NMR study of the modified base resonances of tRNA tyr- coli

220MHz NMR spectra at 28° show several resolved resonances in the high field region for D₂O solutions of tyrosine specific tRNA from E. coli. These resonances are tentatively identified as arising from protons of the modified nucleoside, 2-methylthio-N⁶-(Δ²-isopentenyl)-adenosine and from the modified guanosine of unknown structure in the “wobble position” of the anti codon loop. Assignment of resonances was aided by comparison with spectra of tRNA_su⁺III^tyr, Form II, whose sequence is closely homologous to tRNA_coli^tyr, except for changes in some modified bases. Line widths of resolved resonances indicate that, at 28°, the methyl groups of modified nucleosides are not completely restricted in their motion relative to the overall motion of the macromolecule.     

Interactions of yeast valyl-tRNA synthetase with RNAs and conformational changes of the enzyme.

Different conformations have been identified for the enzyme valyl-tRNA synthetase from yeast inside its complex with one tRNA molecule by neutron scattering. One form is identical to that of the free enzyme in solution; the other form is more contracted, having a radius of gyration which is smaller by 10% and a specific volume which is smaller by 1%. The contracted conformation has been found for the complexes with tRNA^Val and tRNA^Asp in phosphate buffer (pH 6.3) provided the ionic strength is lower than about 150 mm. In higher ionic strength (up to about 500 mm) the enzyme still forms a complex with tRNA^Val but its conformation remains that of the free protein in solution. In the complex with tRNA₃^Leu, the enzyme conformation is that of the free state even at the lowest ionic strength examined (that of the phosphate buffer, 60 mm). The free enzyme is an elongated molecule of radius of gyration 40 Å (a compact protein of the same molecular weight would have a radius of gyration of 30 Å).The positioning within the complex of tRNA^Val, on the one hand, and tRNA₃^Leu, on the other, is very different. The first tRNA is intimately associated with the enzyme, lying predominantly closer to the centre of mass of the complex than the protein. In the complex with tRNA₃^Leu, the tRNA lies further away from the centre of mass of the complex than the protein.Small concentrations of tRNA^Val, tRNA^Asp, tRNA₃^Leu or Escherichia coli 5 S ribosomal RNA cause the enzyme to aggregate into dimers, trimers and higher aggregates provided the ionic strength of the buffer is below 150 mm. In higher ionic strength or for [RNA]: [enzyme] > 1 the aggregates are dissociated to yield the one-to-one RNA-enzyme complex.     

Affinity labelling of tryptophanyl-transfer RNA synthetase

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme.Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, α₂ An ATP analogue, γ-(p-azidoanilide)-ATP does not serve as a substrate for enzymatic aminoacylation of tRNA^Trp but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K₁ = 1 × 10^?3m (K_mfor ATP = 2 × 10^?4m). The covalent photoaddition of azido-ATP³ results in complete loss of enzymatic activity in both the ATP-[³²P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme.Two binding sites for tRNA^Trp have been found from complex formation at pH 5.8 in the presence of Mg²⁺. The two tRNA molecules bind, with K_dis = 3.6 × 10^?8m and K_dis = 0.9 × 10^?6m, respectively, pointing to a strong negative co-operativity between the binding sites for tRNA.N-chlorambucilyl-tryptophanyl-tRNA^Trp and TRSase form a complex with K_dis = 5.5 × 10^?8m at pH 5.8 in the presence of 10 mm-Mg²⁺. This value is similar to the value of K_dis for tryptophanyl-tRNA of 4.8 × 10^?8m. Under the same conditions a 1:1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilyl-Trp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of tRNA^Trp whereas the tRNA devoid of tRNA^Trp does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the complex formation between TRSase and the tRNA analogue. Photoaddition of the azido-ATP completely prevents the reaction of the enzyme with the tRNA analogue, although the non-covalent complex formation is not affected.Exhaustive alkylation of TRSase partially inhibits the reaction of ATP [³²P]pyrophosphate exchange and completely blocks the aminoacylation of tRNA^Trp. Cleavage of the tRNA which is covalently bound to TRSase restores both the ATP-[³²P]pyrophosphate exchange and aminoacylation activity.The TRSase which is covalently-bound to R-Trp-tRNA is able to incorporate only one ATP molecule per dimeric enzyme into the active centre. This doubly modified enzyme is completely enzymatically inactive. Removal of the tRNA residue from the doubly modified enzyme results in the formation of the derivative with one blocked ATP site. Therefore, a “single-site” TRSase may be generated either by alkylation of the enzyme with Cl-R-Trp-tRNA or after the removal of covalently bound tRNA from the doubly labelled protein.Tryptophanyl-tRNA synthetase containing blocked ATP and/or tRNA binding site(s) seems to bo a useful tool for investigation of negative co-operativity and may help in the elucidation of the structure function relationships between the active centres.     

Two forms of methionyl-transfer RNA synthetase from Mycobacterium smegmatis

Two methionyl-transfer RNA synthetases (A and B forms) have been isolated from $\text{Mycobacterium smegmatis}$. The homogeneous preparations of the enzymes showed 1500 fold increase in specific activity in aminoacylation of methionine specific tRNA. The A and B forms differed in their specificity of aminoacylation of tRNA_m^Met and tRNA_f^Met; enzyme B exhibited much higher specificity for tRNA_f^Met. The molecular activities of A and B enzymes for aminoacid and tRNA were identical. The turnover number for aminoacid was 27 fold greater than that for tRNA, while the Km values for tRNA were lower by a factor of 10⁶ as compared to the aminoacid. Both the enzymes catalysed ATP-pyrophosphate exchange reaction to the same extent.     

A critical role of water in the specific cleavage of the anticodon loop of some eukaryotic methionine initiator tRNAs

We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond. A literature search indicated that there is also the case for human initiator tRNA^Met but not for yeast tRNA^Met _i or E. coli tRNA^Met _f. All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop. It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNA^Met _i susceptible to the specific cleavage reaction. Using crystallographic data of tRNA^Met _f of E. coli with U33, we modeled the anticodon loop of this tRNA with C33. We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond. We conclude that a single nucleotide change in the primary structure of tRNA^Met _i made changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.     

Function of Y in codon-anticodon interaction of tRNA Phe

Molar association constants of binding oligonucleotides to the anticodon loops of (yeast) tRNA^Phe, (yeast) tRNA_HCl^Phe and (E. coli) tRNA_F^Met have been determined by equilibrium dialysis. From the temperature dependence of the molar association constants, ΔF, ΔH and ΔS of oligomer-anticodon loop interaction have been determined. The data indicate that the free energy change of codon-anticodon interaction is highly influenced by the presence of a modified purine (tRNA^Phe), of an unmodified purine (tRNA_F^Met) or its absence (tRNA_HCl^Phe). Excision of the modified purine Y in the anticodon loop of tRNA^Phe results in a conformational change of the anticodon loop, which is discussed on the basis of the corresponding changes in ΔF, ΔH and ΔS.     

A new yeast poly(A) polymerase complex involved in RNA quality control

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNA^Met (tRNA_i^Met). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNA_i^Met with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNA_i^Met by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover.     

Primary structure of E. coli alanine transfer RNA: relation to the yeast phenylalanyl tRNA synthetase recognition site

Nucleotide sequence comparison of tRNAs aminoacylated by yeast phenylalanyl tRNA synthetase (PRS) have lead to the proposal that the specific nucleotides of the dihydrouridine (diHU) stem region and adenosine at the fourth position from the 3′ end are involved in the PRS recognition site. Kinetic analysis and enzymatic methylation have shown that the size of the diHU loop and the methylation of guanine at position 10 from the 5′ end both directly affect the PRS aminoacylation kinetics. $\text{E. coli}$ tRNA₁^A1a, which is aminoacylated by PRS, should therefore have 1- the specific nucleotides of the diHU stem region and, 2- adenosine at position 4 from the 3′ end. The PRS aminoacylation kinetics of this tRNA indicates that this molecule 3- has a diHU loop of 8 nucleotides and 4- has an unmethylated guanine at position 10 from the 5′ end. We report here the complete sequence of $\text{E. coli}$ tRNA₁^A1a and confirmation of each of these four predictions.     

PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine

Pyrrolysine is represented by an amber codon in genes encoding proteins such as the methylamine methyltransferases present in some Archaea and Bacteria. Pyrrolysyl-tRNA synthetase (PylRS) attaches pyrrolysine to the amber-suppressing tRNA^Pyl. Archaeal PylRS, encoded by pylS, has a catalytic C-terminal domain but an N-terminal region of unknown function and structure. In Bacteria, homologs of the N- and C-terminal regions of archaeal PylRS are respectively encoded by pylSn and pylSc. We show here that wild type PylS from Methanosarcina barkeri and PylSn from Desulfitobacterium hafniense bind tRNA^Pyl in EMSA with apparent K_d values of 0.12 and 0.13 μm, respectively. Truncation of the N-terminal region of PylS eliminated detectable tRNA^Pyl binding as measured by EMSA, but not catalytic activity. A chimeric protein with PylSn fused to the N terminus of truncated PylS regained EMSA-detectable tRNA^Pyl binding. PylSn did not bind other D. hafniense tRNAs, nor did the competition by the Escherichia coli tRNA pool interfere with tRNA^Pyl binding. Further indicating the specificity of PylSn interaction with tRNA^Pyl, substitutions of conserved residues in tRNA^Pyl in the variable loop, D stem, and T stem and loop had significant impact in binding, whereas those having base changes in the acceptor stem or anticodon stem and loop still retained the ability to complex with PylSn. PylSn and the N terminus of PylS comprise the protein superfamily TIGR03129. The members of this family are not similar to any known RNA-binding protein, but our results suggest their common function involves specific binding of tRNA^Pyl.     

Antico-operative binding of initiator transfer RNAMet to methionyl-transfer RNA synthetase from Escherichia coli: neutron scattering studies.

The interaction of methionyl-tRNA synthetase with initiator tRNA^Met has been investigated by neutron scattering. On the basis of parallel fluorescence measurements, two types of titrations have been performed. (1) In the presence of 10 mm-MgCl₂, a condition which insures antico-operative binding of two tRNA molecules to the enzyme dimer. (2) With saturating amounts of 5′-AMP and l-methioninol, in the presence of 50 mm-MgCl₂, conditions which allow two transfer RNA molecules to bind the dimer with very similar affinities.Varying the solvent density (²H₂O fraction) in the samples has allowed the identification by neutron scattering of changes in the radius of gyration and in the degree of dissociation of the enzyme dimer upon tRNA binding. In buffer containing 10 mm-MgCl₂, at each contrast studied, the binding process involves two steps. Firstly, one tRNA^met_f molecule binds easily to one dimeric enzyme molecule with an associated decrease of the radius of gyration of the enzyme moiety. The centre of mass of this tRNA lies very close to the centre of mass of the protomer with which it associates. Then, at higher tRNA concentration, a second tRNA molecule binds to the enzyme. However, the affinity of this second site is very much weaker. With the binding of the second tRNA, the radius of gyration of the enzyme moiety increases markedly. Concomitant limited dissociation of the dimer is suggested by the experimental data. These observations combined with the fact that, in 50 mm-MgCl₂ both the increased radius of gyration and the partial dissociation of the enzyme are accomplished in the absence of tRNA and remain unaffected upon binding one or two tRNA, confirm that the hindrance to binding a second tRNA in 10 mm-MgCl₂ arises from the constrained conformation of the one tRNA-enzyme complex.     

Antico-operative binding of bacterial and mammalian initiator tRNAMet to methionyl-tRNA synthetase from Escherichia coli

The reaction scheme of methionyl-tRNA synthetase from Escherichia coli with the initiator tRNA_s^Met from E. coli and rabbit liver, respectively, has been resolved. The statistical rate constants for the formation, k_R, and for the dissociation, k_D, of the 1:1 complex of these tRNAs with the dimeric enzyme have been calculated. Identical k_R values of 250 μm^?1 s^?1 reflect similar behaviour for antico-operative binding of both tRNA_s^Met to native methionyl-tRNA synthetase. Advantage was taken of the difference in extent of tryptophan fluorescence-quenching induced by the bacterial and mammalian initiator tRNA_s^Met to measure the mode of exchange of these tRNAs antico-operatively bound to the enzyme. Analysis of the results reveals that antico-operativity does not arise from structural asymmetric assembly of the enzyme subunits. Indeed, both subunits can potentially bind a tRNA molecule. Exchange between tRNA molecules can occur via a transient complex in which both sites are occupied. Either strong and weak sites reciprocate between subunits on the transient complex or occupation of the weak site induces symmetry of this complex. While in the present case, these two alternatives are kinetically indistinguishable, they do account for the observation that, upon increasing the concentration of the competing mammalian tRNA, the rate of exchange of the E. coli initiator tRNA^Met is enhanced, due to its faster rate of dissociation from the transient complex. Finally, it has been verified that in the case of the trypsin-modified methionyl-tRNA synthetase which cannot provide more than one binding site for tRNA, exchange of enzymebound bacterial tRNA by mammalian tRNA does proceed to a limiting rate independent of the mammalian tRNA concentration present in the solution.     

Sequence Relationship of Three Valine Acceptor tRNAs from <Emphasis Type="Italic">Escherichia coli</Emphasis>

THE degree of degeneracy of the genetic code varies for the twenty amino-acids: between one and six different triplets are assigned to a single amino-acid. Four triplets GUU, GUC, GUA, GUG code for the amino-acid valine^1,2. Two valine specific tRNAs have been separated by fractionation of mixed E. coli tRNA³; tRNA^val₁ is specific for GU^A_G and tRNA^val₂ corresponds to GU^U_C (see also ref. 1 for binding properties). Recent studies showed that although both species are recognized by the single activating enzyme present in E. coli, the association constant (Ka) for the minor species, tRNA^val₂ (?20% of total acceptor), is an order of magnitude higher than the association constant of the major species, tRNA^{val 4}₁. As a first step to comparing the structures of these two tRNAs, we analysed the base sequences of the major and minor species. We recently published the nucleotide sequence of tRNA^{val 5}₁; we report here the sequence of two minor subspecies (quite similar to each other) that comprise the tRNA^val₂ acceptor and we comment on the significance of the sequence homologies in relation to the problems of enzyme recognition and tRNA evolution.     

Interaction of manganese with fragments, complementary fragment recombinations, and whole molecules of yeast phenylalanine specific transfer RNA

Binding of Mn²⁺ to the whole molecule, fragments and complementary fragment recombinations of yeast tRNA^Phe, and to synthetic polynucleotides was studied by equilibrium dialysis. The comparison of the binding patterns of the fragments, fragment recombinations and synthetic polynucleotides with that of intact tRNA^Phe permits reasonable conclusions concerning the nature and location of the various classes of sites on tRNA^Phe. Binding of Mn²⁺ to intact tRNA^Phe consists of a co-operative and a non-co-operative phase. There are about 17 “strong” sites and several “weak” ones. Five of the 17 strong sites are associated with the co-operative phase. This phase is completely lacking in the binding of Mn²⁺ to tRNA^Phe fragments (5′-$\text{1}\text{2}$, 3′-$\text{1}\text{2}$, 5′-$\text{3}\text{5}$, 3′-$\text{2}\text{5}$), poly-(A):poly(U) and poly(I):poly(C) helices, and single stranded poly(A) and poly(U). This argues that the co-operative sites arise from the tRNA tertiary structure. This conclusion is further strengthened by the observation that cooperativity is present in a tRNA^Phe molecule which has been split in the anticodon loop, but it is absent in one which has been split in the extra loop. It is in the vicinity of the latter loop, but not the former, that tertiary interactions are seen in the crystal structure. The remaining 12 strong sites are “independent” and appear to be associated with cloverleaf helical sections.     

A simplified method for study of RNA conformation--reaction of formylmethionine transfer RNA with [14C]methylamine-bisulfite

A new chemical method for radioactive labeling of single-stranded regions of RNA has been used to probe the three-dimensional structure of E. coli tRNA^fMet in solution. The procedure involves conversion of cytosine residues to N⁴-[¹⁴C]methylcytosines by treatment with ¹⁴CH₃NH₂ and sodium bisulfite at pH7. Ribonuclease digestion of the modified tRNA produces ¹⁴C-labeled oligonucleotides which comigrate with the corresponding unlabeled oligonucleotides, facilitating structural analysis. By this procedure, E. coli tRNA^fMet has been found to contain only six reactive cytosines: C₁, C₁₆, C₁₇, C₃₅, C₇₅ and C₇₆. In addition, slow reaction at C^m₃₃ was observed. These results are in excellent agreement with previously reported data on the sites of exposed cytosine residues in tRNA^fMet obtained by two other chemical methods. The methylamine-bisulfite procedure is recommended for studying the ordered structure of more complex polyribonucleotides such as viral and ribosomal RNAs.     

Physical mapping of the transfer RNA genes on lambda-h80dglytsu+36

The three Escherichia coli transfer RNA genes of the DNA of the transducing phage λ80cI857S^?t68dglyTsu⁺₃₆tyrTthrT (abbreviated λh80T), which specify the structures of tRNA^Gly₂(su⁺₃₆), tRNA^Tyr₂ and tRNA^Thr₃, have been mapped by hybridizing ferritin-labeled E. coli tRNA to heteroduplexes of λh80T DNA with the DNA of the parental phage (λh80cI857S^?t68) and examining the product in the electron microscope. The DNA of λh80T contains a piece of bacterial DNA of length 0·43 λ unit³ that replaces a piece of phage DNA of length 0·46 λ unit, proceeding left from B · P′ (the junction of bacterial DNA and phage DNA) (i.e. att⁸⁰). A cluster of three ferritin binding sites, and thus of tRNA genes, is seen at a position of 0·24 λ unit (1·1 × 10⁴ nucleotides) to the left of B· P′. The three tRNA genes of the cluster are separated by the unequal spacings of 260 (±30) and 140 (± 30) nucleotides, proceeding left from B·P′. The specific map positions have been identified by hybridization competition between ferritin-labeled whole E. coli tRNA with unlabeled purified tRNA^Tyr₂ and with unlabeled partially purified tRNA^Gly₂. The central gene of the cluster is tRNA^Tyr₂. The tRNA^Gly₂gene is probably the one furthest from B·P′. Thus, the gene order and spacings, proceeding left from B·P′, are: tRNA^Thr₃, 260 nucleotides, tRNA^Try₂, 140 nucleotides, tRNA^Gly₂.     

Clustering of transfer RNA genes of Xenopus laevis

The arrangement of the reiterated DNA sequences complementary to transfer RNA has been studied in Xenopus laevis. Prehybridization of denatured DNA with an excess of unfractionated tRNA results in a small but well-defined increase in the buoyant density of fragments which contain sequences homologous to tRNA. The density increase is smaller than that found for 5 S DNA, but is the same or nearly so for all tRNA coding sequences examined. These results indicate that the majority of tRNA genes are clustered together with spacer DNA, the average size of which is estimated to be approximately 0.5 × 10⁶ daltons (native) DNA.In high molecular weight native DNA preparations, the sequences homologous to unfractionated tRNA, tRNA^Val, tRNA₁^Met and tRNA₂^Met band in CsCl at 1.707, 1.702, 1.708 and 1.711 g cm^?3, respectively. The mean buoyant densities are constant at all molecular weights examined but they do not correspond to the base compositions of the complementary tRNA species. These results indicate that isocoding genes are linked to spacer DNA in separate and extensive gene clusters, and that the different clusters contain different spacer DNA sequences. These clusters form well-defined cryptic DNA satellites which are potentially separable from each other as well as from other chromosomal DNA.