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PubMed1976

Microinjection of tRNA into amphibian oocytes.

The microinjection technique affords us the possibility to introduce purified components into living cells and to answer the question of what effects the change introduced has on cellular metabolism. This technique can therefore be used to test the hypothesis that transfer RNA plays a regulatory role in cellular protein synthesis. Prior to these experiments it is important, however, to test whether transfer RNA microinjected into amphibian oocytes is stable and functional inside this cells. These two questions are answered affirmatively in this report. The stability of tRNA was tested by following the content of TCA precipitable counts inside the oocytes at different times after microinjection of radioactive yeast and E. coli tRNA and by polyacrilamide gel electrophoresis of the material recovered from the cell. The results clearly indicate that tRNAs are resistant to the action of occyte ribonucleases that degrade other RNAs such as 5S RNA. The functionality of the injected tRNA was tested by assaying the intracellular aminoacylation of microinjected yeast tRNA. The aminoacylation of bulk yeast (3H) tRNA introduced into Xenopus laevis oocytes was tested by the capacity of the material recovered 5 hours after injection into the cell to form a ternary complex with wheat protein synthesis elongation factor 1 and GTP. The complex only forms with aminoacyl-tRNA and not with unacylated tRNA. This method showed that at least 80% of the tRNA introduced into the cell was aminoacylated in vivo. A direct assay for internal aminoacylation made use of microinjection of pure tRNAPhe and subsequent determination by phenol extraction of (14C)Phe-tRNA content of oocytes that had been incubated for 2 hours in a medium containing (14C)phenylalanine. The results obtained showed that the oocytes could internally aminoacylate 200-500 times more tRNAPhe that the cell normally contains. Appropiate controls demonstrated that the aminoacylation was aminoacid and tRNA specific and that periodate oxidized tRNAPhe could not be in vivo aminoacylated but tRNAPhe deprived of its Y base could accept the aminoacid. A brief study demonstrated that bulk yeast tRNA and tRNAPhe without its Y base did not inhibit endogenous protein synthesis but a similar amount of tRNAPhe caused 50% inhibition and periodate-oxidized tRNAPhe a 95% inhibition.

Amphibians↗
PubMed1973

Effect of the removal of the Y base on the conformation of yeast tRNA.

The effect of removing the Y base from the anticodon loop of yeast tRNA(Phe) has been examined by high-resolution proton nuclear magnetic resonance spectroscopy and optical melting. Analysis of the changes in the nuclear magnetic resonance spectra indicate that the removal of the Y base produces a small conformational change in the anticodon stem in which all the interbase separations are increased by approximately 0.2 A. The temperature dependence of the nuclear magnetic resonance spectra and the optical melting measurements indicate that the high temperature stability of tRNA(Phe) is decreased by removal of the Y base. With the exception of the anticodon stem, the secondary structure of the other helical stems of the molecule appear to be unaltered when the Y base is excised.

Hydrochloric Acid↗
PubMed1997

Structural and functional considerations of the aminoacylation reaction.

Aminoacyl-tRNA synthetases (aaRS) bind their substrates-ATP, amino acids and tRNA- and stabilize putative transition states in the aminoacylation reaction. Here, we discuss the common and distinguishing structural and functional themes of the 20 known aaRS, which can be divided into two main classes (I and II) and into further subgroups on this basis.

Adenosine Triphosphate↗
PubMedDate not supplied

Nucleoside modifications affect the structure and stability of the anticodon of tRNA(Lys,3).

NMR spectroscopy was used to determine the solution structures of RNA oligonucleotides comprising the anticodon domain of tRNA(Lys,3). The structural effects of the pseudouridine modification at position 39 were investigated and are well correlated with changes in thermodynamic parameters. The loop conformation differs from that seen in tRNA(Phe) and provides an explanation of the critical role of modification in this tRNA.

Anticodon↗
PubMed2006

Genetic code from tRNA point of view.

The possible codon-anticodon pairings follow the standard genetic code, yet in a different mode. The corresponding rules for decoding sequence of the codons in mRNA with tRNA may be called "tRNA code". In this paper we analyse the mutational and translational stability of such tRNA code. Our approach is based on the model of "ambiguous intermediate" and on the study of underlying block structure and Eulerean graph technique. It is shown that the wobble rules and the reduced number of tRNA anticodons strongly affect the mutational and translational stability of the code. The selection of tRNA anticodons, besides the optimization of translation, also ensures the more reliable start and, to a lesser extent, the stop of translation. The attribution of tRNA anticodons to the groups [WWW, WWS, SWW, SWS] and [SSS, SSW, WSS, WSW] as well as [MMM, MMK, KMM, KMK] and [KKK, KKM, MKK, MKM] clearly correlates with class I and class II aminoacyl-tRNA synthetases and obeys the principle of the optimal coding in both cases. Both W-S and M-K groupings also refer to the encoding of amino acids with the large and small side-chain volumes, which may provide such an attribution. The higher variability of tRNA code agrees with the suggestions that the variations in an assignment of tRNA anticodons may serve as the driving force generating the different variants of the genetic code.

Amino Acyl-tRNA Synthetases↗
PubMed2004

Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance.

Expression patterns in Escherichia coli of two small archaeal proteins with a natural content of about 30% rare codons were analyzed. The proteins, a histone-like protein from Sulfolobus shibatae (Ssh10), and a glutaredoxin-like protein from Methanobacterium thermoautotrophicum (mtGrx), were produced with expression plasmids encoding wild-type genes, codon-optimized synthetic, and GST-fusion genes. These constructs were expressed in BL21 (DE3), its LysS derivative, and modified strains carrying copies for rare codon tRNAs or deletions in the RNAseE gene. Both Ssh10 and mtGrx expression levels were constitutively high in BL21(DE3) and its derivatives, with the exception of the LysS phenotype, which prevented high level expression of the Ssh10 wild-type gene. Surprisingly, a codon-optimized mtGrx gene construct displayed undetectable levels of protein production. The translational block observed with the synthetic mtGrx gene could be circumvented by using a synthetic mtGrx-glutathione S-transferase (GST) fusion construct or by in vitro translation. Taken together, the results underscore the importance of mRNA levels and RNA stability, but not necessarily tRNA abundance for efficient heterologous protein production in E. coli.

Archaeal Proteins↗
PubMed1999

Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: quantitative effects on activity, stability and dimerization.

A dense cluster of eight residues was identified at the crossing of two alpha-helices in tyrosyl-tRNA synthetase (TyrRS) from the thermophile Bacillus stearothermophilus. Its mechanism of evolution was characterized. Four residues of this cluster are not conserved in TyrRS from the mesophile Escherichia coli. The corresponding mutations were constructed in TyrRS(Delta1), a derivative of TyrRS from B. stearothermophilus in which the anticodon binding domain is deleted. Mutations I52L (i.e. Ile52 into Leu), M55L and L105V did not affect the activity of TyrRS(Delta1) in the pyrophosphate exchange reaction whereas T51P increased it. The kinetic stabilities of TyrRS(Delta1) and its mutant derivatives at 68.5 degreesC were determined from experiments of irreversible thermal precipitation. They were in the order L105V<I52L<T51P<Wild Type</=M55L; mutation I52L partially compensated L105V in these experiments whereas M55L was coupled neither to I52L nor to L105V. Mutations I52L and L105V affected the stability of the dimeric TyrRS(Delta1) at different steps of its unfolding by urea, monitored under equilibrium conditions by spectrofluorometry or size exclusion chromatography. I52L destabilized the association between the subunits even though residue Ile52 is more than 20 A away from the subunit interface. L105V destabilized the monomeric intermediate of unfolding. The two mutational pathways, going from the wild-type TyrRS(Delta1) to the I52L-L105V double mutant through each of the single mutants were not equivalent for the stability of the monomeric intermediate and for the total stability of the dimer. One pathway contained two neutral steps whereas the other pathway contained a destabilizing step followed by a stabilizing step. Mutation I52L allowed L105V along the first pathway and compensated it along the second pathway. Thus, the effects of I52L and L105V on stability depended on the structural context. The gain in activity due to T51P was at the expense of a slight destabilization.

Amino Acid Substitution↗
PubMed1978

Incomplete aminoacylation of tRNALeu catalyzed in vitro by leucyl-tRNA synthetase from Escherichia coli B.

The extent of esterification of [14C] leucine into Escherichia coli B tRNALeu apparently depends on the concentration of leucyl-tRNA synthetase. The effect is more pronounced at pH 9.0 than at pH 7.4. When reciprocals of leucyl-tRNA concentration at plateau [aa-tRNA]-1 are plotted against reciprocals of initial velocities vo-1 of aminoacylations a straight line is obtained with a slope equal to the rate constant of non-enzymatic deacylation of leucyl-tRNA. Factors which change the stability of leucyl-tRNA, e.g. pH and temperature, also change the shape of the function [aa-tRNA]-1 vs. vo-1. The data are consistent with the idea that the rate constant of spontaneous deacylation of aminoacyl-tRNA is the factor which accounts for the dependence of the level of aminoacylation on initial velocity of aminoacylation.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

[Stabilization of the transition state in the active center of aminoacyl-tRNA-synthetase].

The mechanism for transition state stabilization in the activation of amino acids by aminoacyl-tRNA synthetases is proposed. We carried out a quantum mechanical study on system modelling the attack of the amino acid carboxylate on the alpha-phosphate group of ATP. The activation barrier is reduced by improved hydrogen binding between a phosphoryl oxygen atom and a proton donor group of the enzyme. The relative stabilization of the transition state is found to be about 8 kcal/mol. On the basis of results obtained for the reaction in gas phase, in aqueous solution and in the active site the nature of the enzyme catalysis is discussed.

Amino Acyl-tRNA Synthetases↗
PubMed2002

The affinity of magnesium binding sites in the Bacillus subtilis RNase P x pre-tRNA complex is enhanced by the protein subunit.

The RNA subunit of bacterial ribonuclease P (RNase P) requires high concentrations of magnesium ions for efficient catalysis of tRNA 5'-maturation in vitro. The protein component of RNase P, required for cleavage of precursor tRNA in vivo, enhances pre-tRNA binding by directly contacting the 5'-leader sequence. Using a combination of transient kinetics and equilibrium binding measurements, we now demonstrate that the protein component of RNase P also facilitates catalysis by specifically increasing the affinities of magnesium ions bound to the RNase P x pre-tRNA(Asp) complex. The protein component does not alter the number or apparent affinity of magnesium ions that are either diffusely associated with the RNase P RNA polyanion or required for binding mature tRNA(Asp). Nor does the protein component alter the pH dependence of pre-tRNA(Asp) cleavage catalyzed by RNase P, providing further evidence that the protein component does not directly stabilize the catalytic transition state. However, the protein subunit does increase the affinities of at least four magnesium sites that stabilize pre-tRNA binding and, possibly, catalysis. Furthermore, this stabilizing effect is coupled to the P protein/5'-leader contact in the RNase P holoenzyme x pre-tRNA complex. These results suggest that the protein component enhances the magnesium affinity of the RNase P x pre-tRNA complex indirectly by binding and positioning pre-tRNA. Furthermore, RNase P is inhibited by cobalt hexammine (K(I) = 0.11 +/- 0.01 mM) while magnesium, manganese, cobalt, and zinc compete with cobalt hexammine to activate RNase P. These data are consistent with the hypothesis that catalysis by RNase P requires at least one metal-water ligand or one inner-sphere metal contact.

Bacillus subtilis↗
PubMed1993

Effects of novel polyamines on cell-free polypeptide synthesis catalyzed by Thermus thermophilus HB8 extract.

Effects of novel, naturally occurring polyamines on protein synthesis catalyzed by Thermus thermophilus cell-free extract were investigated. The results revealed the physiological importance of a branched quaternary polyamine, tetrakis(3-aminopropyl) ammonium, in thermophile protein biosynthesis. Longer polyamines than triamine supported the polypeptide synthesis at high temperature, though both the activity and the optimum temperature varied depending on polyamines added. The highest activity was found when tetrakis(3-aminopropyl)ammonium and a tetraamine were simultaneously present. The optimum temperature of the reaction supported by the combination of the branched polyamine and spermine was the highest and in accord with the optimum temperature of the bacterial growth. These results suggested an essential role of the quaternary amine in protein synthesis in vivo. This amine effectively stabilized the ternary complex between ribosomes, the messenger, and phenylalanyl-tRNA, and this stabilization may account, at least in part, for its action on the present reaction. In contrast, another branched polyamine, tris(3-aminopropyl)amine supported the activity only moderately even in the presence of another polyamine, though the tris amine stabilized the ternary complex as effectively as the quaternary amine. This result suggests the presence of another essential site for polyamine action in the thermophile polypeptide synthesis, in addition to the stabilization of the ternary complex. The effects of polyamines on MS2 RNA directed reaction resembled those on poly(U) directed polypeptide synthesis, indicating that polyamines are essential in protein biosynthesis directed by natural messengers in vivo. The quaternary amine inhibited the aminoacylation of tRNA(Phe), and the inhibition was canceled by the addition of another polyamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1984

An unexpected major groove binding of netropsin and distamycin A to tRNA(phe).

Crystalline complexes of yeast tRNA(phe) and the oligopeptide antibiotics netropsin and distamycin A were prepared by diffusing drugs into crystals of tRNA. X-ray structure analyses of these complexes reveal a single common binding site for both drugs which is located in the major or deep groove of the tRNA T-stem. The netropsin-tRNA complex is stabilized by specific hydrogen bonds between the amide groups of the drug and the tRNA bases G51 O(6), U52 O(4) and G53 N(7) on one strand, and is further stabilized by electrostatic interactions between the positively charges guanidino side chain of the drug and the tRNA phosphate P53 on the same strand and the positively charged amidino propyl side chain and the phosphates P61, P62 and P63 on the opposite strand of the double helix. These results are in contrast to the implicated minor groove binding of these drugs to non-guanine sequences in DNA. The binding to the GUG sequence in tRNA implies that major groove binding to certain DNA sequences is possible.

Binding Sites↗
PubMed1995

[A comparative computer analysis of thermodynamic parameters of transport RNA secondary structure].

Thermodynamic parameters of the cloverleaf secondary structure of tRNAs of several major taxons (archaebacteria, eubacteria, eukaryotes, chloroplasts, and mitochondria) were subjected to computer analysis. The distribution of free-energy values was close to normal in all groups (except for the mitochondrial tRNA). In the organisms existing under extreme environmental conditions, the stability of the tRNA secondary structure was higher. Comparative analysis of teh frequency of ¿quasi-complementary¿ GU pairs in stems of randomly generated and real sequences demonstrated preferential fixation of such pairs in the contexts with the most favorable thermodynamics parameters. Noncanonic pairs in tRNA stems set limits on the presence of highly mutable CG dinucleotides. Moreover, the effect of noncanonic pairs other than GU on the frequency of such dinucleotides is far more pronounced than that of GU pairs.

Base Sequence↗
PubMed1999

tRNA(Phe) binds aminoglycoside antibiotics.

Aminoglycoside antibiotics have recently been found to bind to a variety of unrelated RNA molecules, including sequences that are important for retroviral replication. We report the binding of neomycin B, kanamycin A, and Neo-Neo (a synthetic neomycin-neomycin dimer) to tRNA(Phe). Using thermal denaturation studies, fluorescence spectroscopy, Pb2+-mediated tRNA(Phe) cleavage, and gel mobility shift assays, we have established that aminoglycosides interact with yeast tRNA(Phe) and are likely to induce a conformational change. Thermal denaturation studies revealed that aminoglycosides have a substantial stabilizing effect on tRNA(Phe) secondary and tertiary structures, much greater than the stabilization effect of spermine, an unstructured polyamine. Aminoglycoside-induced inhibition of Pb2+-mediated tRNA(Phe) cleavage yielded IC50 values of: 5 microM for Neo-Neo, 100 microM for neomycin B, > 1 mM for kanamycin A, and > 10 mM for spermine. Enzymatic and chemical footprinting indicate that the anticodon stem as well as the junction of the TpsiC and D loops are preferred aminoglycoside binding sites.

Aminoglycosides↗
PubMed2000

tRNA-guanine transglycosylase from Escherichia coli: recognition of noncognate-cognate chimeric tRNA and discovery of a novel recognition site within the TpsiC arm of tRNA(Phe).

tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the posttranscriptional modification of tRNA across the three kingdoms of life. In eukaryotes and eubacteria, TGT is involved in the introduction of queuine into the anticodon of the cognate tRNAs. In archaebacteria, TGT is responsible for the introduction of archaeosine into the D-loop of the appropriate tRNAs. The tRNA recognition patterns for the eubacterial (Escherichia coli) TGT have been studied. These studies are all consistent with a restricted recognition motif involving a U-G-U sequence in a seven-base loop at the end of a helix. While attempting to investigate the potential of negative recognition elements in noncognate tRNAs via the use of chimeric tRNAs, we have discovered a second recognition site for the E. coli TGT in the TpsiC arm of in vitro-transcribed yeast tRNA(Phe). Kinetic analyses of synthetic mutant oligoribonucleotides corresponding to the TpsiC arm of the yeast tRNA(Phe) indicate that the specific site of TGT action is G53 (within a U-G-U sequence at the transition of the TpsiC stem into the loop). Posttranscriptional base modifications in tRNA(Phe) block recognition by TGT, most likely due to a stabilization of the tRNA structure such that G53 is inaccessible to TGT. These results demonstrate that TGT can recognize the U-G-U sequence within a structural context that is different than the canonical U-G-U in the anticodon loop of tRNA(Asp). Although it is unclear if this second recognition site is physiologically relevant, this does suggest that other RNA species could serve as substrates for TGT in vivo.

Base Sequence↗
PubMed1984

Stable tRNA precursors in HeLa cells.

Two tRNA precursors were isolated from 32P-labeled or unlabeled HeLa cells by two dimensional polyacrylamide gel electrophoresis, and were sequenced. These were the precursors of tRNAMet and tRNALeu, and both contained four extra nucleotides including 5'-triphosphates at their 5'-end and nine extra nucleotides including oligo U at their 3'-end. These RNAs are the first naturally occurring tRNA precursors from higher eukaryotes whose sequences have been determined. In these molecules, several modified nucleosides such as m2G, t6A and ac4C in mature tRNAs were undermodified. Two additional hydrogen bonds were formed in the clover leaf structures of these tRNA precursors. These extra hydrogen bonds may be responsible for the stabilities of these tRNA precursors.

Base Sequence↗