A single catalytically active subunit in the multimeric Sulfolobus shibatae CCA-adding enzyme can carry out all three steps of CCA addition

The CCA-adding enzyme ATP(CTP):tRNA nucleotidyltransferase builds and repairs the 3'-terminal CCA sequence of tRNA. Although this unusual RNA polymerase has no nucleic acid template, it can construct the CCA sequence one nucleotide at a time using CTP and ATP as substrates. We found previously that tRNA does not translocate along the enzyme during CCA addition (Yue, D., Weiner, A. M., and Maizels, N. (1998) J. Biol. Chem. 273, 29693-29700) and that a single nucleotidyltransferase motif adds all three nucleotides (Shi, P.-Y., Maizels, N., and Weiner, A. M. (1998) EMBO J. 17, 3197-3206). Intriguingly, the CCA-adding enzyme from the archaeon Sulfolobus shibatae is a homodimer that forms a tetramer upon binding two tRNAs. We therefore asked whether the active form of the S. shibatae enzyme might have two quasi-equivalent active sites, one adding CTP and the other ATP. Using an intersubunit complementation approach, we demonstrate that the dimer is active and that a single catalytically active subunit can carry out all three steps of CCA addition. We also locate one UV light-induced tRNA cross-link on the enzyme structure and provide evidence suggesting the location of another. Our data rule out shuttling models in which the 3'-end of the tRNA shuttles from one quasi-equivalent active site to another, demonstrate that tRNA-induced tetramerization is not required for CCA addition, and support a role for the tail domain of the enzyme in tRNA binding.     

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.     

Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains.

RNA microhelices that recreate the acceptor stems of transfer RNAs are charged with specific amino acids. Here we identify a two-helix pair in alanyl-tRNA synthetase that is required for RNA microhelix binding. A single point mutation at an absolutely conserved residue in this motif selectively disrupts RNA binding without perturbation of the catalytic site. These results, and findings of similar motifs in the proximity of the active sites of other tRNA synthetases, suggest that two-helix pairs are widespread and provide a structural framework important for contacts with bound RNA substrates.     

The crystal structure of the ternary complex of T.thermophilus seryl-tRNA synthetase with tRNA(Ser) and a seryl-adenylate analogue reveals a conformational switch in the active site.

The low temperature crystal structure of the ternary complex of Thermus thermophilus seryl-tRNA synthetase with tRNA(Ser) (GGA) and a non-hydrolysable seryl-adenylate analogue has been refined at 2.7 angstrom resolution. The analogue is found in both active sites of the synthetase dimer but there is only one tRNA bound across the two subunits. The motif 2 loop of the active site into which the single tRNA enters interacts within the major groove of the acceptor stem. In particular, a novel ring-ring interaction between Phe262 on the extremity of this loop and the edges of bases U68 and C69 explains the conservation of pyrimidine bases at these positions in serine isoaccepting tRNAs. This active site takes on a significantly different ordered conformation from that observed in the other subunit, which lacks tRNA. Upon tRNA binding, a number of active site residues previously found interacting with the ATP or adenylate now switch to participate in tRNA recognition. These results shed further light on the structural dynamics of the overall aminoacylation reaction in class II synthetases by revealing a mechanism which may promote an ordered passage through the activation and transfer steps.     

Cell growth inhibition by sequence-specific RNA minihelices.

RNA minihelices which reconstruct the 12 base pair acceptor-T psi C domains of transfer RNAs interact with their cognate tRNA synthetases. These substrates lack the anticodons of the genetic code and, therefore, cannot participate in steps of protein synthesis subsequent to aminoacylation. We report here that expression in Escherichia coli of either of two minihelices, each specific for a different amino acid, inhibited cell growth. Inhibition appears to be due to direct competition between the minihelix and its related tRNA for binding to their common synthetase. This competition, in turn, sharply lowers the pool of the specific charged tRNA for protein synthesis. Inhibition is relieved by single nucleotide changes which disrupt the minihelix-synthetase interaction. The results suggest that sequence-specific RNA minihelix substrates bind to cognate synthetases in vivo and can, in principle, act as cell growth regulators. Naturally occurring non-tRNA substrates for aminoacylation may serve a similar purpose.     

Crystal structure of paromomycin docked into the eubacterial ribosomal decoding A site

BACKGROUND: Aminoglycoside antibiotics interfere with translation in both gram-positive and gram-negative bacteria by binding to the tRNA decoding A site of the 16S ribosomal RNA. RESULTS: Crystals of complexes between oligoribonucleotides incorporating the sequence of the ribosomal A site of Escherichia coli and the aminoglycoside paromomycin have been solved at 2.5 A resolution. Each RNA fragment contains two A sites inserted between Watson-Crick pairs. The paromomycin molecules interact in an enlarged deep groove created by two bulging and one unpaired adenines. In both sites, hydroxyl and ammonium side chains of the antibiotic form 13 direct hydrogen bonds to bases and backbone atoms of the A site. In the best-defined site, 8 water molecules mediate 12 other hydrogen bonds between the RNA and the antibiotics. Ring I of paromomycin stacks over base G1491 and forms pseudo-Watson-Crick contacts with A1408. Both the hydroxyl group and one ammonium group of ring II form direct and water-mediated hydrogen bonds to the U1495oU1406 pair. The bulging conformation of the two adenines A1492 and A1493 is stabilized by hydrogen bonds between phosphate oxygens and atoms of rings I and II. The hydrophilic sites of the bulging A1492 and A1493 contact the shallow groove of G=C pairs in a symmetrical complex. CONCLUSIONS: Water molecules participate in the binding specificity by exploiting the antibiotic hydration shell and the typical RNA water hydration patterns. The observed contacts rationalize the protection, mutation, and resistance data. The crystal packing mimics the intermolecular contacts induced by aminoglycoside binding in the ribosome.     

RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase

In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.     

In vitro selection of RNAs that undergo autolytic cleavage with Pb2+.

An in vitro selection method has been developed to obtain RNA molecules that specifically undergo autolytic cleavage reactions by Pb2+ ion. The method utilizes a circular RNA intermediate which is regenerated following the cleavage reaction to allow amplification and multiple cycles of selection. Pb2+ is known to catalyze a specific cleavage reaction between U17 and G18 of yeast tRNA(Phe). Starting from pools of RNA molecules which have a random distribution of sequences at nine or ten selected positions in the sequence of yeast tRNA(Phe), we have isolated many RNA molecules that undergo rapid and specific self-cleavage with Pb2+ at a variety of different sites. Terminal truncation experiments suggest that most of these self-cleaving RNA molecules do not fold like tRNA. However, two of the variants are cleaved rapidly with Pb2+ at U17 even though they lack the highly conserved nucleotides G18 and G19. Both specific mutations and terminal truncation experiments suggest that the D and T loops of these two variants interact in a manner similar to that of tRNA(Phe) despite the absence of the G18U55 and G19C56 tertiary interactions. A model for an alternate tertiary interaction involving a U17U55 pair is presented. This model may be relevant to the structure of about 100 mitochondrial tRNAs that also lack G18 and G19. The selection method presented here can be directly applied to isolate catalytic RNAs that undergo cleavage in the presence of other metal ions, modified nucleotides, or sequence-specific nucleases.     

Determination of the free-energy coupling between ATP and an affinity label attached to rabbit muscle phosphofructokinase

The smallest enzymatically active form of rabbit muscle phosphofructokinase is a tetramer of four identical or nearly identical monomers. The enzyme is inhibited by ATP, and this inhibition by ATP is relieved by the activating adenine nucleotides adenosine cyclic 3',5'-phosphate, AMP, and ADP. Each monomer contains one binding site specific for the inhibitor ATP and another site specific for the activating adenine nucleotides. The enzyme can also be activated by covalently labeling the activating adenine nucleotide binding sites with the affinity label 5'-[p-(fluorosulfonyl)benzoyl]adenosine. These activator binding sites on the enzyme have been covalently labeled to various degrees, ranging from an average value of less than one label per tetramer to four labels per tetramer, and the free-energy coupling, delta Gxy, between the covalently bound affinity label and ATP binding at the inhibitory site was determined. For enzyme preparations containing four labels per tetramer, delta Gxy is approximately 1 kcal/mol at pH 6.95 and 25 degrees C. A very significant free-energy coupling is observed in those preparations containing an average of one label per tetramer and less, and the change in delta Gxy in going from native tetramers to ones containing an average of two labels per tetramer is twice as great as the change in delta Gxy observed in going from tetramers containing an average of two labels per tetramer to ones containing four labels per tetramer, suggesting that modification of the final two monomers in the tetramer contributes much less to the antagonistic effect on ATP binding than does modification of the first two monomers in the tetramer.     

Neutron scattering study of the binding of tRNAPhe to Escherichia coli phenylalanyl-tRNA synthetase

Escherichia coli phenylalanyl-tRNA synthetase has been characterized by small-angle neutron scattering. In solution (20 mM imidazole hydrochloride, pH 7.6, 10 mM 2-mercaptoethanol, and 0.1 mM ethylenediaminetetraacetic acid), this enzyme has a molecular weight of 227K +/- 20K with a radius of gyration of 48.3 +/- 0.6 A, independent of the presence of MgCl2 up to 50 mM. The change of the scattering upon adding tRNAPhe to the enzyme has been followed with 10 mM MgCl2 present in the buffer. One enzyme molecule is capable of binding two tRNAPhe molecules with affinity constants larger than 10(6) M-1. Parallel titration experiments in 73% 2H2O, close to the matching point of tRNA, show that the RG of the enzyme is not changed by the binding of one or two tRNAPhe molecules. These results are compared with quasi-electric light scattering studies [Holler, E., Wang, C. C., & Ford, N.C., Jr. (1981) Biochemistry 20, 861-867] where the addition of either MgCl2 or tRNAPhe was shown to cause dramatic changes of the apparent translational diffusion constant of phenylalanyl-tRNA synthetase.     

In vitro genetic analysis of the structural features of the pre-tRNA required for determination of the 3' splice site in the intron excision reaction.

During processing of intron-containing pre-tRNAs, the Xenopus laevis splicing endonuclease binds the precursor and cleaves it at both the 5' and 3' splice sites. In vitro selection was used to determine structural features characteristic of precursor tRNA molecules that are active in this reaction. We performed two types of selection, one for molecules that are not cut, the other for molecules that are cut at only one site. The results shed light on various aspects of the intron excision reaction, including the importance of the three-dimensional structure of the mature domain for recognition and binding of the enzyme, the active role played by the single-stranded region of the intron, and the importance of the cardinal positions which, although not necessarily occupied by the same base in all precursors, nevertheless play a fundamental role in the splicing reaction. A precursor can be cut at the 3' site if a base in the single-stranded loop of the intron is allowed to pair (A-I pair) with the base of the 5' exon situated at the position immediately following the anticodon stem [first cardinal position (CP1)]. The nature of the bases involved in the A-I pair is important, as is the position of the base in the single-stranded loop of the intron. We discuss the role of the cardinal positions in the reaction.     

Probing the dynamics of restriction endonuclease NgoMIV‐DNA interaction by single‐molecule FRET

Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single‐molecule Förster resonance energy transfer (smFRET) of surface‐immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein‐induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA ‐ NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA ‐ protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites (“slow” trajectories) or by semi‐specific interactions of two DNA‐bound NgoMIV tetramers (“fast” trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules.     

5-methyltetrahydrofolate is bound in intersubunit areas of rat liver folate-binding protein glycine N-methyltransferase

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. It is abundant in the liver, where it uses excess S-adenosylmethionine (AdoMet) to methylate glycine to N-methylglycine (sarcosine) and produces S-adenosylhomocysteine (AdoHcy), thereby controlling the methylating potential of the cell. GNMT also links utilization of preformed methyl groups, in the form of methionine, to their de novo synthesis, because it is inhibited by a specific form of folate, 5-methyltetrahydrofolate. Although the structure of the enzyme has been elucidated by x-ray crystallography of the apoenzyme and in the presence of the substrate, the location of the folate inhibitor in the tetrameric structure has not been identified. We report here for the first time the crystal structure of rat GNMT complexed with 5-methyltetrahydrofolate. In the GNMT-folate complex, two folate binding sites were located in the intersubunit areas of the tetramer. Each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr5, Leu207, and Met215 residues of all subunits. Binding experiments in solution also confirm that one GNMT tetramer binds two folate molecules. For the enzymatic reaction to take place, the N-terminal fragments of GNMT must have a significant degree of conformational freedom to provide access to the active sites. The presence of the folate in this position provides a mechanism for its inhibition.     

Binding of C5 Protein to P RNA Enhances the Rate Constant for Catalysis for P RNA Processing of Pre-tRNAs Lacking a Consensus G(+ 1)/C(+ 72) Pair

The RNA subunit of the ribonucleoprotein enzyme ribonuclease P (RNase P (P RNA) contains the active site, but binding of Escherichia coli RNase P protein (C5) to P RNA increases the rate constant for catalysis for certain pre-tRNA substrates up to 1000-fold. Structure-swapping experiments between a substrate that is cleaved slowly by P RNA alone (pre-tRNA^f-met605) and one that is cleaved quickly (pre-tRNA^met608) pinpoint the characteristic C(+ 1)/A(+ 72) base pair of initiator tRNA^f-met as the sole determinant of slow RNA-alone catalysis. Unlike other substrate modifications that slow RNA-alone catalysis, the presence of a C(+ 1)/A(+ 72) base pair reduces the rate constant for processing at both correct and miscleavage sites, indicating an indirect but nonetheless important role in catalysis. Analysis of the Mg²⁺ dependence of apparent catalytic rate constants for pre-tRNA^met608 and a pre-tRNA^met608 (+ 1)C/(+ 72)A mutant provides evidence that C5 promotes rate enhancement primarily by compensating for the decrease in the affinity of metal ions important for catalysis engendered by the presence of the CA pair. Together, these results support and extend current models for RNase P substrate recognition in which contacts involving the conserved (+ 1)G/C(+ 72) pair of tRNA stabilize functional metal ion binding. Additionally, these observations suggest that C5 protein has evolved to compensate for tRNA variation at positions important for binding to P RNA, allowing for tRNA specialization.     

Solution conformations of unmodified and A(37)N(6)-dimethylallyl modified anticodon stem-loops of Escherichia coli tRNA(Phe)

The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells. tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications. The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis. The enzyme dimethylallyl (Delta(2)-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A(37) in several tRNA species. Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA(Phe), we have investigated the structural and dynamic changes introduced by the dimethylallyl group. The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop. This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA(Phe). The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA(Phe) anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation. Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation. The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon-anticodon helix.     

G-1:C73 recognition by an arginine cluster in the active site of Escherichia coli histidyl-tRNA synthetase

Aminoacylation of a transfer RNA (tRNA) by its cognate aminoacyl-tRNA synthetase relies upon the recognition of specific nucleotides as well as conformational features within the tRNA by the synthetase. In Escherichia coli, the aminoacylation of tRNA(His) by histidyl-tRNA synthetase (HisRS) is highly dependent upon the recognition of the unique G-1:C73 base pair and the 5'-monophosphate. This work investigates the RNA-protein interactions between the HisRS active site and these critical recognition elements. A homology model of the tRNA(His)-HisRS complex was generated and used to design site-specific mutants of possible G-1:C73 contacts. Aminoacylation assays were performed with these HisRS mutants and N-1:C73 tRNA(His) and microhelix(His) variants. Complete suppression of the negative effect of 5'-phosphate deletion by R123A HisRS, as well as the increased discrimination of Q118E HisRS against a 5'-triphosphate, suggests a possible interaction between the 5'-phosphate and active-site residues Arg123 and Gln118 in which these residues create a sterically and electrostatically favorable pocket for the binding of the negatively charged phosphate group. Additionally, a network of interactions appears likely between G-1 and Arg116, Arg123, and Gln118 because mutation of these residues significantly reduced the sensitivity of HisRS to changes at G-1. Our studies also support an interaction previously proposed between Gln118 and C73. Defining the RNA-protein interactions critical for efficient aminoacylation by E. coli HisRS helps to further characterize the active site of this enzyme and improves our understanding of how the unique identity elements in the acceptor stem of tRNA(His) confer specificity.     

A single base pair affects binding and catalytic parameters in the molecular recognition of a transfer RNA

A single G3.U70 base pair in the acceptor helix is a major determinant of the identity of an alanine transfer RNA. Alteration of this base pair to A.U or G.C prevents aminoacylation with alanine. We show here that, at approximate physiological conditions (pH 7.5, 37 degrees C), high concentrations of the mutant A3.U70 species do not inhibit aminoacylation of a wild-type alanine tRNA. The observation suggests that, under these conditions, the G3 to A3 substitution increases Km for tRNA by more than 30-fold. Other experiments at pH 7.5 show that no aminoacylation of A3.U70, G3.C70, or U3.G70 mutant tRNAs occurs with substrate levels of enzyme. This suggests that kcat for these mutant tRNAs is sharply reduced as well and that the catalytic defect is not due to slow release of charged mutant tRNAs from the enzyme. Investigations were also done at pH 5.5, where association of tRNAs with synthetases is generally stronger and where binding can be conveniently measured apart from aminoacylation. Under these conditions, the binding of the A3.U70 and G3.C70 species is readily detected and is only 3-5-fold weaker than the binding of the wild-type tRNA. Although the A3.U70 species was demonstrated to compete with the wild-type tRNA for the same site on the enzyme, no aminoacylation could be detected. Thus, even when conditions are adjusted to obtain strong competitive binding, a sharp reduction in kcat prevents aminoacylation of a tRNA(Ala) species with a substitution at position 3.70.     

Tertiary core rearrangements in a tight binding transfer RNA aptamer

Guided by an in vitro selection experiment designed to obtain tight binding aptamers of Escherichia coli glutamine specific tRNA (tRNAGln) for glutaminyl-tRNA synthetase (GlnRS), we have engineered a tRNA mutant in which the five-nucleotide variable loop sequence 5'-44CAUUC48-3' is replaced by 5'-44AGGU48-3'. This mutant tRNA binds to GlnRS with 30-fold improved affinity compared to the wild type. The 2.7 A cocrystal structure of the RNA aptamer-GlnRS complex reveals major rearrangements in the central tertiary core of the tRNA, while maintaining an RNA-protein interface identical to the wild type. The repacked RNA core features a novel hydrogen bonding arrangement of the trans Levitt pair G15-U48, a new sulfate binding pocket in the major groove, and increased hydrophobic stacking interactions among the bases. These data suggest that enhanced protein binding to a mutant globular RNA can arise from stabilization of RNA tertiary interactions rather than optimization of RNA-protein contacts.