Glutamyl transfer ribonucleic acid synthetase of Escherichia coli. II. Interaction with intact glutamyl transfer ribonucleic acid.
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Adenosine Triphosphate↗
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The interconvertibility of various bacterial transfer ribonucleic acids between an active and an inactive stable configuration.
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Acetates↗
Interaction of lupin isoleucine-tRNA synthetase with isoleucine transfer ribonucleic acid measured by the thermal inactivation method.
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Adenosine↗
[Possibilities of forming a hydrogen-bonded cytosine-adenine pair in the structure of transfer ribonucleic acid and at the wobble-position of the codon-anticodon complex].
220 nucleotide sequences of tRNAs were investigated to elucidate the frequency of appearance of C-A pairs in their main two-stranded regions, in the positions 26--44 and 15--48. It was supposed, that in the formation of C-A pairs on antiparallel polynucleotide chains the atomic groups--N4-H and -N3 of cytosine make up H-bonds with the groups N7- and H-N6--of adenine. On parallel chains, H-bonds, probably, form -N6-H and --N1 groups of adenine with N3- and H-N4--of cytosine. The calculation results predicted a significant energy of ineraction between cytosine and adenine. By the investigation of the molecular models it was shown that the formation of H-bonded C-A pairs requires considerable changes of conformation in rebosephosphate chains. In addition a theoretical analysis revealed the possibility of formation of C-A pairs at the wobble-position of codon-anticodon complex. The significance of this nucleotide pair in the processes of genetic coding proved to depend on the stability of the codon-anticodon complex, modification of cytosine 34 and structural features of the distant regions of the tRNA.
Adenine↗
Evolution of the genome and the genetic code: selection at the dinucleotide level by methylation and polyribonucleotide cleavage.
Noting the scarcity of CpG dinucleotide in total genomic DNA derived from higher organisms and the scarcity of TpA dinucleotide in total genomic DNA derived from most life forms, we examined the distribution of these dinucleotides in sequences derived from functionally distinct types of human DNA, including mitochondrial DNA, intergenic DNA, intron DNA, and DNA destined to be represented in the cytoplasm as mRNA, tRNA, or rRNA. While CpG frequency has fallen to its lowest levels in DNA that is transcriptionally silent, TpA is most stringently excluded in DNA destined to be expressed as mRNA in the cytosol. This observation suggests that the selective pressures leading to the removal of CpG and TpA operate at different levels. With respect to TpA, dinucleotide scarcity may reflect a requirement for mRNA stability and may indicate the action of UpA-selective ribonucleases. We propose that, by reason of its instability, UpA must have been very rare in primordial RNA. Therefore, tRNA with the anticodon for this dinucleotide may have failed to evolve, making UpA the primordial doublet "stop" codon. The modern triplet code has faithfully conserved this arrangement in the two universal stop codons, UAA and UAG.
Base Sequence↗
Synthesis and aminoacylation of 3'-amino-3'-deoxy transfer RNA and its activity in ribosomal protein synthesis.
3'-amino-3'-deoxy adenosine was enzymatically converted into 3'-amino-3'-deoxy ATP. This ATP analogue was incorporated into the 3'-terminal adenosine position of Escherichia coli tRNA. The modified tRNA was aminoacylated with phenylalanine by use of E. coli phenylalanyl tRNA synthetase. The phenylalanine was attached to the 3'-amino group of the tRNA, as shown by its high resistance to base-catalyzed hydrolysis in contrast with the normal lability of phenylalanyl-tRNA. Aminoacyl tRNA synthetases charge the 3'-amino-3'-deoxy tRNA with kinetics that are similar to those of the charging reaction in which normal tRNA is the substrate. When phenylalanyl-3'-amino-3'-deoxy tRNA is used in a protein-synthesizing system directed by poly(U) in vitro, this molecule is capable of receiving an acetyl-phenylalanine from the donor site of the ribosome. However, the ribosome is unable to cleave the amide bond connecting phenylalanine to the tRNA molecule; hence, the phenylalanyl-3'-amino-3'-deoxy tRNA has acceptor but not donor activity in protein synthesis. Failure of the ribosome to cleave the amide bond may be due to its greater stability relative to the normal ester bond. However, it may also be due to the fact that the isomerization of the peptidyl chain between the 2' and 3' positions of adenosine is prevented due to the amide bond at the 3' position, and cleavage may normally occur with the peptidyl chain on the 2' position of adenosine.
Adenosine↗
tRNA methyltransferase activity in neonatal and mature mammalian neural tissue.
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Aging↗
Activities of tRNA-embedded dimeric minizymes.
Ribozymes have been shown to be potent inhibitors of gene expression and viral function. Effects of ribozyme-mediated repression to target gene in living cells are correlated with the amounts of expression and stabilities of ribozyme molecules. In our previous study, it was demonstrated that a minimized hammerhead ribozyme, minizyme, with high activity forms a dimeric structure with a common stem II. We constructed dimeric minizymes that could cleave the BCR-ABL chimeric (b2a2) mRNA which had been difficult target for conventional hammerhead ribozymes without damaging the normal ABL mRNA. In order to achieve high expression of these dimeric minizymes in vivo for the treatment of CML, we embedded the dimeric minizyme portion downstream of a tRNA(Val) promoter sequence which could be recognized by RNA polymerase III. We determined cleavage activities of tRNA-embedded dimeric minizymes and compared the activities between tRNA-embedded hammerhead ribozyme and tRNA-embedded dimeric minizymes. All tRNA-embedded dimeric minizymes tested were capable of cleavage the target substrate. The activity of the tRNA-embedded dimeric minizyme targeted at BCR-ABL mRNA was almost the same as that of the naked dimeric minizymes. Interestingly, the cleavage activity of tRNA-embedded dimeric minizymes was higher than that of tRNA-embedded conventional hammerhead ribozyme.
Base Sequence↗
Closely spaced and divergent promoters for an aminoacyl-tRNA synthetase gene and a tRNA operon in Escherichia coli. Transcriptional and post-transcriptional regulation of gltX, valU and alaW.
The transcription of the gltX gene encoding the glutamyl-tRNA synthetase and of the adjacent valU and alaW tRNA operons of Escherichia coli K-12 has been studied. The alaW operon containing two tRNA(GGCAla) genes, is 800 base-pairs downstream from the gltX terminator and is transcribed from the same strand. The valU operon, containing three tRNA(UACVal) and one tRNA(UUULys) (the wild-type allele of supN) genes, is adjacent to gltX and is transcribed from the opposite strand. Its only promoter is upstream from the gltX promoters. The gltX gene transcript is monocistronic and its transcription initiates at three promoters, P1, P2 and P3. The transcripts from one or more of these promoters are processed by RNase E to generate two major species of gltX mRNA, which are stable and whose relative abundance varies with growth conditions. The stability of gltX mRNA decreases in an RNase E- strain and its level increases with growth rate about three times more than that of the glutamyl-tRNA synthetase. The 5' region of these mRNAs can adopt a stable secondary structure (close to the ribosome binding site) that is similar to the anticodon and part of the dihydroU stems and loops of tRNA(Glu), and which might be involved in translational regulation of GluRS synthesis. The gltX and valU promoters share the same AT-rich and bent upstream region, whose position coincides with the position of the upstream activating sequences of tRNA and rRNA promoters to which they are similar. This suggests that gltX and valU share transcriptional regulatory mechanisms.
Amino Acyl-tRNA Synthetases↗
Peptide bond formation does not involve acid-base catalysis by ribosomal residues.
Ribosomes catalyze the formation of peptide bonds between aminoacyl esters of transfer RNAs within a catalytic center composed of ribosomal RNA only. Here we show that the reaction of P-site formylmethionine (fMet)-tRNA(fMet) with a modified A-site tRNA substrate, Phelac-tRNA(Phe), in which the nucleophilic amino group is replaced with a hydroxyl group, does not show the pH dependence observed with small substrate analogs such as puromycin and hydroxypuromycin. This indicates that acid-base catalysis by ribosomal residues is not important in the reaction with the full-size substrate. Rather, the ribosome catalyzes peptide bond formation by positioning the tRNAs, or their 3' termini, through interactions with rRNA that induce and/or stabilize a pH-insensitive conformation of the active site and provide a preorganized environment facilitating the reaction. The rate of peptide bond formation with unmodified Phe-tRNA(Phe) is estimated to be >300 s(-1).
Acids↗
The structural basis for seryl-adenylate and Ap4A synthesis by seryl-tRNA synthetase.
BACKGROUND: Seryl-tRNA synthetase is a homodimeric class II aminoacyl-tRNA synthetase that specifically charges cognate tRNAs with serine. In the first step of this two-step reaction, Mg.ATP and serine react to form the activated intermediate, seryl-adenylate. The serine is subsequently transferred to the 3'-end of the tRNA. In common with most other aminoacyl-tRNA synthetases, seryl-tRNA synthetase is capable of synthesizing diadenosine tetraphosphate (Ap4A) from the enzyme-bound adenylate intermediate and a second molecule of ATP. Understanding the structural basis for the substrate specificity and the catalytic mechanism of aminoacyl-tRNA synthetases is of considerable general interest because of the fundamental importance of these enzymes to protein biosynthesis in all living cells. RESULTS: Crystal structures of three complexes of seryl-tRNA synthetase from Thermus thermophilus are described. The first complex is of the enzyme with ATP and Mn2+. The ATP is found in an unusual bent conformation, stabilized by interactions with conserved arginines and three manganese ions. The second complex contains seryl-adenylate in the active site, enzymatically produced in the crystal after soaking with ATP, serine and Mn2+. The third complex is between the enzyme, Ap4A and Mn2+. All three structures exhibit a common Mn2+ site in which the cation is coordinated by two active-site residues in addition to the alpha-phosphate group from the bound ligands. CONCLUSIONS: Superposition of these structures allows a common reaction mechanism for seryl-adenylate and Ap4A formation to be proposed. The bent conformation of the ATP and the position of the serine are consistent with nucleophilic attack of the serine carboxyl group on the alpha-phosphate by an in-line displacement mechanism leading to the release of the inorganic pyrophosphate. A second ATP molecule can bind with its gamma-phosphate group in the same position as the beta-phosphate of the original ATP. This can attack the seryl-adenylate with the formation of Ap4A by an identical in-line mechanism in the reverse direction. The divalent cation is essential for both reactions and may be directly involved in stabilizing the transition state.
Adenosine Monophosphate↗
No Effect of 5-Fluorouracil on the Properties of Purified alpha-Amylase from Barley Half-seeds.
alpha-Amylase has been purified from de-embryonated seeds of barley (Hordeum vulgare L. cv. Betzes) which have been incubated on 10(-6)m gibberellic acid (GA(3)) following 3 days of imbibition in buffer. Incubation of the half-seeds in up to 10(-2)m 5-fluorouracil (5-FU) during the entire incubation period, including imbibition, had no effect on any of the following characteristics of purified alpha-amylase: thermal stability in the absence of calcium, molecular weight of the enzyme, isozyme composition, specific activity, or the amount of alpha-amylase synthesized by the aleurone tissue. The synthesis of rRNA and tRNA was strongly inhibited by 5-FU, indicating that the analog had entered the aleurone cells. These results are not in agreement with those of Carlson (Nature New Biology 237: 39-41 [1972]) who found that treatment of barley aleurone with 10(-4)m 5-FU prior to the addition of GA(3) resulted in decreased thermal stability of GA(3)-induced alpha-amylase and who interpreted this as evidence that the mRNA for alpha-amylase was synthesized during the imbibition of the aleurone tissue and independently of gibberellin action. Results of the present experiments indicate that the thermal stability of highly purified alpha-amylase is not altered by treatment of barley half-seeds with 5-FU, and that 5-FU cannot be used as a probe to examine the timing of alpha-amylase mRNA synthesis.
Journal Article↗
Molecular and functional properties of an archaeal phenylalanyl-tRNA synthetase from the hyperthermophile Sulfolobus solfataricus.
An archaeal phenylalanyl-tRNA synthetase (FRS) has been purified from the hyperthermophile Sulfolobus solfataricus (Ss). This enzyme is a heterotetramer made of two different subunits whose molecular mass is 56 kDa and 64 kDa, respectively. As thought, SsFRS is essential for the in vitro poly(Phe) synthesis. Interestingly, the enzyme is able to aminoacylate only endogenous tRNA but it does not seem to be a strictly ATP-dependent synthetase. SsFRS interacts with the elongation factor 1alpha isolated from the same source; this caused a significant enhancement of the SstRNA aminoacylation efficiency, thus indicating that, as well as in eukarya, in this archaeon a tRNA channelling mechanism should occur. The overall results presented in this paper show that the archaeal SsFRS behaves as the analogous enzymes isolated from eukaryal sources rather than those from eubacterial organisms.
Adenosine Triphosphate↗
The T loop structure is dispensable for substrate recognition by tRNase ZL.
tRNA 3'-processing endoribonucleases (tRNase Z, or 3'-tRNase; EC 3.1.26.11) are enzymes that remove 3'-trailers from pre-tRNAs. An about 12-base-pair stem, a T loop-like structure, and a 3'-trailer were considered to be the minimum requirements for recognition by the long form (tRNase ZL) of tRNase Z; tRNase ZL can recognize and cleave a micro-pre-tRNA or a hooker/target RNA complex that resembles a micro-pre-tRNA. We examined four hook RNAs containing systematically weakened T stems for directing target RNA cleavage by tRNase ZL. As expected, the cleavage efficiency decreased with the decrease in T stem stability, and to our surprise, even the hook RNA that forms no T stem-loop-directed slight cleavage of the target RNA, suggesting that the T stem-loop structure is important but dispensable for substrate recognition by tRNase ZL. To analyze the effect of the T loop on substrate recognition, we compared the cleavage reaction for a micro-pre-tRNA with that for a 12-base-pair double-stranded RNA, which is the same as the micro-pre-tRNA except for the lack of the T loop structure. The observed rate constant value for the double-stranded RNA was comparable with that for the micro-pre-tRNA, whereas the K(d) value for the complex with the double-stranded RNA was much higher than that for the complex with the micro-pre-tRNA. These results suggest that the T loop structure is not indispensable for the recognition, although the interaction between the T loop and the enzyme exists. Cleavage assays for such double-stranded RNA substrates of various lengths suggested that tRNase ZL can recognize and cleave double-stranded RNA substrates that are longer than 5 base pairs and shorter than 20 base pairs. We also showed that double-stranded RNA is not a substrate for the short form of tRNase Z.
Animals↗
A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 A.
The three-dimensional crystal structure of seryl-transfer RNA synthetase from Escherichia coli, refined at 2.5 A resolution, is described. It has an N-terminal domain that forms an antiparallel alpha helical coiled-coil, stretching 60 A out into the solvent and stabilized by interhelical hydrophobic interactions and an active-site alpha-beta domain based around a seven-stranded antiparallel beta sheet. Unlike the three other known synthetase structures, the enzyme contains no classical nucleotide-binding fold, and is the first representative of a second class of aminoacyl-tRNA synthetase structures.
Amino Acid Sequence↗
The plant aminoacyl-tRNA synthetases. 2'-DeoxyATP and ATP in reactions catalysed by yellow lupin aminoacyl-tRNA synthetases.
2'-Deoxyadenosine triphosphate (dATP) is not a substrate for valyl-tRNA, leucyl-tRNA and isoleucyl-tRNA synthetases from yellow lupin seeds. Yellow lupin seryl-tRNA, phenylalanyl-tRNA, tyrosyl-tRNA, arginyl-tRNA, lysyl-tRNA and methionyl-tRNA synthetases use dATP as a substrate both in aminoacyl deoxyadenylate formation and tRNA aminoacylation reactions. Generally, dATP is a poorer substrate, being 3 - 50% as effective as ATP. None of the other nucleoside triphosphates tested was shown to be a substrate for these enzymes. Specificity (k cat/Km) towards ATP is greater in tRNA aminoacylation than in pyrophosphate exchange reaction catalysed by seryl-tRNA synthetase. Energy of activation is the same (Ea = 18.5 kcal) for dATP- and ATP-dependent pyrophosphate exchange. Both dATP- and ATP-dependent tRNA aminoacylation reactions exhibit the same temperature dependence with Ea = 9.0 kcal. Half lives of the enzyme-bound seryl adenylate and deoxyadenylate are 2 min and 4 min, respectively (pH 8.0, 25 degrees C). Both enzyme-bound phenylalanyl adenylate and deoxyadenylate exhibit the same stability (half lives 0.3 min), and enzyme-bound tyrosyl deoxyadenylate is hydrolysed faster (half life 0.6 min) than tyrosyl adenylate (half life 2.5 min). Serine is transferred both from enzyme-bound seryl adenylate and deoxyadenylate to tRNA with the same 60% efficiency. One mol of ATP or 1.1 mol of dATP is hydrolysed per one mol of seryl-tRNA formed during aminoacylation in the complete ATP- or dATP-dependent system, respectively. AMP concentrations (up to 0.5 mM), which do not affect the equilibrium of the ATP-dependent tRNA esterification with serine, significantly change the equilibrium of the dATP-dependent reaction.
Adenosine Triphosphate↗
Multiple forms of tryptophanyl-tRNA synthetase from beef pancreas.
Three different forms of tryptophanyl-tRNA synthetase can be isolated from pancreatic extracts. Structural, immunological and catalytic properties of these various forms have been compared. The native enzyme is a dimeric molecule of molecular weight 108000. Two other forms, of molecular weight 85000 and 82000, are composed of two polypeptide chains identical with the carboxyl terminal region of the native subunits. These molecules are supposed to derive from the original protein by removal, from the amino-terminal part of each subunit, of a fragment of 11000 to 13000 molecular weight. Such removal modifies the shape and the stability of the molecule and decreases its specific acitvity. The origin of the derived forms is attributed to proteolysis. In fact, limited proteolysis of purified tryptophanyl-tRNA synthetase, in its native form, by elastase, results in the formation of an active compound, similar to one of the tryptophanyl-tRNA synthetase derived forms. Furthermore, incubation with "elastolytic fractions" prepared from pancreatic extracts presenting a particularly high level of proteolytic activity produces the same cleavage in tryptophanyl-tRNA synthetase polypeptide chain.
Amino Acids↗
A uniform response to mismatches in codon-anticodon complexes ensures ribosomal fidelity.
Ribosomes take an active part in aminoacyl-tRNA selection by distinguishing correct and incorrect codon-anticodon pairs. Correct codon-anticodon complexes are recognized by a network of ribosome contacts that are specific for each position of the codon-anticodon duplex and involve A-minor RNA interactions. Here, we show by kinetic analysis that single mismatches at any position of the codon-anticodon complex result in slower forward reactions and a uniformly 1000-fold faster dissociation of the tRNA from the ribosome. This suggests that high-fidelity tRNA selection is achieved by a conformational switch of the decoding site between accepting and rejecting modes, regardless of the thermodynamic stability of the respective codon-anticodon complexes or their docking partners at the decoding site. The forward reactions on mismatched codons were particularly sensitive to the disruption of the A-minor interactions with 16S rRNA and determined the variations in the misreading efficiency of near-cognate codons.
Animals↗