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PubMed1989

Spectrin Tunis (Sp alpha I/78), an elliptocytogenic variant, is due to the CGG----TGG codon change (Arg----Trp) at position 35 of the alpha I domain.

Spectrin Tunis (Sp alpha I/78) is an alpha l domain variant that causes asymptomatic elliptocytosis in the heterozygote state. It is manifested by a reduction of spectrin dimer self-association and by the development of a major 78-Kd fragment at the expense of the alpha l 80-Kd fragment upon spectrin-limited digestion. Amino acid sequence analysis, following peptide transfer onto Immobilon membranes, showed that the 78-Kd fragment results from a sensitized cleavage after lysyl residue 10. Using a 13.5-kb genomic alpha-spectrin probe and the Xbal, Pvull, and Mspl polymorphic sites detected with this probe, we concluded that spectrin Tunis is associated with the + - + haplotype (in the above order). Twenty mer oligonucleotides, complementary to genomic segments from introns 2 and 3, respectively, were synthesized. We then performed DNA amplification and sequencing. In the two investigated carriers of spectrin Tunis, we found the C----T base substitution of the codon corresponding to position 35 of the alpha l domain (CGG----TGG; Arg----Trp). The mutation lies in the last part of an alpha helix that extends from residues 9 to 44 of partial repeat alpha 1' and is comparable with helix 3 of full repeats 1 to 5. The modified proteolytic site, located 25 amino acid residues upstream, occurs at the beginning of the helix.

Amino Acid Sequence↗

PubMed1999

Two novel point mutations of mitochondrial tRNA genes in histologically confirmed Parkinson disease.

Mutations in mitochondrially encoded tRNA genes have been described in a variety of neurological disorders. One such mutation, the A to G transition at nucleotide position 4336 of the mitochondrial tRNA(Gln) gene, has been associated with both Alzheimer and Parkinson disease. We have now performed a complete sequence analysis of all 22 mitochondrially encoded tRNA genes in 20 cases of histologically proven idiopathic Parkinson disease. Genomic DNA extracted from the substantia nigra of frozen or formalin-fixed and paraffin-embedded brains was used for amplification by polymerase chain reaction followed by automated sequencing. Two new homoplasmic point mutations were detected in the genes for tRNA(Thr) (15950 G/A) and tRNA(Pro) (15965 T/C) in 1 patient each. Restriction enzyme digestion revealed absence of the 15950 G/A mutation in 96 controls and in 40 cases of neuropathologically confirmed Alzheimer disease. The 15965 T/C mutation was shown to be absent from 100 control subjects and 47 Alzheimer cases. In addition to the two novel mutations, six known sequence variants were detected in a total of 6 different patients in the genes for tRNA(Asp) (G7521A, 1), tRNA(Arg) (T10463C, 1), tRNA(LeuCUN) (A12308G, 2), and tRNA(Thr) (A15924G, 1; G15928A, 2), including 1 patient carrying the tRNA(Gln) (A4336G) mutation. The G15950A transition affects position 70 of the aminoacyl acceptor stem of tRNA(Thr), which has been implicated as a recognition element for threonyl-tRNA synthetase and, at least in some tRNAs, in the processing of primary mitochondrial transcripts. The T15965C point mutation in the mitochondrial tRNA(Pro) gene alters position 64 of the TpsiC stem. The corresponding nucleotide in bacterial aminoacyl-tRNAs is involved in the interaction with elongation factor Tu. Thus, the two novel mutations are likely to be of functional relevance and could contribute to dopaminergic nerve cell death in affected individuals.

Aged↗

PubMed1983

Different regions of aminoacyl-tRNA regulate the function of elongation factor Tu.

In this work we show that intact aminoacyl-tRNA (aa-tRNA) and its 3' half-molecule, but not its 3' C-C-A-aa fragment, require selective ionic conditions for stimulating the mRNA-independent GTPase of elongation factor Tu (EF-Tu) in the presence of ribosomes.l Stimulation by aa-tRNA and its 3' half-molecule is only observed at 20 and 30 mM Mg2+ and not at 10 mM, where they exert inhibitory activity; by contrast, C-C-A-aa enhances the GTPase activity at all three of these Mg2+ concentrations. Ammonium ion is needed for stimulation by C-C-A-aa, whereas it inhibits the stimulation by aa-tRNA and its 3' half-molecule. The concentration of aminoacylated fragments needed for half-maximum stimulation follows this order: A-Val much greater than C-A-Val greater than C-C-A-Val much greater than 3' Val-tRNA1Val half-molecule greater than Val-tRNA1Val. The extent of maximum stimulation of the EF-Tu GTPase in the presence of ribosomes varies moderately depending on the aa-tRNA species; a clear dependence on the nature of the aminoacyl side chain is observed in the effects of their respective C-C-A-aa fragments tested (C-C-A-Arg, C-C-A-Val, C-C-A-Phe, C-C-A-Met, C-C-A-Lys). In the absence of ribosomes and at low [Mg2+], the one-round GTP hydrolysis by EF-Tu is enhanced by C-C-A-aa fragments, whereas it is inhibited by the corresponding aa-tRNAs. Our results suggest that besides the 3' aminoacylated extremity another region(s) of the aa-tRNA molecule controls the GTPase of EF-Tu. The "unspecific" stimulation by C-C-A-aa and the "specific," aa-tRNA-like effect of the 3' aa-tRNA half-molecule point to the importance of the T chi C loop and stem, as well as of the adjacent regions for the regulation of this function.

Escherichia coli↗

PubMed2000

Evolution of arginine biosynthesis in the bacterial domain: novel gene-enzyme relationships from psychrophilic Moritella strains (Vibrionaceae) and evolutionary significance of N-alpha-acetyl ornithinase.

In the arginine biosynthetic pathway of the vast majority of prokaryotes, the formation of ornithine is catalyzed by an enzyme transferring the acetyl group of N-alpha-acetylornithine to glutamate (ornithine acetyltransferase [OATase]) (argJ encoded). Only two exceptions had been reported-the Enterobacteriaceae and Myxococcus xanthus (members of the gamma and delta groups of the class Proteobacteria, respectively)-in which ornithine is produced from N-alpha-acetylornithine by a deacylase, acetylornithinase (AOase) (argE encoded). We have investigated the gene-enzyme relationship in the arginine regulons of two psychrophilic Moritella strains belonging to the Vibrionaceae, a family phylogenetically related to the Enterobacteriaceae. Most of the arg genes were found to be clustered in one continuous sequence divergently transcribed in two wings, argE and argCBFGH(A) ["H(A)" indicates that the argininosuccinase gene consists of a part homologous to known argH sequences and of a 3' extension able to complement an Escherichia coli mutant deficient in the argA gene, encoding N-alpha-acetylglutamate synthetase, the first enzyme committed to the pathway]. Phylogenetic evidence suggests that this new clustering pattern arose in an ancestor common to Vibrionaceae and Enterobacteriaceae, where OATase was lost and replaced by a deacylase. The AOase and ornithine carbamoyltransferase of these psychrophilic strains both display distinctly cold-adapted activity profiles, providing the first cold-active examples of such enzymes.

Acetyltransferases↗

PubMed1994

Functional and morphological abnormalities of mitochondria in human cells containing mitochondrial DNA with pathogenic point mutations in tRNA genes.

mtDNA with a point mutation in the tRNA(Ile) gene at nucleotide position 4269 found in a patient with fatal cardiomyopathy and mtDNA with a point mutation in the tRNA(Arg) gene at 10410 found in a patient with Alpers disease were transferred cytoplasmically to rho zero HeLa cells (HeLa cells lacking mtDNA) to determine whether these novel mtDNA mutations in the tRNA genes are responsible for the defects in mitochondrial respiration function observed in these diseases. Cybrid clones (clones of rho zero HeLa cells with mtDNA from the patients) were isolated, and respiratory function and morphology of the mitochondria of the cybrid clones containing wild-type mtDNA and mutant mtDNA predominantly were compared. The results showed that accumulation of mutant mtDNA at 4269 alone without defects in the nuclear genome was sufficient to produce a disease phenotype, while mutant mtDNA at 10410 was not related to pathogenesis and reflected one of the rare polymorphic sites of human mtDNA. Moreover, we found that mitochondria in living cells were significantly swollen only when they contained predominantly the pathogenic mutant mtDNA, suggesting that the functional abnormality of mitochondria induced by pathogenic mtDNA mutations in tRNA genes is always associated with their swollen structure.

Cardiomyopathies↗

PubMed2004

Structural inhibition of the colicin D tRNase by the tRNA-mimicking immunity protein.

Colicins are toxins secreted by Escherichia coli in order to kill their competitors. Colicin D is a 75 kDa protein that consists of a translocation domain, a receptor-binding domain and a cytotoxic domain, which specifically cleaves the anticodon loop of all four tRNA(Arg) isoacceptors, thereby inactivating protein synthesis and leading to cell death. Here we report the 2.0 A resolution crystal structure of the complex between the toxic domain and its immunity protein ImmD. Neither component shows structural homology to known RNases or their inhibitors. In contrast to other characterized colicin nuclease-Imm complexes, the colicin D active site pocket is completely blocked by ImmD, which, by bringing a negatively charged cluster in opposition to a positively charged cluster on the surface of colicin D, appears to mimic the tRNA substrate backbone. Site-directed mutations affecting either the catalytic domain or the ImmD protein have led to the identification of the residues vital for catalytic activity and for the tight colicin D/ImmD interaction that inhibits colicin D toxicity and tRNase catalytic activity.

Amino Acid Sequence↗

PubMed1987

Differential ability of various plasmid DNAs to sequester inhibitors of RNA polymerase III transcription.

Deletion mutants of the Drosophila tRNA(Arg) gene that lack A-box promoter sequences are not transcribed in several cell-free systems; however, they are actively expressed in vivo in Xenopus oocytes (Sharp et al., 1983a). We show that two A-box deletion mutants of the tRNA(Arg) gene can be transcribed by a HeLa cell-free transcription system if it is preincubated with various DNAs, indicating that an inhibitor is responsible for the lack of mutant tRNA gene transcription. Optimal mutant transcription rescue, and presumably optimal binding of inhibitor, is facilitated by the presence of an active RNA polymerase II promoter in the preincubating DNA. Plasmid DNAs containing RNA polymerase III or weak RNA polymerase II promoters are of intermediate rescue efficiency, and pBR322 DNA is least efficient. Competition studies indicate that the stability of the inhibitor-DNA complex formed initially is apparently increased if the preincubating DNA contains an active RNA polymerase II promoter. Thus, HeLa whole-cell lysates contain a specific inhibitor(s) of RNA polymerase III transcription that primarily affects weakened RNA polymerase III promoters (e.g., A-box deletion mutants) and binds preferentially to DNAs containing an active RNA polymerase II promoter. Yet this apparent sequestration of inhibitor by Class II templates does not appear to inhibit their subsequent transcription by RNA polymerase II. These data raise the possibility that there may be interactions between the RNA polymerase II and III transcription machinery.

Animals↗

PubMed1992

Evidence that axonal tRNAs are resistant to RNase and ATPase and can be aminoacylated in the absence of exogenous ATP.

A high molecular weight (HMW) fraction of the 150,000 g supernatant of rat brain homogenates contains protein-tRNA complexes which are able to incorporate [3H]Arg and [3H]Lys into tRNA. The aminoacylation of tRNA(Arg) was found to be dependent on ATP and inhibited by RNase. Conversely, the aminoacylation of tRNA(Lys) did not require exogenous ATP and was resistant to RNase and ATPase. In HMW fractions of regenerating rat sciatic nerves, the charging of both tRNA(Arg) and tRNA(Lys) was resistant to RNase and ATPase and did not require exogenous ATP. Because sciatic nerves are rich in axoplasm and tRNAs are known to be present in axons, we tested the hypothesis that degradative enzyme-resistant, ATP-tRNA complexes were of axonal origin. In HMW fractions from rat liver (containing no axons), both tRNA(Arg) and tRNA(Lys) were sensitive to RNase and required exogenous ATP for charging. But, in similar fractions of axoplasm obtained from the giant axon of squid, both tRNAs were insensitive to RNase and ATPase and did not require exogenous ATP for charging. These results suggest that tRNAs in axons are present in protected HMW complexes and contain endogenous stores of ATP. The presence of ATP in the HMW complexes was demonstrated by the luciferase-luciferin assay for ATP. The nature of the protection of tRNAs from RNases was examined by dissociating proteins from HMW complexes by boiling, treating with proteinase K, or overhomogenizing the tissue. These procedures failed to render brain tRNA(Lys) susceptible to RNase. But phenol-extracted, ethanol-precipitated brain tRNA(Lys) was sensitive to RNase, suggesting that the protection of tRNA(Lys) may be by a protease- and heat-resistant polypeptide or by a nonproteinaceous mechanism.

Adenosine Triphosphatases↗

PubMed1995

tRNAs as primer of reverse transcriptases.

Genetic elements coding for proteins that present amino acid identity with the conserved motifs of retroviral reverse transcriptases constitute the retroid family. With the exception of reverse transcriptases encoded by mitochondrial plasmids of Neurospora, all reverse transcriptases have an absolute requirement for a primer to initiate DNA synthesis. In retroviruses, plant pararetroviruses, and retrotransposons (transposons containing long terminal repeats), DNA synthesis is primed by specific tRNAs. All these retroelements contain a primer binding site presenting a Watson-Crick complementarity with the primer tRNA. The tRNAs most widely used as primers are tRNA(Trp), tRNA(Pro), tRNA(1,2Lys), tRNA(3Lys), tRNA(iMet). Other tRNAs such as tRNA(Gln), tRNA(Leu), tRNA(Ser), tRNA(Asn) and tRNA(Arg) are also occasionally used as primers. In the retroviruses and plant pararetroviruses, the primer binding site is complementary to the 3' end of the primer tRNA. In the case of retrotransposons, the primer binding site is either complementary to the 3' end or to an internal region of the primer tRNA. Additional interactions taking place between the primer tRNA and the retro-RNA outside of the primer binding site have been evidenced in the case of Rous sarcoma virus, human immunodeficiency virus type I, and yeast retrotransposon Ty1. A selective encapsidation of the primer tRNA, probably promoted by interactions with reverse transcriptase, occurs during the formation of virus or virus-like particles. Annealing of the primer tRNA to the primer binding site appears to be mediated by reverse transcriptase and/or the nucleocapsid protein. Modified nucleosides of the primer tRNA have been shown to be important for replication of the primer binding site, encapsidation of the primer (in the case of Rous sarcoma virus), and interaction with the genomic RNA (in the case of human immunodeficiency virus type I).

Binding Sites↗

PubMed2004

Molecular epidemiology of childhood mitochondrial encephalomyopathies in a Finnish population: sequence analysis of entire mtDNA of 17 children reveals heteroplasmic mutations in tRNAArg, tRNAGlu, and tRNALeu(UUR) genes.

OBJECTIVES: Many heteroplasmic point mutations in tRNA genes of mitochondrial DNA (mtDNA) have been associated with human diseases. We recently reported on a prospective 7-year study in which we enrolled 116 consecutive children with undefined encephalomyopathy. Seventeen of them were found to have both a defect in the mitochondrial respiratory chain and abnormal ultrastructure of muscle mitochondria, suggesting a clinically probable mitochondrial encephalopathy. METHODS: We determined the frequency of mtDNA mutations in these 17 children by analyzing the entire sequence of mtDNA by conformation-sensitive gel electrophoresis and sequencing. RESULTS: Three heteroplasmic tRNA mutations that were considered to be pathogenic were detected. Two of the mutations were novel transitions, 10438A>G in the tRNA(Arg) gene and 14696A>G in the tRNA(Glu) gene, whereas the third one was 3243A>G, the common MELAS mutation. The mutant load was very high in the blood and skeletal muscle of the patients and markedly lower in the blood of asymptomatic maternal relatives. The 10438A>G mutation changes the nucleotide flanking the anticodon, whereas 14696A>G changes a nucleotide in the stem of the pseudouridine loop, creating a novel base pair and reducing the wobble. CONCLUSIONS: Our results emphasize that the analysis of the entire sequence of mtDNA is worthwhile in the diagnostic evaluation of patients with clinically probable mitochondrial encephalomyopathy. The frequency of pathogenic mtDNA mutations was found to be 18% among children with biochemically and histologically defined mitochondrial disease, suggesting that the likelihood of nuclear DNA mutations in such a group is several times higher than that of mtDNA mutations.

Base Sequence↗

PubMed2005

Structural basis for autoinhibition and mutational activation of eukaryotic initiation factor 2alpha protein kinase GCN2.

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2. The autoinhibited form of GCN2 is activated in cells starved of amino acids by binding of uncharged tRNA to a histidyl-tRNA synthetase-like domain. Replacement of Arg-794 with Gly in the PK domain (R794G) activates GCN2 independently of tRNA binding. Crystal structures of the GCN2 protein kinase domain have been determined for wild-type and R794G mutant forms in the apo state and bound to ATP/AMPPNP. These structures reveal that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving Arg794. This conformational change is associated with intradomain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation.

Adenosine Triphosphate↗

PubMed1989

The complete nucleotide sequence, gene organization, and genetic code of the mitochondrial genome of Paracentrotus lividus.

The 15,697-nucleotide sequence of Paracentrotus lividus mitochondrial DNA is reported. This genome codes for 2 rRNAs, 22 tRNAs, and 12 mRNAs which specify 13 subunits of the mitochondrial inner membrane respiratory complexes. The gene arrangement differs from that of other animal species. The two ribosomal genes 16 S and 12 S are separated by a stretch of about 3.3 kilobase pairs which contains the ND1 and ND2 genes and a cluster of 15 tRNA genes. The ND4L coding sequence is not contained in the ND4 mRNA but has its own mRNA which maps between the tRNA(Arg) and the Co II genes. The main noncoding region, located in the tRNA gene cluster, is only 132 nucleotides long, but contains sequences homologous to the mammalian displacement loop. Other short noncoding sequences are interspersed in the genome: they contain a conserved AT consensus which probably has a role in transcription or RNA processing. As regards the mitochondrial genetic code, the codons AGA and AGG specify serine and are recognized by a tRNA with a GCU anticodon, whereas AUA and AAA code for isoleucine and asparagine rather than for methionine and lysine. Except for ND4L which starts with AUC and ATPase 8 which starts with GUG, AUG is used as the initiation codon. In 11 out of 13 cases the genes terminate with the canonical stop codons UAA or UAG. These observations suggest that during invertebrate evolution each lineage developed its own mechanism of mitochondrial DNA replication and transcription and of RNA processing and translation.

Amino Acid Sequence↗

PubMed1989

Evolution of a tRNA operon in gamma purple bacteria.

Genomic DNA from eubacteria belonging to the gamma-3 subdivision of purple bacteria, as classified by Woese (C.R. Woese, Microbiol. Rev. 51:221-271, 1987), were probed with the argT operon of Escherichia coli encoding 5'-tRNA(Arg)-tRNA(His)-tRNA(Leu)-tRNA(Pro)-3'. The homologous operon from Vibrio harveyi was isolated and sequenced. Comparison of the five available sequences of this tRNA cluster from members of the families Enterobacteriaceae, Aeromonadaceae, and Vibrionaceae led to the conclusion that variations in different versions of this operon arose not only by point mutations but also by duplication and addition-deletion of entire tRNA genes. This data base permitted the formulation of a proposal dealing with the evolutionary history of this operon and suggested that DNA regions containing tRNA genes are active centers (hot spots) of recombination. Finally, since the operon from V. harveyi was not highly repetitive and did not contain tRNA pseudogenes, as in the Photobacterium phosphoreum operon, hybridization of genomic DNAs from different photobacterial strains with probes specific for the repeated pseudogene element was performed. We conclude that the phylogenetic distribution of the repetitive DNA is restricted to strains of P. phosphoreum.

Base Sequence↗

PubMed2006

Comparison of codon usage and tRNAs in mitochondrial genomes of Candida species.

To gain insight into the nature of the mitochondrial genomes (mtDNA) of different Candida species, the synonymous codon usage bias of mitochondrial protein coding genes and the tRNAs in C. albicans, C. parapsilosis, C. stellata, C. glabrata and the closely related yeast Saccharomyces cerevisiae were analyzed. Common features of the mtDNA in Candida species are a strong A+T pressure on protein coding genes, and insufficient mitochondrial tRNA species are encoded to perform protein synthesis. The wobble site of the anticodon is always U for the NNR (NNA and NNG) codon families, which are dominated by A-ending codons, and always G for the NNY (NNC and NNU) codon families, which is dominated by U-ending codons, and always U for the NNN (NNA, NNU, NNC and NNG) codon families, which are dominated by A-ending codons and U-ending codons. Patterns of synonymous codon usage of Candida species can be classified into three groups: (1) optimal codon-anticodon usage, Glu, Lys, Leu (translated by anti-codon UAA), Gln, Arg (translated by anti-codon UCU) and Trp are containing NNR codons. NNA, whose corresponding tRNA is encoded in the mtDNA, is used preferentially. (2) Non-optimal codon-anticodon usage, Cys, Asp, Phe, His, Asn, Ser (translated by anti-codon GCU) and Tyr are containing NNY codons. The NNU codon, whose corresponding tRNA is not encoded in the mtDNA, is used preferentially. (3) Combined codon-anticodon usage, Ala, Gly, Leu (translated by anti-codon UAG), Pro, Ser (translated by anti-codon UGA), Thr and Val are containing NNN codons. NNA (tRNA encoded in the mtDNA) and NNU (tRNA not encoded in the mtDNA) are used preferentially. In conclusion, we propose that in Candida species, codons containing A or U at third position are used preferentially, regardless of whether corresponding tRNAs are encoded in the mtDNA. These results might be useful in understanding the common features of the mtDNA in Candida species and patterns of synonymous codon usage.

Algorithms↗

PubMed1985

Inhibition of the aminoacylation of selected tRNA molecules by an estrogen-regulated factor on uterine ribosomes. Regulation of aminoacylation of tRNA by estrogens.

Administration of estradiol to ovariectomized mature rats for 1 h induces a transient increase in the peptide elongation rate on uterine ribosomes. An inhibitor of the peptide elongation rate, which appears to be regulated by estrogen treatment in vivo, can be extracted from ribosomes of estrogen-deprived rats. The extracted inhibitor or a native inhibitor-ribosome complex affects the rate of the peptide elongation reaction in a uterine cell-free protein synthesis system by inhibiting the ability of selected tRNAs in the assay to be charged with amino acids by their respective aminoacyl-tRNA synthetases. The degree of inhibition of charging of the affected tRNAs ranges from 22% to 78%, the order of inhibition being Pro greater than Val greater than Arg greater than Try greater than Leu greater than Glu greater than Ile greater than Gly greater than His greater than Ser greater than Lys. Inhibition results from a specific dose-dependent, and presumably reversible, effect of the inhibitor on tRNA, but not on the aminoacyl-tRNA synthetase. The effect does not result from removal of A-C-C terminal nucleotides from the 3' end of tRNA, but does inhibit the ability of selected tRNAs to bind to the aminoacyl-tRNA synthetases. We propose that regulation of the peptide elongation rate on uterine ribosomes by estradiol occurs through the estradiol-induced inactivation of a ribosome-associated inhibitor, which causes a reversible alteration to selected tRNAs. The modified tRNAs are unable to bind to their respective aminoacyl-tRNA synthetase to become charged with an amino acid thus causing the availability of selected aminoacyl-tRNAs to become rate-limiting in the sequential elongation of peptides.

Amino Acyl-tRNA Synthetases↗

PubMed2000

Reproducing tna operon regulation in vitro in an S-30 system. Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Catabolite repression regulates transcription initiation, whereas excess tryptophan induces antitermination at Rho factor-dependent termination sites in the leader region of the operon. Synthesis of the leader peptide, TnaC, is essential for antitermination. BoxA and rut sites in the immediate vicinity of the tnaC stop codon are required for termination. In this paper we use an in vitro S-30 cell-free system to analyze the features of tna operon regulation. We show that transcription initiation is cyclic AMP (cAMP)-dependent and is not influenced by tryptophan. Continuation of transcription beyond the leader region requires the presence of inducing levels of tryptophan and synthesis of the TnaC leader peptide. Using a tnaA'-'trpE fusion, we demonstrate that induction results in a 15-20-fold increase in synthesis of the tryptophan-free TnaA-TrpE fusion protein. Replacing Trp codon 12 of tnaC by an Arg codon, or changing the tnaC start codon to a stop codon, eliminates induction. Addition of bicyclomycin, a specific inhibitor of Rho factor action, substantially increases basal level expression. Analyses of tna mRNA synthesis in vitro demonstrate that, in the absence of inducer transcription is terminated and the terminated transcripts are degraded. In the presence of inducer, antitermination increases the synthesis of the read-through transcript. TnaC synthesis is observed in the cell-free system. However, in the presence of tryptophan, a peptidyl-tRNA also appears, TnaC-tRNA(Pro). Our findings suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA. The ribosome associated with this newly synthesized peptidyl-tRNA presumably stalls at the tnaC stop codon, blocking Rho's access to the BoxA and rut sites, thereby preventing termination. 1-Methyltryptophan also is an effective inducer in vitro. This tryptophan analog is not incorporated into TnaC.

Bacterial Proteins↗

PubMed1985

Analysis of translational fidelity of ribosomes with protamine messenger RNA as a template.

A novel method was developed to estimate the translational fidelity of mammalian ribosomes in vitro with protamine mRNA of rainbow trout as template. Protamines are mixtures of basic proteins consisting of only seven types of amino acids (Arg, Ile, Val, Ser, Pro, Ala, and Gly), arginine (codon, AGR and CGN) being abundant. Taking advantage of the absence of lysine (codon, AAG) in the proteins, we determined the misincorporation of this amino acid into protamines in a cell-free translation system consisting of mouse liver ribosomes, protamine mRNA, [3H]lysine, [14C]arginine, and seven unlabeled amino acids: Ile, Val, Ser, Pro, Ala, Gly, and Met. After the reaction, translation products were analyzed by either sucrose gradient centrifugation or polyacrylamide gel electrophoresis. In the former method, radioactive protamines are mostly found on monosomes, but not on polysomes, probably because of the basic nature of the proteins. The error frequency was calculated from the molar ratio of [3H]lysine to [14C]arginine incorporated into protamines with an appropriate correction. The frequency was found to be 0.0006-0.002. This method enabled us to determine the frequency of misrecognition of purine bases at the second position of arginine codons in mRNA.

Amino Acid Sequence↗

PubMed1993

Unassigned or nonsense codons in Micrococcus luteus.

We previously reported that in Micrococcus luteus, a Gram-positive eubacterium with high genomic G + C content, certain codons ending with A did not appear in coding frames, including termination sites, and tRNAs that translate these codons were not detected. These facts suggest that at least some of them are unassigned (nonsense) codons, i.e. not assigned to any amino acid or to any stop signal. We have investigated whether AGA and AUA, universal Arg and Ile codons, respectively, are really unassigned codons by using a cell-free extract prepared from M. luteus and synthetic messenger RNAs. Translation of synthetic mRNA containing in-frame AGA codons does not result in "read-through" to codons beyond the AGA codons, i.e. translation ceases at codon AGA. Essentially the same result was obtained with mRNA containing AUA in-frame. A sucrose-gradient centrifugation profile of the reaction mixture has shown that practically all of the peptides that have been synthesized are attached to 70 S ribosomes. When in-frame AGA or AUA codons are replaced by UGA codons in mRNA, no read-through occurs beyond UGA, just as in the case of AGA or AUA. However, the synthesized peptide is released from the 70 S ribosomes. These data suggest that AGA and AUA are unassigned codons and differ from UGA in that they are not used for termination.

Adenine Nucleotides↗