The translational efficiency of tRNA is a property of the anticodon arm

We have reciprocally transplanted the anticodon arm sequences of a set of amber suppressor tRNA genes, using recombinant DNA techniques. By this means, a very efficient suppressor may be converted to a poor one, and the poorest tRNA to the efficiency of the best one. In tRNA molecules of normal 2 degrees and 3 degrees structure, the suppressor efficiencies of different composite tRNAs having the same anticodon arm sequence are approximately the same. Large numbers of simultaneous changes throughout the rest of the molecule do not affect the efficiency. Selective nucleotide modification as a result of varied anticodon arm sequences cannot explain these efficiencies. Efficiencies are also unlikely to differ because of selective aminoacylation. Measurement of in vivo tRNA shows, however, that tRNA levels do vary if the anticodon arm sequence is changed. If tRNA levels are normalized, the anticodon arm effect on the translational efficiency remains. Therefore, different anticodon arms, all of normal secondary structure, are not equivalent in translation. The most efficient sequences in this series resemble those found in natural tRNAs associated with similar anticodons, as is proposed in the extended anticodon theory (Yarus, M. (1982) Science 218, 646-652). These molecules also provide some information on the specificity of nucleotide modification enzymes and on determinants of the steady-state tRNA level.     

Preference for guanosine at first codon position in highly expressed Escherichia coli genes. A relationship with translational efficiency.

The variation in base composition at the three codon sites in relation to gene expressivity, the latter estimated by the Codon Adaptation Index, has been studied in a sample of 1371 Escherichia coli genes. Correlation and regression analyses show that increasing expression levels are accompanied by higher frequencies of base G at first, of base A at second and of base C at third codon positions. However, correlation between expressivity and base compositional biases at each codon site was only significant and positive at first codon position. The preference for G-starting codons as gene expression level increases is discussed in terms of translational optimization.     

Modified nucleoside, 5-carbamoylmethyluridine, located in the first position of the anticodon of yeast valine tRNA

A novel modified nucleoside located in the first position of the anticodon of yeast tRNAVal2a was isolated and its chemical structure was characterized as 5-carbamoylmethyluridine by means of ultraviolet absorption spectrum, mass spectrum, and nuclear magnetic resonance spectrum.     

A novel lysine-substituted nucleoside in the first position of the anticodon of minor isoleucine tRNA from Escherichia coli

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium ("lysidine"), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.     

Identification of a modified nucleoside located in the first position of the anticodon of Torulopsis utilis tRNAPro as 5-carbamoylmethyluridine

An unknown nucleoside in the first position of the anticodon of Torulopsis utilis tRNAPro has been isolated. The UV, 1H NMR and secondary ion mass spectra indicated that this nucleoside is a uridine derivative, 5-carbamoylmethyluridine. The structure was completely established by comparison of the instrumental analysis results and chromatographic behavior of the isolated nucleoside with those of a synthetic sample.     

Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading.

Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-->C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes.     

5-(carboxymethylaminomethyl)-2-thiouridine, a new modified nucleoside found at the first letter position of the anticodon.

The structure of a modified uridine derivative which was detected at the first letter position of the anticodon of Bacillus subtilis tRNA1Lys was determined to be 5-(carboxymethylaminomethyl)-2-thiouridine. The determination was mainly based in this ultraviolet absorption spectra and mass spectrometric analysis of the trimethylsilyl derivative.     

Mutations upstream of the ribosome-binding site affect translational efficiency

The DNA coding for the major outer membrane lipoprotein of Escherichia coli has been fused to the coding region of the beta-galactosidase gene to measure the effect of various mutations on the efficiency of translation initiation. The various mutants were made by either inserting or deleting a small number of nucleotides into or from a region just upstream of the ribosome-binding site. These small mutations dramatically affect translation initiation as measured by the production of beta-galactosidase. We postulate that these mutations affect translation initiation by altering the secondary structure of the messenger RNA. In one case, we predict that a stem and loop just upstream of the Shine-Dalgarno sequence sterically hinders the binding of the ribosome to the mRNA.     

5-methoxyuridine: a new minor constituent located in the first position of the anticodon of tRNAAla, tRNAThr, and tRNAVal from Bacillus subtilis.

The sequences of the anticodon of tRNAAla, tRNAThr, and tRNAVal from Bacillus subtilis W 168 were N-G-C, N-G-U, and N-A-C, respectively. A new minor constituent, N, occupied the first position of the anticodon of each tRNA. N was indentified as 5-methoxyuridine (mo5U, Figure 1) by comparison of its UV absorption spectra, Rf values in thin-layer chromatography using several solvent systems and mass spectra with those of chemically synthesized specimen.     

Lanthanum reduces the excitation efficiency in fly photoreceptors

Lanthanum (La3+), a known inhibitor of Ca2+ binding proteins, was applied to the extracellular space of fly retina. Shot noise analysis indicated that a combination of intense light and La3+ caused a large (down to zero) reduction in the rate of occurrence of the quantal responses to single photons (quantum bumps) which sum to produce the photoreceptor potential. Light in the presence of La3+ also increased the effective bump duration. These effects are very similar to the effects of the mutations trp of Drosophila and nss of Lucilia flies on the quantum bump rate and duration. La3+ applied to the nss mutant caused only a small reduction in the bump rate, suggesting that La3+ may affect the nss gene product which is deficient in the mutant. The close similarity in the properties of the receptor potential of the La(3+)-treated photoreceptor of the wild type and of the nss mutant together with existing evidence for the highly reduced intracellular Ca2+ ([Ca2+]i) level in nss photoreceptors suggest that both La3+ and the mutation cause a severe reduction in [Ca2+]i. This effect may arise from an inhibition of a Ca2+ transporter protein located in the surface membrane that normally replenishes Ca2+ pools in the photoreceptors, a process essential for light excitation.     

Altering the position of the first horizontal cleavage furrow of the amphibian (Xenopus) egg reduces embryonic survival.

The animal/vegetal cleavage ratio (AVCR), defined as the ratio of the height of the animal blastomere to the height of the Xenopus embryo at the 8 cell stage, can be shifted by placing embryos in novel gravitational fields: clinostating (microgravity simulation) increases AVCR, and centrifugation (hypergravity simulation) reduces AVCR. This report contributes to an understanding of the subcellular mechanism responsible for the furrow relocation and assesses its significance. Embryo inversion and D2O immersion were found to increase AVCR, and cold shock was found to reduce AVCR. Based on the additive or antagonistic effects of combined treatments, it is postulated that the primary cause of AVCR changes is an alteration in the distribution of yolk platelets and the rearrangement of microtubule arrays. Embryos with a decreased AVCR exhibited reduced survival in early developmental stages, indicating serious difficulties in cleavage, blastulation and/or gastrulation. Cold-shocked embryos with a reduced AVCR could be rescued by D2O pretreatment or clinostating, an observation which supports the notion that changes accompanying AVCR modifications represent the primary cause of the reduction in percent survival.     

Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy.

Effects on translation in vivo by modification deficiencies for 2-methylthio-N6-isopentenyladenosine (ms2i6A) (Escherichia coli) or 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) (Salmonella typhimurium) in tRNA were studied in mutant strains. These hypermodified nucleosides are present on the 3' side of the anticodon (position 37) in tRNA reading codons starting with uridine. In E. coli, translational error caused by tRNA was strongly reduced in the case of third-position misreading of a tryptophan codon (UGG) in a particular codon context but was not affected in the case of first-position misreading of an arginine codon (CGU) in another codon context. Misreading of UGA nonsense codons at two different positions was codon context dependent. The efficiencies of some tRNA nonsense suppressors were decreased in a tRNA-dependent manner. Suppressor tRNA which lacks ms2i6A-ms2io6A becomes more sensitive to codon context. Our results therefore indicate that, besides improving translational efficiency, ms2i6A37 and ms2io6A37 modifications in tRNA are also involved in decreasing the intrinsic codon reading context sensitivity of tRNA. Possible consequences for regulation of gene expression are discussed.