Non-bridging phosphate oxygen atoms within the tRNA anticodon stem-loop are essential for ribosomal A site binding and translocation

The conformation of the anticodon stem-loop of tRNAs required for correct decoding by the ribosome depends on intramolecular and intermolecular interactions that are independent of the tRNA nucleotide sequence. Non-bridging phosphate oxygen atoms have been shown to be critical for the structure and function of several RNAs. However, little is known about the role they play in ribosomal A site binding and translocation of tRNA to the P site. Here, we show that non-bridging phosphate oxygen atoms within the tRNA anticodon stem-loop at positions 33, 35, and 37 are important for A site binding. Those at positions 34 and 36 are not necessary for binding, but are essential for translocation. Our results correlate with structural data, indicating that position 34 interacts with the highly conserved 16S rRNA base G966 and position 36 interacts with the universally conserved tRNA base U33 during translocation to the P site.     

Structural and functional role of bases 32 and 33 in the anticodon loop of yeast mitochondrial tRNAIle

Previous work has demonstrated the usefulness of the yeast model to investigate the molecular mechanisms underlying defects due to base substitutions in mitochondrial tRNA genes, and to identify suppressing molecules endowed with potential clinical relevance. The present paper extends these investigations to two human equivalent yeast mutations located at positions 32 and 33 in the anticodon loop of tRNA(Ile). Notwithstanding the proximity of the two T>C base substitutions, the effects of these mutations have been found to be quite different in yeast, as they are in human. The T32C substitution has a very severe effect in yeast, consisting in a complete inhibition of growth on nonfermentable substrates. Conversely, respiratory defects caused by the T33C mutation could only be observed in a defined genetic context. Analyses of available sequences and selected tRNA three-dimensional structures were performed to provide explanations for the different behavior of these adjacent mutations. Examination of the effects of previously identified suppressors demonstrated that overexpression of the TUF1 gene did not rescue the defective phenotypes determined by either mutation, possibly as a consequence of the lack of interactions between EF-Tu and the tRNA anticodon arm in known structures. On the contrary, both the cognate IleRS and the noncognate LeuRS and ValRS are endowed with suppressing activities toward both mutations. This allows us to extend to the tRNA(Ile) mutants the cross-suppression activity of aminoacyl-tRNA synthetases previously demonstrated for tRNA(Leu) and tRNA(Val) mutants.     

Adaptation to tRNA acceptor stem structure by flexible adjustment in the catalytic domain of class I tRNA synthetases

Class I aminoacyl-tRNA synthetases (aaRSs) use a Rossmann-fold domain to catalyze the synthesis of aminoacyl-tRNAs required for decoding genetic information. While the Rossmann-fold domain is conserved in evolution, the acceptor stem near the aminoacylation site varies among tRNA substrates, raising the question of how the conserved protein fold adapts to RNA sequence variations. Of interest is the existence of an unpaired C-A mismatch at the 1-72 position unique to bacterial initiator tRNA(fMet) and absent from elongator tRNAs. Here we show that the class I methionyl-tRNA synthetase (MetRS) of Escherichia coli and its close structural homolog cysteinyl-tRNA synthetase (CysRS) display distinct patterns of recognition of the 1-72 base pair. While the structural homology of the two enzymes in the Rossmann-fold domain is manifested in a common burst feature of aminoacylation kinetics, CysRS discriminates against unpaired 1-72, whereas MetRS lacks such discrimination. A structure-based alignment of the Rossmann fold identifies the insertion of an α-helical motif, specific to CysRS but absent from MetRS, which docks on 1-72 and may discriminate against mismatches. Indeed, substitutions of the CysRS helical motif abolish the discrimination against unpaired 1-72. Additional structural alignments reveal that with the exception of MetRS, class I tRNA synthetases contain a structural motif that docks on 1-72. This work demonstrates that by flexible insertion of a structural motif to dock on 1-72, the catalytic domain of class I tRNA synthetases can acquire structural plasticity to adapt to changes at the end of the tRNA acceptor stem.     

Thermodynamic contribution of nucleoside modifications to yeast tRNA(Phe) anticodon stem loop analogs.

The determination of the structural and functional contributions of natural modified nucleosides to tRNA has been limited by lack of an approach that can systematically incorporate the modified units. We have produced a number of oligonucleotide analogs, of the anticodon of yeast tRNA(Phe) by, combining standard automated synthesis for the major nucleosides with specialty chemistries for the modified nucleosides. In this study, both naturally occurring and unnatural modified nucleotides were placed in native contexts. Each oligonucleotide was purified and the nucleoside composition determined to validate the chemistry. The RNAs were denatured and analyzed to determine the van't Hoff thermodynamic parameters. Here, we report the individual thermodynamic contributions for Cm, Gm, m1G, m5C, psi. In addition m5m6U, m1psi, and m3psi, were introduced to gain additional understanding of the physicochemical contribution of psi and m5C at an atomic level. These oligonucleotides demonstrate that modifications have measurable thermodynamic contributions and that loop modifications have global contributions.     

A magnesium-induced conformational transition in the loop of a DNA analog of the yeast tRNA(Phe) anticodon is dependent on RNA-like modifications of the bases of the stem.

Two single-stranded DNA heptadecamers corresponding to the yeast tRNA(Phe) anticodon stem-loop were synthesized, and the solution structures of the oligonucleotides, d(CCAGACTGAAGATCTGG) and d(CCAGACTGAAGAU-m5C-UGG), were investigated using spectroscopic methods. The second, or modified, base sequence differs from that of DNA by RNA-like modifications at three positions; dT residues were replaced at positions 13 and 15 with dU, and the dC at position 14 with d(m5C), corresponding to positions where these nucleosides occur in tRNA(Phe). Both oligonucleotides form intramolecular structures at pH 7 in the absence of Mg2+ and undergo monophasic thermal denaturation transitions (Tm = 47 degrees C). However, in the presence of 10 mM Mg2+, the modified DNa adopted a structure that exhibited a biphasic "melting" transition (Tm values of 23 and 52 degrees C) whereas the unmodified DNA structure exhibited a monophasic denaturation (Tm = 52 degrees C). The low-temperature, Mg(2+)-dependent structural transition of the modified DNA was also detected using circular dichroism (CD) spectroscopy. No such transition was exhibited by the unmodified DNA. This transition, unique to the modified DNA, was dependent on divalent cations and occurred most efficiently with Mg2+; however, Ca2+ also stabilized the alternative conformation at low temperature. NMR studies showed that the predominant structure of the modified DNA in sodium phosphate (pH 7) buffer in the absence of Mg2+ was a hairpin containing a 7-nucleotide loop and a stem composed of 3 stable base pairs. In the Mg(2+)-stabilized conformation, the loop became a two-base turn due to the formation of two additional base pairs across the loop.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of 5-methylcytidine in the anticodon arm of yeast tRNA(Phe): site-specific Mg2+ binding and coupled conformational transition in DNA analogs.

The tDNA(Phe)AC, d(CCAGACTGAAGAU13m5C14U15GG), with a DNA sequence similar to that of the anticodon stem and loop of yeast tRNA(Phe), forms a stem and loop structure and has an Mg(2+)-induced structural transition that was not exhibited by an unmodified tDNA(Phe)AC d(T13C14T15) [Guenther, R. H., Hardin, C. C., Sierzputowska-Gracz, H., Dao, V., & Agris, P. F. (1992) Biochemistry (preceding paper in this issue)]. Three tDNA(Phe)AC molecules having m5C14, tDNA(Phe)AC d(U13m5C14U15), d(U13m5C14T15), and d(T13,5C14U15), also exhibited Mg(2+)-induced structural transitions and biphasic thermal transitions (Tm approximately 23.5 and 52 degrees C), as monitored by CD and UV spectroscopy. Three other tDNA(Phe)AC, d(T13C14T15), d(U13C14U15), and d(A7;U13m5C14U15) in which T7 was replaced with an A, thereby negating the T7.A10 base pair across the anticodon loop, had no Mg(2+)-induced structural transitions and only monophasic thermal transitions (Tm of approximately 52 degrees C). The tDNA(Phe)AC d(U13m5C14U15) had a single, strong Mg2+ binding site with a Kd of 1.09 x 10(-6) M and a delta G of -7.75 kcal/mol associated with the Mg(2+)-induced structural transition. In thermal denaturation of tDNA(Phe)AC d(U13m5C14U15), the 1H NMR signal assigned to the imino proton of the A5.dU13 base pair at the bottom of the anticodon stem could no longer be detected at a temperature corresponding to that of the loss of the Mg(2+)-induced conformation from the CD spectrum. Therefore, we place the magnesium in the upper part of the tDNA hairpin loop near the A5.dU13 base pair, a location similar to that in the X-ray crystal structure of native, yeast tRNA(Phe).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleation and the transition state of the SH3 domain

We present a verified computational model of the SH3 domain transition state (TS) ensemble. This model was built for three separate SH3 domains using experimental phi-values as structural constraints in all-atom protein folding simulations. While averaging over all conformations incorrectly considers non-TS conformations as transition states, quantifying structures as pre-TS, TS, and post-TS by measurement of their transmission coefficient ("probability to fold", or p(fold)) allows for rigorous conclusions regarding the structure of the folding nucleus and a full mechanistic analysis of the folding process. Through analysis of the TS, we observe a highly polarized nucleus in which many residues are solvent-exposed. Mechanistic analysis suggests the hydrophobic core forms largely after an early nucleation step. SH3 presents an ideal system for studying the nucleation-condensation mechanism and highlights the synergistic relationship between experiment and simulation in the study of protein folding.     

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.     

Conservation of helical structure contributes to functional metal ion interactions in the catalytic domain of ribonuclease P RNA

Like protein enzymes, catalytic RNAs contain conserved structure motifs important for function. A universal feature of the catalytic domain of ribonuclease P RNA is a bulged-helix motif within the P1-P4 helix junction. Here, we show that changes in bulged nucleotide identity and position within helix P4 affect both catalysis and substrate binding, while a subset of the mutations resulted only in catalytic defects. We find that the proximity of the bulge to sites of metal ion coordination in P4 is important for catalysis; moving the bulge distal to these sites and deleting it had similarly large effects, while moving it proximal to these sites had only a moderate effect on catalysis. To test whether the effects of the mutations are linked to metal ion interactions, we used terbium-dependent cleavage of the phosphate backbone to probe metal ion-binding sites in the wild-type and mutant ribozymes. We detect cleavages at specific sites within the catalytic domain, including helix P4 and J3/4, which have previously been shown to participate directly in metal ion interactions. Mutations introduced into P4 cause local changes in the terbium cleavage pattern due to alternate metal ion-binding configurations with the helix. In addition, a bulge deletion mutation results in a 100-fold decrease in the single turnover cleavage rate constant at saturating magnesium levels, and a reduced affinity for magnesium ions important for catalysis. In light of the alternate terbium cleavage pattern in P4 caused by bulge deletion, this decreased ability to utilize magnesium ions for catalysis appears to be due to localized structural changes in the ribozyme's catalytic core that weaken metal ion interactions in P4 and J3/4. The information reported here, therefore, provides evidence that the universal conservation of the P4 structure is based in part on optimization of metal ion interactions important for catalysis.     

Biochemical and physiological properties of the DNA binding domain of AraC protein

Intact AraC protein is poorly soluble and difficult to purify, whereas its dimerization domain is the opposite. Unexpectedly, the DNA binding domain of AraC proved also to be soluble in cells when overproduced and is easily purified to homogeneity. The DNA binding affinity of the DNA binding domain for its binding site could not be measured by electrophoretic mobility shift because of its rapid association and dissociation rates, but its affinity could be measured with a fluorescence assay and was found to have a dissociation constant of 1 x 10(-8)M in 100 mM KCl. The binding of monomers of the DNA binding domain to adjacent half-sites occurs without substantial positive or negative cooperativity. A simple analysis relates the DNA binding affinities of monomers of DNA binding domain and normal dimeric AraC protein.     

Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA-binding domain as studied by amide proton exchange.

Backbone amide proton exchange rates in the DNA-binding domain of GAL4 have been determined using 1H-15N heteronuclear correlation NMR spectroscopy. Three forms of the protein were studied-the native Zn-containing protein, the Cd-substituted protein, and a Zn-GAL4/DNA complex. Exchange rates in the Zn-containing protein are significantly slower than in the Cd-substituted protein. This shows that Cd-substituted GAL4 is destabilized relative to the native Zn-containing protein. Upon DNA binding, global retardation of amide proton exchange with solvent was observed, indicating that internal fluctuations of the DNA-recognition module are significantly reduced by the presence of DNA. In all forms of the protein, the internal dyad symmetry of the DNA-recognition module of GAL4 is reflected by the backbone amide proton exchange rates.     

Reconstruction of the src-SH3 protein domain transition state ensemble using multiscale molecular dynamics simulations

We use an integrated computational approach to reconstruct accurately the transition state ensemble (TSE) for folding of the src-SH3 protein domain. We first identify putative TSE conformations from free energy surfaces generated by importance sampling molecular dynamics for a fully atomic, solvated model of the src-SH3 protein domain. These putative TSE conformations are then subjected to a folding analysis using a coarse-grained representation of the protein and rapid discrete molecular dynamics simulations. Those conformations that fold to the native conformation with a probability (P(fold)) of approximately 0.5, constitute the true transition state. Approximately 20% of the putative TSE structures were found to have a P(fold) near 0.5, indicating that, although correct TSE conformations are populated at the free energy barrier, there is a critical need to refine this ensemble. Our simulations indicate that the true TSE conformations are compact, with a well-defined central beta sheet, in good agreement with previous experimental and theoretical studies. A structured central beta sheet was found to be present in a number of pre-TSE conformations, however, indicating that this element, although required in the transition state, does not define it uniquely. An additional tight cluster of contacts between highly conserved residues belonging to the diverging turn and second beta-sheet of the protein emerged as being critical elements of the folding nucleus. A number of commonly used order parameters to identify the transition state for folding were investigated, with the number of native Cbeta contacts displaying the most satisfactory correlation with P(fold) values.     

Protein folding kinetics beyond the phi value: using multiple amino acid substitutions to investigate the structure of the SH3 domain folding transition state

The SH3 domain folding transition state structure contains two well-ordered turn regions, known as the diverging turn and the distal loop. In the Src SH3 domain transition state, these regions are stabilized by a hydrogen bond between Glu30 in the diverging turn and Ser47 in the distal loop. We have examined the effects on folding kinetics of amino acid substitutions at the homologous positions (Glu24 and Ser41) in the Fyn SH3 domain. In contrast to most other folding kinetics studies which have focused primarily on non-disruptive substitutions with Ala or Gly, here we have examined the effects of substitutions with diverse amino acid residues. Using this approach, we demonstrate that the transition state structure is generally tolerant to amino acid substitutions. We also uncover a unique role for Ser at position 41 in facilitating folding of the distal loop, which can only be replicated by Asp at the same position. Both these residues appear to accelerate folding through the formation of short-range side-chain to backbone hydrogen bonds. The folding of the diverging turn region is shown to be driven primarily by local interactions. The diverging turn and distal loop regions are found to interact in the transition state structure, but only in the context of particular mutant backgrounds. This work demonstrates that studying the effects of a variety of amino acid substitutions on protein folding kinetics can provide unique insights into folding mechanisms which cannot be obtained by standard Phi value analysis.     

The role of the catalytic domain of E. coli GluRS in tRNA discrimination

Discrimination of tRNA^Gln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA^Gln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA^Gln. Our results demonstrate that in addition to the anticodon-binding domain, tRNA^Gln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.     

The 1.2 Å crystal structure of an E. coli tRNA acceptor stem microhelix reveals two magnesium binding sites

tRNA identity elements assure the correct aminoacylation of tRNAs by the cognate aminoacyl-tRNA synthetases. tRNA^Ser belongs to the so-called class II system, in which the identity elements are rather simple and are mostly located in the acceptor stem region, in contrast to ‘class I’, where tRNA determinants are more complex and are located within different regions of the tRNA.The structure of an Escherichia coli tRNA^Ser acceptor stem microhelix was solved by high resolution X-ray structure analysis. The RNA crystallizes in the space group C2, with one molecule per asymmetric unit and with the cell constants a = 35.79, b = 39.13, c = 31.37 Å, and β = 111.1°. A defined hydration pattern of 97 water molecules surrounds the tRNA^Ser acceptor stem microhelix. Additionally, two magnesium binding sites were detected in the tRNA^Ser aminoacyl stem.     

Evidence for cooperative effects in the binding of polyvalent metal ions to pure phosphatidylcholine bilayer vesicle surfaces

An internal NMR monitor for the study of lanthanide ion (Ln³⁺) binding to phospholipid bilayer membranes has been developed. The dimethylphosphate anion, DMP^?, forms labile complexes with Ln³⁺ in aqueous solution and in solutions also containing bilayer dispersions. The hyperfine shift in the DMP^? resonance induced by Pr³⁺ ions has been used to determine the overall thermodynamic formation constants for the Pr(DMP)²⁺ and Pr(DMP)₂⁺ complexes: 81 (M^?1) and 349 (M^?2) at 52°C; the limiting hyperfine shift (³¹P) at 52°C is 91.5 ppm downfield. These parameters, applied to the observed DMP^? hyperfine shift in the presence of the membrane, establish both the free Pr³⁺ concentration and the amount of Pr³⁺ bound to the phospholipid surface. Extensive data for the binding of Pr³⁺ to the outer surfaces of sonicated vesicles yield a limiting hyperfine shift per Pr³⁺ of 181.6 ppm downfield for the dipalmitoylphosphatidylcholine ³¹P resonance at 52°C, clearly demonstrating that the binding stoichiometry is two DPPCs per Pr³⁺. A Hill analysis indicates that the binding data are more anti-cooperative than a realistic Langmuir isotherm, yet more cooperative than a Stern isotherm incorporating electrostatic considerations at the Debye-Hückel level. Fittings to specific models lead to a cooperative model in which tense (T) sites, with low affinity for Pr³⁺, present in the absence of metal ions, quickly give way to relaxed (R) sites (two DPPCs per site), with much higher affinity for Pr³⁺, as the amount of Pr³⁺ bound to the surface increases. The intrinsic equilibrium constants for the binding of Pr³⁺ to DPPC vesicles are 2 M^?1 and 3 000 M^?1 for the T and R sites, respectively, at 52°C. The distribution coefficient between these sites ([R]/[T]) in the absence of Ln³⁺ is 0.14 at 52°C. We picture the binding site conversion as a head-group conformational change involving mostly the choline moiety. Sketchy results for binding on the inside vesicle surface indicate that the overall affinity for Pr³⁺ is significantly greater and suggest that the site stoichiometry may be different.