, H., Gehrke, C., Kuo, K. C., and Agris, P. F. (1984) J. Biol. Chem. 259, 2856-2862), but the mutations reside in different genes. In vivo 35S-labeled tRNA from the parental suppressor strain sup3, the antisuppressor strains sin3 and sin4, and the double mutant sin3 sin4 has been digested to nucleosides and analyzed with high performance liquid chromatography methods. The major sulfur-carrying nucleoside in wild-type S. pombe tRNA is mcm5s2U. It is reduced in the mutant strains. Two other thiolated nucleosides are also present: 2-thiouridine and a nucleoside of unknown structure. Neither was affected by the antisuppressor mutations. Thiocytidine has not been found. Independent from their effect on suppressors, the two mutations sin3 and sin4 reduce the growth rate of cells, and sin3 also increases cell length. In vivo decoding of the serine codon UCG by the UCA reading serine tRNA is not promoted by the two antisuppressor mutations.

Anticodon↗
PubMed2001

Ribosomal crystallography: from poorly diffracting microcrystals to high-resolution structures.

The cellular organelles translating the genetic code into proteins, the ribosomes, are large, asymmetric, flexible, and unstable ribonucleoprotein assemblies, hence they are difficult to crystallize. Despite two decades of intensive effort and thorough searches for suitable sources, so far only three crystal types have yielded high-resolution structures: two large subunits (from an archaean and from a mesophilic eubacterium) and one thermophilic small subunit. These structures have added to our understanding of decoding, have revealed dynamic aspects of the biosynthetic process, and have indicated the strategies adopted by ribosomes for interacting between themselves as well as with inhibitors, factors and substrates.

Archaea↗
PubMed2001

Anatomical risk factors for phonological dyslexia.

Successful behavioral genetic studies require precise definition of a homogenous phenotype. This study searched for anatomical markers that might restrict variability in the reading disability phenotype. The subjects were 15 college students (8 male/7 female) diagnosed with a reading disability (RD) and 15 controls (8 males/7 females). All subjects completed a cognitive and reading battery. Only 11 of the RD subjects had a phonological deficit [phonological dyslexia (PD): pseudo word decoding scores < 90 (27th percentile)]. Thirteen RD (9 PD) and 15 controls received a volumetric MRI scan. Four anatomical measures differentiated the PD group from the remainder of the subjects: (i) marked rightward cerebral asymmetry, (ii) marked leftward asymmetry of the anterior lobe of the cerebellum, (ii) combined leftward asymmetry of the planum and posterior ascending ramus of the sylvian fissure, and (iv) a large duplication of Heschl's gyrus on the left. When these four measures were normalized and summed, the resulting variable predicted short- and long-term phonological memory. By contrast, oral and written comprehension skills were predicted by a different anatomical variable: low cerebral volume. These findings provide neurobiological support for an RD phenotype characterized by phonological deficits in the presence of normal or superior comprehension. The study of individual variation in cortical structure may provide a useful link between genotype and behavior.

Adult↗
PubMed2025

CROPseq-multi: a universal solution for multiplexed perturbation in high-content pooled CRISPR screens.

Forward genetic screens seek to dissect complex biological systems by systematically perturbing genetic elements and observing the resulting phenotypes. While standard screening methodologies introduce individual perturbations, multiplexing perturbations improves the performance of single-target screens and enables combinatorial screens for the study of genetic interactions. Current tools for multiplexing perturbations are limited by technical challenges and do not offer compatibility across diverse screening methodologies, including enrichment, single-cell sequencing, and optical pooled screens. Here, we report the development of CROPseq-multi (CSM), a CROPseq1-inspired lentiviral system to multiplex Streptococcus pyogenes (Sp) Cas9-based perturbations with versatile readout compatibility and high performance for both perturbation and barcode identification. CSM has equivalent per-guide activity to CROPseq and low lentiviral recombination frequencies. Dual-guide CSM libraries are constructed in a single, facile molecular cloning step that facilitates the use of unique molecular identifiers. CSM is compatible with enrichment screening methodologies, single-cell RNA-sequencing readouts, and optical pooled screens. For optical pooled screens, an optimized and multiplexed in situ detection protocol improves barcode counts 10-fold (for mRNA detection), enables detection of recombination events, and reduces the number of sequencing cycles required for decoding by 3-fold relative to CROPseq. CROPseq-multi-v2 (CSMv2) adds compatibility for detection methods based on T7 RNA polymerase in vitro transcription2-5. CSM provides a single system for CRISPR screens that is compatible with individual and combinatorial perturbations, diverse SpCas9-based perturbation technologies, and multiple high-content, single-cell phenotypic readouts.

CRISPR Cas9↗
PubMed1998

Possible evolution of factors involved in protein biosynthesis.

The elongation factors of protein biosynthesis are well preserved through out evolution. They catalyze the elongation phase of protein biosynthesis, where on the ribosome amino acids are added one at a time to a growing peptide according to the genetic information transcribed into mRNA. Elongation factor Tu (EF-Tu) provides the binding of aminoacylated tRNA to the ribosome and protects the aminoester bond against hydrolysis until a correct match between the codon on mRNA and the anticodon on tRNA can be achieved. Elongation factor G (EF-G) supports the translocation of tRNAs and of mRNA on the ribosome so that a new codon can be exposed for decoding. Both these factors are GTP binding proteins, and as such exist in an active form with GTP and an inactive form with GDP bound to the nucleotide binding domain. Elongation factor Ts (EF-Ts) will catalyze the exchange of nucleotide on EF-Tu. This review describes structural work on EF-Tu performed in our laboratory over the last eight years. The structural results provide a rather complete picture of the major structural forms of EF-Tu, including the so called ternary complex of aa-tRNA:EF-Tu:GTP. The structural comparison of this ternary complex with the structure of EF-G:GDP displays an unexpected macromolecular mimicry, where three domains of EF-G mimick the shape of the tRNA in the ternary complex. This observation has initiated much speculation on the evolution of all factors involved in protein synthesis, as well as on the details of the ribosomal function in one part of elongation.

Amino Acid Sequence↗
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↗
PubMed2026

Cross-ancestry proteome-wide Mendelian randomization prioritizes 12 plasma protein candidates for breast cancer risk.

The plasma proteome provides a molecular bridge between genetic variation and disease risk, yet its contribution to breast cancer susceptibility across ancestries remains unclear. We conducted a proteome-wide Mendelian randomization (MR) study of 2,923 plasma proteins using cis-protein quantitative trait loci from 34,557 European participants in the UK Biobank Pharma Proteomics Project, integrated with genome-wide association studies of 156,901 breast cancer cases and 204,634 controls of European, East Asian, and African ancestries. Cross-ancestry meta-analysis identified 12 candidate proteins associated with breast cancer risk (P < 2.5&#xd7;10-5), including six previously reported and six newly implicated in MR studies. DNPH1 showed cross-ancestry heterogeneity, with a risk-increasing association in European populations and a nominally inverse association in East Asian populations. CASP8, RALB, and USP28 displayed subtype-differentiated associations. Orthogonal validation provided variable support: six demonstrated strong evidence of statistical colocalization; four replicated in an independent European proteomic dataset (deCODE, n = 35,559); two replicated in an independent East Asian proteomic dataset (JCTF, n = 1,384); and four were supported by polygenic-score analyses in the ancestrally diverse All of Us cohort (9,250 cases, 214,857 controls). These findings prioritize a high-confidence subset of plasma proteins, including LRRC25, PARK7, and LRRC37A2, for future mechanistic and translational investigation.

Mendelian randomization↗
PubMed1988

Molecular genetics of the apolipoprotein B gene in pigs in relation to atherosclerosis.

Immunologically defined alleles of the pig apolipoprotein B (ApoB) locus (apoB) are correlated with different blood cholesterol levels and predisposition towards premature coronary heart disease. We show here that these alleles are associated with differences in the apoB gene by identifying six restriction fragment length polymorphisms at the pig apoB locus. We have sequenced a 2.4-kb fragment encompassing exons 11 through 14 of one allele, and 7.1 kb from the 3' one-third of exon 26 to about 1 kb past the last exon from another allele. The decoded amino acids of the pig and human ApoB proteins are identical at 70% of these positions. One region close to the C-terminus of the protein is surprisingly different in pigs and humans (57% identity) but the C-terminal region is relatively well conserved (74% identity). Neither of the two putative low-density lipoprotein (LDL) receptor-binding sites is completely conserved in pigs and humans, but identical stretches of amino acids occur near these sites more frequently than in the other sequenced regions. We compare the nucleotide sequences of the region encompassing the putative LDL receptor-binding sites from four pig alleles, including one implicated directly in atherosclerosis. None of the differences appears to account for the hypercholesterolemic phenotype. We conclude that significant differences in the physiology of LDL particles result from changes outside the putative receptor-binding region.

Alleles↗
PubMed1995

UGA suppression by a mutant RNA of the large ribosomal subunit.

A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 168 rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations. In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA (UGA211) of E. coli. The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination. The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon. Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA. Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L11, or 16S rRNA. Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon.

Base Sequence↗
PubMed1995

tRNA genes transcribed from the plastid-like DNA of Plasmodium falciparum.

Besides their mitochondrial genome, malarial parasites contain a second organellar DNA. This 35 kb circular molecule has a number of features reminiscent of plastid DNAs. Sequence analysis shows that along with other genes the circle codes for 25 different tRNAs all of which are transcribed. Six of the tRNAs have some unusual features, and one has an intron, the only one found so far on the circle. Comparison of codon and anticodon usage indicates that the 25 tRNAs are sufficient to decode all the protein genes present on the circle. The maintenance of such a parsimonious but complete translation system is further evidence for the functionality of the circle.

Animals↗
PubMed1995

The CUG codon is decoded in vivo as serine and not leucine in Candida albicans.

Previous studies have shown that the yeast Candida albicans encodes a unique seryl-tRNA(CAG) that should decode the leucine codon CUG as serine. However, in vitro translation of several different CUG-containing mRNAs in the presence of this unusual seryl-tRNA(CAG) result in an apparent increase in the molecular weight of the encoded polypeptides as judged by SDS-PAGE even though the molecular weight of serine is lower than that of leucine. A possible explanation for this altered electrophoretic mobility is that the CUG codon is decoded as modified serine in vitro. To elucidate the nature of CUG decoding in vivo, a reporter system based on the C. albicans gene (RBP1) encoding rapamycin-binding protein (RBP), coupled to the promoter of the C. albicans TEF3 gene, was utilized. Sequencing and mass-spectrometry analysis of the recombinant RBP expressed in C. albicans demonstrated that the CUG codon was decoded exclusively as serine while the related CUU codon was translated as leucine. A database search revealed that 32 out of the 65 C. albicans gene sequences available have CUG codons in their open reading frames. The CUG-containing genes do not belong to any particular gene family. Thus the amino acid specified by the CUG codon has been reassigned within the mRNAs of C. albicans. We argue here that this unique genetic code change in cellular mRNAs cannot be explained by the 'Codon Reassignment Theory'.

Amino Acid Sequence↗
PubMed2002

The transorientation hypothesis for codon recognition during protein synthesis.

During decoding, a codon of messenger RNA is matched with its cognate aminoacyl-transfer RNA and the amino acid carried by the tRNA is added to the growing protein chain. Here we propose a molecular mechanism for the decoding phase of translation: the transorientation hypothesis. The model incorporates a newly identified tRNA binding site and utilizes a flip between two tRNA anticodon loop structures, the 5'-stacked and the 3'-stacked conformations. The anticodon loop acts as a three-dimensional hinge permitting rotation of the tRNA about a relatively fixed codon-anticodon pair. This rotation, driven by a conformational change in elongation factor Tu involving GTP hydrolysis, transorients the incoming tRNA into the A site from the D site of initial binding and decoding, where it can be proofread and accommodated. The proposed mechanisms are compatible with the known structures, conformations and functions of the ribosome and its component parts including tRNAs and EF-Tu, in both the GTP and GDP states.

Anticodon↗
PubMed2001

Regulation of gene expression by action potentials: dependence on complexity in cellular information processing.

Nervous system development and plasticity are regulated by neural impulse activity, but it is not well understood how the pattern of action potential firing could regulate the expression of genes responsible for long-term adaptive responses in the nervous system. Studies on mouse sensory neurons in cell cultures equipped with stimulating electrodes show that specific genes can be regulated by different patterns of action potentials, and that the temporal dynamics of intracellular signalling cascades are critical in decoding and integrating information contained in the pattern of neural impulse activity. Functional consequences include effects on neurite outgrowth, cell adhesion, synaptic plasticity and axon-glial interactions. Signalling pathways involving Ca2+, CaM KII, MAPK and CREB are particularly important in coupling action potential firing to the transcriptional regulation of both neurons and glia, and in the conversion of short-term to long-term memory. Action potentials activate multiple convergent and divergent pathways, and the complex network properties of intracellular signalling and transcriptional regulatory mechanisms contribute to spike frequency decoding.

Action Potentials↗
PubMed2001

Seryl-tRNA synthetase is not responsible for the evolution of CUG codon reassignment in Candida albicans.

A number of Candida species translate the standard leucine-CUG codon as serine using a novel ser-tRNA(CAG). This tRNA, which has an unusual anticodon stem-loop structure, has been implicated in the evolution of this codon reassignment. However, such a sense codon reassignment might also require a change in the specificity of the cognate aminoacyl tRNA-synthetase, in this case the ser-tRNA synthetase. Here we describe the cloning and sequence analysis of the C. albicans seryl aminoacyl-tRNA synthetase (CaSerRS) gene (CaSES1). The predicted CaSerRS sequence shows a significant level of amino acid identity to SerRs from other organisms and fully complements a S. cerevisiae SerRS null strain without any apparent defect in growth rate. This suggests that the SerRS recognizes and charges S. cerevisiae ser-tRNAs with similar efficiency to that of the S. cerevisiae SerRS. Using an antibody raised against CaSerRS, we also demonstrate the presence of SerRS in a range of Candida spp. showing CUG codon reassignment. We conclude that the key element in CUG reassigment in Candida spp. is the tRNA that decodes the CUG codon rather than a SerRS structural change. The nucleotide sequence of the CaSES1 gene has been deposited at GenBank under Accession No. AF290915.

Amino Acid Sequence↗
PubMed2000

Inhibition of selenoprotein synthesis by selenocysteine tRNA[Ser]Sec lacking isopentenyladenosine.

A common posttranscriptional modification of tRNA is the isopentenylation of adenosine at position 37, creating isopentenyladenosine (i(6)A). The role of this modified nucleoside in protein synthesis of higher eukaryotes is not well understood. Selenocysteyl (Sec) tRNA (tRNA([Ser]Sec)) decodes specific UGA codons and contains i(6)A. To address the role of the modified nucleoside in this tRNA, we constructed a site-specific mutation, which eliminates the site of isopentenylation, in the Xenopus tRNA([Ser]Sec) gene. Transfection of the mutant tRNA([Ser]Sec) gene resulted in 80% and 95% reduction in the expression of co-transfected selenoprotein genes encoding type I and II iodothyronine deiodinases, respectively. A similar decrease in type I deiodinase synthesis was observed when transfected cells were treated with lovastatin, an inhibitor of the biosynthesis of the isopentenyl moiety. Neither co-transfection with the mutant tRNA gene nor lovastatin treatment reduced type I deiodinase mRNA levels. Also, mutant tRNA expression did not alter initiation of translation or degradation of the type I deiodinase protein. Furthermore, isopentenylation of tRNA([Ser]Sec) was not required for synthesis of Sec on the tRNA. We conclude that isopentenylation of tRNA([Ser]Sec) is required for efficient translational decoding of UGA and synthesis of selenoproteins.

Animals↗
PubMed1983

Anticodon shift in tRNA: a novel mechanism in missense and nonsense suppression.

In a previous publication, an unusual UGG-reading missense suppressor caused by insertion of an extra adenylate residue in the anticodon loop of an Escherichia coli glycine tRNA was described. In this study, we provide in vivo evidence that the additional nucleotide causes an "anticodon shift" by one nucleotide in the 3' direction and that the "new" anticodon can explain the unanticipated coding properties of the suppressor. We converted the UGG suppressor with ethyl methanesulfonate, a base-substitution mutagen, to suppressors that read codons related to UGG by a single base change. Sequence analysis of each mutant tRNA revealed that its mutational alteration was an anticipated base change in one of the three nucleotides of the "new" anticodon. Although the new suppressors read codons beginning with A or U, the mutant tRNAs lack the customary hypermodified nucleosides on the 3' side of the anticodon. As determined on the basis of their in vivo coding specificities, the new mutant tRNAs do not continue to utilize the original anticodon triplet for decoding. Furthermore, the failure of the UGG suppressor to correct frameshift mutations throughout each of three genes of the trp operon suggests that the addition of a nucleotide to the anticodon loop of a tRNA does not necessarily result in out-of-frame decoding by the tRNA. Therefore, a "frameshift" mutation in a tRNA has principally changed the triplet codon recognition properties of the molecule.

Anticodon↗