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PubMed1991

Activation of methionine by Escherichia coli methionyl-tRNA synthetase.

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed1997

Nucleotide G-1207 of 18S rRNA is an essential component of the human 80S ribosomal decoding center.

mRNA analogues-derivatives of oligoribonucleotides consisting of two different codons and bearing an aryl azide group at the 5'-phosphates-were crosslinked to human 80S ribosomes by UV-irradiation of the various model complexes obtained in the presence of the cognate tRNAs. Three sequences, namely pUUUGUU (coding for Phe and Val), pUUCUAAA (first triplet coding for Phe and second being stop-codon), and pGUGUUU (coding for Val and Phe), have been used. Sequences of 18S rRNA containing nucleotides crosslinked to the mRNA analogues were examined by hydrolysis with RNase H in the presence of various cDNA probes. Crosslinked nucleotides were identified by primer extension. In all cases, only nucleotide G-1207 (equivalent to G-926 in Escherichia coli 16S rRNA) has been detected as crosslinked. Crosslinking of the mRNA analogues to the large ribosomal subunit was negligible.

Benzoates↗

PubMed1982

Template-free ribosomal synthesis of polypeptides from aminoacyl-tRNAs.

In the present paper it has been demonstrated that Escherichia coli ribosomes in the absence of messenger polynucleotides are capable of synthesizing some polypeptides from aminoacyl-tRNAs as substrates. EF-Tu induced binding of aminoacyl-tRNA, ribosomal peptidyl transferase and EF-G-promoted translocation are strictly required for this template-free elongation. Typical ribosomal inhibitors such as tetracycline, chloramphenicol, phenylboric acid, fusidic acid have been shown to inhibit the template-free synthesis of polypeptides. The synthesis requires GTP cleavage; a non-cleavable analog of GTP, guanyl-5'-yl methylenediphosphonate does not maintain the synthesis. Among sixteen different aminoacyl-tRNAs studied as substrates for the ribosomal template-free synthesis of polypeptides Lys-tRNA, Ser-tRNA, Thr-tRNA and Asp-tRNA were the best. Gly-tRNA, Glu-tRNA, Val-tRNA, Arg-tRNA, Ala-tRNA and Leu-tRNA as substrates gave relatively low levels of the polypeptide synthesis on nonprogrammed ribosomes. Pro-tRNA, Phe-tRNA, Asn-tRNA, Met-tRNA, Ile-tRNA and Gln-tRNA were practically inactive as substrates for the template-free elongation. No correlation has been found between the abilities of the aminoacyl-tRNAs to serve as substrates for the template-free elongation and their activities in template-free binding to ribosomes.

Escherichia coli↗

PubMed1971

Initial dipeptide formation in hemoglobin biosynthesis.

Initiation factors M(1) + M(2) from reticulocyte ribosomes bind Met-tRNA(F) to rabbit reticulocyte ribosomes containing endogenous hemoglobin mRNA. The initial binding of Met-tRNA(F) appears to be to the small ribosomal subunit. The Met-tRNA(F) is able to participate in what is presumed to be the first peptide bond in the formation of hemoglobin, namely the synthesis of a methionyl-valine dipeptide. The formation of this methionyl-valine dipeptide requires Met-tRNA(F), initiation factors M(1), M(2), and M(3), as well as Val-tRNA and T(1). No synthesis of methionyl-valine dipeptide takes place if Met-tRNA(F) is replaced by Met-tRNA(M), or if initiation factor M(3) is omitted. Thus, Met-tRNA(F) appears to be the initiator tRNA for hemoglobin biosynthesis and M(3), although required for the synthesis of the first peptide bond of hemoglobin, does not appear to be necessary, under the experimental conditions studied, for Met-tRNA(F) binding.

Animals↗

PubMed1975

The primary structure of the major cytoplasmic valine tRNA of mouse myeloma cells.

This paper describes the derivation of the primary structure of the major valine tRNA in the cytoplasm of mouse myeloma cells. Approximately 75% of the nucleotide sequence of this tRNA is also shared by the tRNA1-Val of yeast, this homology serving as a further indication of the extreme conservation of the structures of the tRNAs of different eukaryotic organisms. A novel feature of mouse myeloma tRNA1-Val is its loop IV sequence: -U-PSI-C-G-M1A-A-A-. This particular loop IV sequence has not previously been found in a tRNA structure. In addition, tRNA1-Val possesses some unusual nucleoside modifications. 5-Methyluridine (T) was not found to occur within loop IV of this tRNA, although this minor nucleoside is also absent from certain other mammalian tRNAs. Only one other tRNA, mammalian tRNAf-Met, has been found to possess 2-methylguanosine (m2G) in the position between the (b) and (c) stems of the cloverleaf. Numerous tRNAs have m2-2G in this location, and it would appear that the second methylation of this guanosine is characteristically absent from certain mammalian tRNA species.

Animals↗

PubMed1981

A small-angle X-ray scattering study of the complex formation between elongation factor Tu . GTP and valyl-tRNA Val I from Escherichia coli.

The complex formation between elongation factor Tu (EF-Tu) . GTP and valyl-tRNA Val 1 has been investigated using the small-angle X-ray scattering titration technique. The main species observed is a 1:1 complex with a stability constant log K greater than or equal to 6. The corresponding interaction between EF-Tu . GTP and non-aminoacylated tRNA appears to be much weaker with an estimated log K approximately equal to 4. The radius of gyration determined for the EF-Tu . GTP -- valyl-tRNA Val 1 complex is larger (R = 3.6 nm) than that of EF-Tu . GTP (R = 2.5 nm). Likewise, the maximum distance within this complex is larger (Dmax = 12.5 nm) than the one within EF-Tu . GTP (Dmax = 8.5 nm). These data as well as the p(r) curve are consistent with a multiellipsoid model for the complex. From this model it is indicated that the acceptor stem of tRNA is attached to EF-Tu and that the anticodon stem and loop protrude into the solution.

Bacterial Proteins↗

PubMed2000

Defects in tRNA processing and nuclear export induce GCN4 translation independently of phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2.

Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNA(Met) binding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNA(AAC)(Val) (tRNA(Val*)) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded by PUS4 also leads to translational derepression of GCN4 (Gcd(-) phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd(-) phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate that PUS4 overexpression did not diminish functional initiator tRNA(Met) levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd(-) phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5'-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNA(Tyr) that cannot be processed by RNase P had a Gcd(-) phenotype. Interestingly, the Gcd(-) phenotype of hcPUS4 also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Delta cells have a Gcd(-) phenotype. Overproduced PUS4 appears to impede 5'-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNA(Val*) showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNA(Met) binding to the ribosome.

Alcohol Oxidoreductases↗

PubMed2005

Escape from the interferon response associated with RNA interference using vectors that encode long modified hairpin-RNA.

In mammalian cells, siRNAs have been used to induce RNA interference (RNAi) in an attempt to prevent nonspecific effects (including the interferon (IFN) response) which are caused by long double-stranded RNAs (dsRNAs) of more than 30 bp. In this report, we describe a novel and simple strategy for avoiding activation of the IFN response by dsRNA. We show that modified hairpin-RNAs (mhRNAs) of more than 100 bp, with multiple specific point-mutations within the sense strand and transcribed from the U6 or tRNA(Val) promoters, can cause RNAi without inducing the IFN pathway genes. Moreover, we demonstrate that the 50-bp mhRNA vector could effectively suppress the replication of multiple hepatitis C viruses (the genomes of which differ slightly, thus the 21-bp siRNA vector failed to suppress one of them). Our findings should enhance the exploitation of RNAi in mammalian cells, especially in the field of RNAi therapy against pathogenic viruses.

Base Sequence↗

PubMed1983

A quantitative regional analysis of amino acids involved in rat brain protein synthesis by high performance liquid chromatography.

A biochemical method is described for the simultaneous quantitative estimation of unidirectional blood-brain amino acid influx and protein biosynthesis in individual structures of the rat brain. The method involved a double labeling experiment started by the administration of [14C]carboxyl-labeled amino acids and terminated 2 min after infusion of 3H-labeled amino acids, each at tracer quantities, the total labeling period being 45 min. Specific radioactivities of 14C- or 3H-labeled phenylalanine, tyrosine, leucine, isoleucine, and valine were determined in plasma and in small brain tissue samples for free amino acids, aminoacyl-tRNAs, and proteins. Amino acids were converted to their corresponding 5-dimethylamino-naphthalenesulfonyl (Dns, dansyl) derivatives and separated on HPLC C18 reversed-phase columns isocratically according to a newly developed optimizing procedure. The order of influx values between the neutral amino acids in relation to each other was Leu greater than Tyr greater than Ile greater than Phe greater than Val in every structure examined. Although aminoacylation of tRNAs was found to proceed to a comparable degree for neutral amino acids in all regions investigated, the specific radioactivity of amino acids attached to tRNAs differed substantially from that in the free amino acid pool, especially for leucine and valine. The results indicate the necessity of aminoacyl-tRNA determinations for tracer incorporation studies in protein synthesis analysis. Relative protein synthesis rates in the halothane-anesthetized rat were determined to be 30 and 67-91 pmol total amino acid incorporation/min/mg tissue for white and gray matter, respectively.

Amino Acids↗

PubMed2006

Visualizing bz1 missense suppression in Zea mays: an assay for monocot tRNA expression and utilization.

Bombardment of a highly expressed dicot tRNA(ala)(GAC) gene into Zea mays bz-E2 or bz-E5 coleoptiles causes suppression of an Ala(458 )-->Val missense mutation, visualized by the development of anthocyanin pigment. Missense suppression is blocked by mutation of tRNA(ala)(GAC) at a site that prevents aminoacylation by the dicot alanyl-tRNA synthetase, indicating that features identified for expression and utilization of dicot tRNAs also function in monocots. This assay of the expression and utilization of tRNA(ala)(GAC) also can be used to study a variety of tRNAs and their genes, most of which can be relatively easily altered to be charged by alanyl tRNA synthetase.

Cotyledon↗

PubMedDate not supplied

[Study of the biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. I. Introduction of directed mutations into the alkaline phosphatase gene].

Various mutations in E. coli alkaline phosphatase gene were obtained by oligonucleotide-directed mutagenesis. They result in amino acid substitutions in the signal peptide cleavage site [Val for Ala(-1)] and in the N terminus of mature polypeptide chain: Ala for Arg(+1) and Gln for Glu(+4); Gln for Glu(+4). Enzyme activity was observed in all E. coli strains transformed by plasmids with cloned mutant genes. In addition, an amber mutation was introduced into the Arg(+1) position, and the synthesis of mutant alkaline phosphatase was shown in E. coli strains containing suppressor tRNAs specific for Ser, Gln, Tyr, Leu, Ala, Glu, Phe, Gly, His, Pro, and Cys.

Alkaline Phosphatase↗

PubMed1978

A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function.

The biosynthesis of a secretory protein and a transmembrane viral glycoprotein are compared by two different experimental approaches. (a) NH2-terminal sequence analysis has been performed on various forms of the transmembrane glycoprotein of vesicular stomatitis virus synthesized in cell-free systems. The sequence data presented demonstrate that the nascent precursor of the glycoprotein contains a "signal sequence" of 16 amino acids at the NH2 terminus, whose sequence is Met-Lys-Cys-Leu-Leu-Tyr-Leu-Ala-Phe-Leu-Phe-Ile-(His-Val-Asn)-Cys. This signal sequence is proteolytically cleaved during the process of insertion into microsomal membranes prior to chain completion. The new NH2 terminus of the inserted, cleaved, and glycosylated membrane protein is located within the lumen of the microsomal vesicles and is identical to that of the authentic glycoprotein from virions. (b) Nascent chain competition experiments were performed between this glycoprotein, bovine pituitary prolactin (a secretory protein), and rabbit globin (a cytosolic protein). It was found that the nascent membrane glycoprotein, but not nascent globin, competed with nascent prolactin for membrane sites involved in the early biosynthetic event of transfer across membranes. These data suggest that an initially common pathway is involved in the biogenesis of secretory proteins and at least one class of integral membrane proteins.

Amino Acid Sequence↗

PubMed1997

Limited proteolysis of angiogenin by elastase is regulated by plasminogen.

Human neutrophil elastase cleaves angiogenin at the Ile-29/Met-30 peptide bond to produce two major disulfide-linked fragments with apparent molecular weights of 10,000 and 4000, respectively. Elastase-cleaved angiogenin has slightly increased ribonucleolytic activity, but has lost its ability to undergo nuclear translocation in endothelial cells, a process essential for angiogenic activity. Cleavage appears to alter the cell-binding properties of angiogenin, despite the fact that it occurs some distance from the putative receptor-binding site, since the elastase-cleaved protein fails to compete with its native counterpart for nuclear translocation in endothelial cells. Plasminogen specifically accelerates elastase proteolysis of angiogenin. It does not enhance elastase activity toward ribonuclease A or the synthetic peptide substrate MeOSuc-Ala-Ala-Pro-Val-pNA. Plasminogen-accelerated inactivation of angiogenin by elastase might be a significant event in the process of angiogenin-induced angiogenesis since (i) angiogenin and plasminogen circulate in plasma at high concentrations, (ii) angiogenin, especially when bound to actin, activates tissue plasminogen activator to generate plasmin from plasminogen, and (iii) elastase cleaves plasminogen to produce angiostatin, a potent inhibitor of angiogenesis and metastasis. Interrelationships among angiogenin, plasminogen, plasminogen activators, elastase, and angiostatin may provide a sensitive regulatory system to balance angiogenesis and antiangiogenesis.

Amino Acid Sequence↗

PubMed1975

Tertiary structure in E. coli tRNA Arg and tRNA Val.

300 MHz proton NMR has been used to demonstrate that both Escherichia coli tRNA Arg and tRNA(1) Val have a tertiary structure base pair between 4-thiouridine (s-4U) at position 8 and A at position 14. Formation of this s-4U(8)-A(14) base pair leads to a very low field (14.8 ppm) resonance in the spectra of both molecules. When s-4U is converted to U the 14.8 resonance is replaced by a new resonance which appears at 14.3 ppm. The presence of the tertiary structure s-4U(8)-A(14) base pair greatly constrains the folding of these tRNAs in solution.

Arginine↗

PubMed1994

A nuclear tRNA gene cluster in the protozoan Leishmania tarentolae and differential distribution of nuclear-encoded tRNAs between the cytosol and mitochondria.

All mitochondrial tRNAs in the protozoan Leishmania are believed to be encoded in the nuclear genome and imported selectively into the mitochondria by an as yet unknown mechanism. Previously, we reported that two tRNAs whose genes are tightly linked were imported by mitochondria. In contrast, a tRNA encoded by a lone tRNA gene was not detectable in mitochondria. The lone tRNA gene had flanking sequences that were different from the linked genes. These studies implied a possible correlation between tRNA gene organization and gene flanking sequence, and selective tRNA import into mitochondria. Here, we report the identification of a cluster of 10 tRNA genes and show the distribution of the corresponding tRNAs in cytosolic and mitochondrial fractions. tRNA(leu)(CAG) and tRNA2(arg)(TCG) are abundant in the cytosol, but relatively scarce in mitochondria. Conversely, tRNA(ile)(TAT) and tRNA1(lys)(TTT) are abundant in mitochondria, but relatively scarce in the cytosol. tRNA(val)(TAC) and tRNA2(thr)(TGT) are barely detectable in either cellular compartment, while tRNA(gln)(TTG), tRNA1(arg)(ACG), tRNA(gly)(TCC), and tRNA(trp)(CCA) are detected in approximately equal levels in both compartments. Sequencing of the 2600 bp that comprise the tRNA gene cluster also encoding the genes for 5S RNA and URNAB RNA indicates that nucleotide composition, length, and location of genes within the cluster do not clearly correlate with import characteristics. The unexpected presence of the tRNA(trp)(CCA)-gene transcript in mitochondria is also reported. Evidence suggests that this tRNA may have unidentified base modifications at the anticodon triplet.

Animals↗

PubMed1981

The protein synthetic activity in vitro of ribosomes differing in the extent of phosphorylation of their ribosomal proteins.

We describe a re-examination of the cell-free protein synthetic activity of eukaryotic ribosomes having proteins phosphorylated to different extents. Ribosomal 40 S subunits were isolated both from a variety of cells in which there is relatively little phosphorylation of ribosomal protein S6, and from cells subjected in vivo to different stimuli that promote the extensive phosphorylation of protein S6. The ability of these subunits to bind Met-tRNA as well as the second amino acyl-tRNA (Val-tRNA) was compared in the presence of highly purified initiation factors, elongation factor EF-1 at various concentrations of 60S subunits, 9 S globin mRNA and potassium ions. The ability of the subunits to synthesize polyphenylalanine was also studied using highly purified elongation factors. In no case was any significant difference in activity observed between ribosomes with protein S6 phosphorylated to different extents. Similar, though less extensive, studies were preformed comparing 60 S ribosomal subunits differing in the extent of phosphorylation of the acidic phosphoprotein, L gamma , and of L14. No difference in activity was observed between these ribosomes.

Animals↗

PubMed1983

Interaction between the different domains of aminoacyl-tRNA and the elongation-factor-Tu x kirromycin complex.

In this work, we have studied the effect of aa-tRNA and derived 3' aminoacylated fragments on the EF-Tu GTPase in the presence of kirromycin, using two systems: without and with ribosomes. The aa-tRNA fragments were obtained by enzymatic digestion. Procedures for the enzymatic preparation of C-A-Val and Val-tRNA Val1 3' half molecule, as well as a purification method for short 3' aminoacylated fragments based on the amino group charge, were newly developed for this work. Aminoacyl-adenosine was found to be able to stimulate the EF-Tu x kirromycin GTPase, but only to a very small extent. Increasing the length of the aminoacylated fragments increased the stimulatory effect as follow: A-Val much less than C-A-Val less than C-C-A-Val less than 3' valyladenosine dodecanucleotide much less than Val-tRNA Val1 3' half molecule less than Val-tRNA Val1. The presence of ribosomes did not affect the order of effectiveness, but increased the basic GTPase activity of EF-Tu x kirromycin and the stimulation by aa-tRNA, its 3' half molecule and even more by its 3' short fragments. The effect of aa-tRNA and derived 3' fragments in the absence of ribosomes was not influenced by MgCl2 concentrations of 5-30 mM whereas, in the presence of ribosomes, low concentrations of MgCl2 (5 mM) greatly reduced the stimulation of aa-tRNA and, to a lesser extent, also the effect of the C-C-A-aa as well as the basic activity of the EF-Tu x kirromycin GTPase. The extent of the stimulation by aa-tRNA, and even more by C-C-A-aa, depends on the nature of the amino acid. Among the aminoacyl side chains tested (Arg-, Phe-, Val-, Met-, Leu-, Lys-) arginine was found to be the most active and leucine the least. Our results show that (a) the 3' aminoacylated extremity is of prime importance for the stimulation of the EF-Tu GTPase, (b) in the 3' extremity there are critical sequences for the interaction with EF-Tu and (c) other domains of the aa-tRNA molecule are capable of influencing this reaction: one of the most important is the region including the T psi C loop and stem.

Cations↗

PubMed1990

Dual effects of the ricin A chain on protein synthesis in rabbit reticulocyte lysate. Inhibition of initiation and translocation.

Ricin A chain caused inhibition of protein synthesis by reticulocyte lysate with concomitant depurination of 28S rRNA. The partial reaction(s) of protein synthesis inhibited was investigated by following the appearance of [35S]methionine from initiator [35S]Met-tRNA into 40S ribosomal subunits, 80S monosomes and polysomes. Ricin A chain caused an accumulation of [35S]Met in monosomes which did not enter polysomes. In these respects the effects of the ricin A chain resembled those of diphtheria toxin, an inhibitor of elongation-factor-2-catalyzed translocation. This is consistent with the previously proposed site of action of ricin as an inhibitor of elongation. However, the inhibitory effects of the ricin A chain and diphtheria toxin are not equivalent because we observed that the rate of formation of the 80S initiation complex was reduced approximately sixfold with the ricin A chain relative to diphtheria toxin. Analysis of methionine-containing peptides bound to 80S monosomes in ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, programmed with globin mRNA, revealed a predominance of Met-Val, suggesting that the elongation cycle is inhibited at the translocation step. Translocation was also implicated as the step blocked in both the ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, by the finding that nascent peptide chains were unreactive towards puromycin. It is concluded that ricin-A-chain-modified ribosomes are deficient in two protein synthesis partial reactions: the formation of the 80S initiation complex during initiation and the translocation step of the elongation cycle.

Animals↗