Insulin-induced translocation of protein kinase B to the plasma membrane in rat adipocytes

Translation termination in vivo was studied in the yeast Saccharomyces cerevisiae using a translation-assay system. Codon changes that were made at position -2 relative to the stop codon, gave a 3.5-fold effect on termination in a release-factor-defective (sup45) mutant strain, in line with the effect observed in a wild-type strain. The influence of the -2 codon could be correlated to the charge of the corresponding amino acid residue in the nascent peptide; an acidic residue favoring efficient termination. Thus, the C-terminal end of the nascent peptide influences translation termination both in the bacterium Escherichia coli and to a lesser extent in the yeast S. cerevisiae. However, the sensitivity to the charge of the penultimate amino acid is reversed when the E. coli and S. cerevisiae are compared. Changing - 1 (P-site) codons in yeast gave a 10-fold difference in effect on the efficiency of termination. This effect could not be related to any property of the encoded last amino acid in the nascent peptide. Iso-codons read by the same tRNA (AAA/G, GAA/G) gave similar readthrough values. Codons for glutamine (CAA/G), glutamic acid (GAA/G) and isoleucine (AUA/C) that are read by different isoaccepting tRNAs are associated with an approximately twofold difference in each case in termination efficiency. This suggests that the P-site tRNA is able to influence termination at UGAC in yeast.     

The methyl group of the N6-methyl-N6-threonylcarbamoyladenosine in tRNA of Escherichia coli modestly improves the efficiency of the tRNA

tRNA species that read codons starting with adenosine (A) contain N6-threonylcarbamoyladenosine (t6A) derivatives adjacent to and 3' of the anticodons from all organisms. In Escherichia coli there are 12 such tRNA species of which two (tRNA(Thr1)GGU and tRNA(Thr3)GGU) have the t6A derivative N6-methyl-N6-threonylcarbamoyladenosine (m6t6A37). We have isolated a mutant of E. coli that lacks the m6t6A37 in these two tRNA(Thr)GGU species. These tRNA species in the mutant are likely to have t6A37 instead of m6t6A37. We show that the methyl group of m6t6A37 originates from S-adenosyl-L-methionine and that the gene (tsaA) which most likely encodes tRNA(m6t6A37)methyltransferase is located at min 4.6 on the E. coli chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA(Thr)GGU to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m6t6A37-containing tRNA(Thr)GGU species. We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m6t6A37 in tRNA(Thr)GGU slightly reduces the efficiency of this tRNA to read cognate codon ACC.     

Quantitative analysis of in vivo ribosomal events at UGA and UAG stop codons

An in vivo translation assay system has been designed to measure, in one and the same assay, the three alternatives for a ribosome poised at a stop codon (termination, read-through and frameshift). A quantitative analysis of the competition has been done in the presence and absence of release factor (RF) mutants, nonsense suppressors and an upstream Shine-Dalgarno-like sequence. The ribosomal +1 frameshift product is measurable when the stop codon is decoded by wild-type or mutant RF (prf A1 or prf B2) and also in the presence of competing suppressor tRNAs. Frameshift frequency appears to be influenced by RF activity. The amount of frameshift product decreases in the presence of competing suppressor tRNAs, however, this decrease is not in proportion to the corresponding increase in the suppression product. Instead, there is an increase in the total amount of protein expressed from the gene, perhaps due to the purging of queued ribosomes. Mutated RFs reduce the total output of the reporter gene by reducing the amount of all three protein products. The nascent peptide has earlier been shown to influence the translation termination process by interacting with the RFs. At 42 degrees C in a temperature-sensitive RF mutant strain, protein measurements indicate that the nascent peptide seems to influence the binding efficiencies of the RFs.     

Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons

Prokaryotic translational release factors, RF1 and RF2, catalyze polypeptide release at UAG/UAA and UGA/UAA stop codons, respectively. In this study, we isolated a bacterial RF2 mutant (RF2*) containing an E167K substitution that restored the growth of a temperature-sensitive RF1 strain of Escherichia coli and the viability of a chromosomal RF1/RF2 double knockout. In both in vivo and in vitro polypeptide termination assays, RF2* catalyzed UAG/UAA termination, as does RF1, as well as UGA termination, showing that RF2* acquired omnipotent release activity. This result suggests that the E167K mutation abolished the putative third-base discriminator function of RF2. These findings are interpreted as indicating that prokaryotic and eukaryotic release factors share the same anticodon moiety and that only one omnipotent release factor is sufficient for bacterial growth, similar to the eukaryotic single omnipotent factor.     

Crystal structures of three misacylating mutants of Escherichia coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP

Three previously described mutant Escherichia coli glutaminyl-tRNA synthetase (GlnRS) proteins that incorrectly aminoacylate the amber suppressor derived from tRNATyr (supF) with glutamine were cocrystallized with wild-type tRNAGln and their structures determined. In two of the mutant enzymes studied, Asp235, which contacts base pair G3-C70 in the acceptor stem, has been changed to asparagine in GlnRS7 and to glycine in GlnRS10. These mutations result in changed interactions between Asn235 of GlnRS7 and G3-C70 of the tRNA and an altered water structure between Gly235 of GlnRS10 and base pair G3-C70. These structures suggest how the mutant enzymes can show only small changes in their ability to aminoacylate wild-type cognate tRNA on the one hand and yet show a lack of discrimination against a noncognate U3-A70 base pair on the other. In contrast, the change of Ile129 to Thr in GlnRS15 causes virtually no change in the structure of the complex, and the explanation for its ability to misacylate supF is unclear.     

Major groove binding of the tRNA/mRNA complex to the 16 S ribosomal RNA decoding site

We propose a detailed three-dimensional model, with atomic detail, for the structure of the Escherichia coli 16 S rRNA decoding site in a complex with mRNA and the A and P-site tRNAs. Model building began with four primary assumptions: (1) A and P-site tRNA conformations are identical with those seen in the tRNA crystal structure; (2) A and P-site tRNAs adopt an S-type orientation upon binding mRNA in the ribosome; (3) A1492 and A1493 bind non-specifically to the mRNA through a series of hydrogen bonds; and (4) C1400 lies in close proximity to the P-site tRNA wobble base in order to satisfy a UV-induced photocrosslink formed between the two residues. We have models with both major groove and minor groove binding of the tRNA/mRNA complex to the decoding site RNA, and conclude that major groove binding is more likely. Both classes of models maintain structural features reported in the NMR structure of the A-site region of the decoding site RNA with bound paromomycin. We also present models for the tRNA/mRNA complex bound to the decoding site RNA in the presence of the aminoglycoside paromomycin. We discuss possible mechanisms for ribosomal proof reading and antibiotic disruption of this proofreading.     

Epidemiology of IgA nephropathy in central and eastern Kentucky for the period 1975 through 1994. Central Kentucky Region of the Southeastern United States IgA Nephropathy DATABANK Project

Bacillus subtilis has been thought to have a high readthrough rate at the UGA stop codon because no opal suppressor tRNA has been isolated so far [Lovett et al. (1991) J. Bacteriol. 173, 1810-1812]. To examine whether a tRNATrp which we have characterized [Matsugi et al. (1992) Nucleic Acids Res. 20, 3514] has the ability to read the UGA codon, in vitro translation was performed with a synthetic mRNA containing a test codon, UGA, UAG, UAA, or UGG, in a reading frame. Addition of Trp-tRNATrp to the system significantly increased the readthrough rate only in the case of UGA. This suggests that this tRNATrp has a dual recognition pattern in B. subtilis, i.e., for the canonical tryptophan codon and for readthrough at the UGA stop codon.     

Maternal smoking and body composition of the newborn

Translational stop signals are defined in the genetic code as UAA, UAG and UGA, although the mechanism of their decoding via protein factors is clearly different from that of the other codons. There are strong biases in the upstream and downstream nucleotides surrounding stop codons. Experimental tests have shown that termination-signal strength is strongly influenced by the identity of the nucleotide immediately downstream of the codon (+4), with a correlation between the strength of this four-base signal and its occurrence at termination sites. The +4 nucleotide and other biases downstream of the stop codon may reflect sites of contact between the release factor and the mRNA, whereas upstream biases may be due to coding restrictions, with the release factor perhaps recognizing the final tRNA and the last two amino acids of the polypeptide undergoing synthesis. This means that the translational stop signal is probably larger than the triplet codon, but its exact length will be clearer when it is known which nucleotides are in direct contact with the release factor. Ultimately it will be defined exactly when a crystal structure of the release factor with its recognition substrate becomes available.     

tRNA imbalance promotes -1 frameshifting via near-cognate decoding

tRNAGly1 is the Escherichia coli glycine tRNA specific for GGG codons. A genetic selection for multicopy suppressors of a frameshift mutation has shown that increased levels of wild-type tRNAGly1 causes -1 frameshifting. Analysis of the suppression spectrum of this multicopy suppressor and peptide sequencing of the suppressed protein product showed that it promoted GG doublet decoding at the near-cognate GGA codons. It is proposed that increasing the concentration of the GGG-specific tRNAGly1 relative to the cognate GGA-decoding tRNAGly2 allows the near-cognate tRNA to read GGA codons. Near-cognate decoding of GGA codons by tRNAGly1 can occur by a two-out-of-three reading mechanism, in which only the first two bases of the GGA codon are paired with the anticodon, thus permitting doublet translocations. In mycoplasmas, a single tRNA typically decodes all four triplets of a codon family and introduction of a feature of the Mypoplasma mycoides tRNAGly responsible for non-discriminate decoding, a C at position 32, into the anticodon E. coli tRNAGly1, enhanced the efficiency of doublet decoding.     

Analysis of effects of tRNA:message stability on frameshift frequency at the Escherichia coli RF2 programmed frameshift site

The codon that is in-frame prior to +1 frameshifting at the E.coli prfB (RF2 gene) frameshift site is randomized to create thirty-two variants. These alleles vary 1000-fold in frameshift-dependent expression in fusions to lacZ. Frameshifting is more frequent at sites where the in-frame codon ends in uridine, as if third position wobble pairs to message uridine facilitate slippage into the +1 frame. Consistent with other studies of programmed frameshift sites, efficient frameshifting depends on stable message:tRNA base pairs after rephasing. For complexes with mispairs, frameshift frequency depends on the nature, number, and position of mispairs. Central purine:purine mispairs are especially inhibitory. Relative stabilities of +1 rephased complexes are estimated from published data on the stabilities of tRNA:tRNA complexes. Stability correlates with frameshifting over its entire range, which suggests that stability is an important determinant of the probability of translation of the rephased complex.     

Random mutagenesis into the conserved Gly154 of subtilisin E: isolation and characterization of the revertant enzymes

We analyzed the role played by the conserved Gly154, a constituent of the P1 substrate-binding pocket of Bacillus subtilis subtilisin E, in the catalytic properties of the protease. Using an Escherichia coli expression system, the termination codon at position 154 in subtilisin E was first introduced to abolish the catalytic activity through truncation of the C-terminus from amino acid residues 154-275. We then attempted to obtain revertants with substitutions of various amino acids at position 154 by the polymerase chain reaction using a mixture of oligonucleotides. In addition to the Gly residue (wild-type), six amino acid substitutions (Ala, Arg, Leu, Phe, Pro and Thr) gave caseinolytic activity. When assayed with synthetic peptide substrates, most of the revertants showed a considerable decrease in specific activity and a P1 specificity similar to that of the wild-type enzyme. An Ala154 mutant purified from the periplasmic space in E. coli, however, resulted in an up to 2.3-fold preference for Val rather than Pro as a P2 substrate relative to the wild-type. Further, a significant 2-10-fold increase in the catalytic efficiency occurred in the Gly127Ala plus Gly154Ala combination variant, relative to the single Gly127Ala variant, without any change in the restricted specificity. The kinetic data and molecular modeling analysis demonstrate the important role of position 154 in the catalytic efficiency as well as in the substrate specificity of subtilisin E.     

Complete sequence of the amphioxus (Branchiostoma lanceolatum) mitochondrial genome: relations to vertebrates

The complete nucleotide sequence of the mitochondrial DNA of the amphioxus Branchiostoma lanceolatum has been determined. This mitochondrial genome is small (15 076 bp) because of the short size of the two rRNA genes and the tRNA genes. In addition, this genome contains a very short non-coding region (57 bp) with no sequence reminiscent of a control region. The organisation of the coding genes, as well as of the two rRNA genes, is identical to that of the sea lamprey. Some differences in the repartition of the tRNA genes occur when compared to the lamprey. The mitochondrial codon usage of the amphioxus is reminiscent of that of urochordates since the AGA codon is read as a glycine and not as a stop codon as in vertebrates. Moreover, the base composition at the wobble positions of the codon is strongly biased toward guanine. Altogether, these data clearly emphasise the close relationships between amphioxus and vertebrates, and reinforce the notion that prochordates may be viewed as the brother group of vertebrates.     

Novel temperature-sensitive mutants of Escherichia coli that are unable to grow in the absence of wild-type tRNA6Leu

Escherichia coli has only a single copy of a gene for tRNA6Leu (Y. Komine et al., J. Mol. Biol. 212:579-598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O2-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA4Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O2-methyluridine) as its anticodon, tRNA6Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA6Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su+6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA6Leu. These tRNA6Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA4Leu restored the growth of group I mutants at 42 degrees C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA4Leu poorly recognizes the UUG codon at 42 degreesC and that the wild-type tRNA6Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA6Leu remains to be investigated.     

Initiation of protein synthesis in mammalian cells with codons other than AUG and amino acids other than methionine

Protein synthesis is initiated universally with the amino acid methionine. In Escherichia coli, studies with anticodon sequence mutants of the initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, however, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding raised the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we studied the activities, in initiation, of four different anticodon sequence mutants of human initiator tRNA in mammalian COS1 cells, using reporter genes carrying mutations in the initiation codon that are complementary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, respectively. CAG initiates protein synthesis with glutamine but extremely poorly, whereas UAG could not be used to initiate protein synthesis with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons other than AUG for studying the many interesting aspects of protein synthesis initiation in mammalian cells.