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At least 343 records · Page 19Linked to original sources

PubMed2006

Conformational transitions in RNA single uridine and adenosine bulge structures: a molecular dynamics free energy simulation study.

Extra unmatched nucleotides (single base bulges) are common structural motifs in folded RNA molecules and can participate in RNA-ligand binding and RNA tertiary structure formation. Often these processes are associated with conformational transitions in the bulge region such as flipping out of the bulge base from an intrahelical stacked toward a looped out state. Knowledge of the flexibility of bulge structures and energetics of conformational transitions is an important prerequisite to better understand the function of this RNA motif. Molecular dynamics simulations were performed on single uridine and adenosine bulge nucleotides at the center of eight basepair RNA molecules and indicated larger flexibility of the bulge bases compared to basepaired regions. The umbrella sampling method was applied to study the bulge base looping out process and accompanying conformational and free energy changes. Looping out toward the major groove resulted in partial disruption of adjacent basepairs and was found to be less favorable compared to looping out toward the minor groove. For both uridine and adenosine bulges, a positive free energy change for full looping out was obtained which was approximately 1.5 kcal mol-1 higher in the case of the adenosine compared to the uridine bulge system. The simulations also indicated stable partially looped out states with the bulge bases located in the RNA minor groove and forming base triples with 5'-neighboring basepairs. In the case of the uridine bulge this state was more stable than the intrahelical stacked bulge structure. Induced looping out toward the minor groove involved crossing of an energy barrier of approximately 3.5 kcal mol-1 before reaching the base triple state. A continuum solvent analysis of intermediate bulge states indicated that electrostatic interactions stabilize looped out and base triple states, whereas van der Waals interactions and nonpolar contributions favor the stacked bulge conformation.

Adenine↗

PubMed1972

Proton dispersion forces. Secondary-structure stabilizing forces between the hydrogen bonds of the polynucleotides.

In the double helix formed by the semiprotonated polycytidylic acid (poly C), both strands are linked via NH(+)...N hydrogen bonds. It is a known fact that such symmetrical hydrogen bonds with a double minimum potential well are extremely polarizable. This polarizability causes interaction effects, in particular the proton dispersion forces between such hydrogen bonds. These forces result in a shift of the energy levels and a continuum is observed in the infrared (IR) spectra of solutions in which such hydrogen bonds are present. The continuum occurs in the IR spectrum of the semiprotonated poly C, when the former is present in coiled state. If the double helix forms, an extremely broad band of the NH stretching vibration is observed instead of the continuum, since in the double helix all hydrogen bonds are oriented equally to one another and polarize each other mutually to a strong degree. The proton dispersion forces between the hydrogen bonds balance a considerable part of the electrostatic repulsion of the protons and hence enable the double helix to form. It is conceivable that an unsymmetrical double minimum potential well is present in the NH...N bonds in the DNA and RNA. Such bonds may likewise be considerably more polarizable than electron systems and thus, in this case too, proton dispersion forces would contribute to helix stabilization.

Cytosine Nucleotides↗

PubMed2005

Variational electrostatic projection (VEP) methods for efficient modeling of the macromolecular electrostatic and solvation environment in activated dynamics simulations.

New methods for the calculation of electrostatic interactions between the active dynamical region and surrounding external solvated macromolecular environment in hybrid quantum mechanical/molecular mechanical (QM/MM) simulations are presented. The variational electrostatic projection (VEP) method, and related variational reverse-mapping procedure (VEP-RVM) utilize an expansion in Gaussian surface elements for representation of electrostatic interactions. The use of a discretized surface that surrounds the active dynamical region greatly reduces the number of interactions with the particles of the external environment. The methods are tested on two catalytic RNA systems: the hammerhead and the hairpin ribozymes. It is shown that with surface elements numbering from 302 to 1202 points the direct VEP and VEP-RVM methods are able to obtain relative force errors in the range of 0.5-0.05% and 0.09-0.0001%, respectively, using a 4.0 A projection buffer. These results are encouraging and provide an essential step in the development of new variational macromolecular solvent-boundary methods for QM/MM calculations of enzyme reactions.

Computer Simulation↗

PubMed1991

How does RNase H recognize a DNA.RNA hybrid?

The mechanism of RNase H substrate recognition is proposed from a model of a chemically modified DNA.RNA hybrid Escherichia coli RNase H complex. Site-directed mutagenesis of the enzyme and substrate titration observed by heteronuclear two-dimensional NMR spectra have been carried out. A model complex has been built, based on free structures of the enzyme and the substrate independently determined by x-ray crystallography and NMR distance geometry, respectively. In addition to steric and electrostatic complementarities between the molecular surfaces of the enzyme and the minor groove of the hybrid in the model, putative hydrogen bonds between the polar groups in the enzyme and 2'-oxygens of the RNA strand of the hybrid fix the hybrid close to the active site of the enzyme. The enzymatic activities of the mutant proteins and the changes in NMR spectra during the course of substrate titration are consistent with the present model. Moreover, the specific cleavage of the RNA strand in DNA.RNA hybrids can be explained as well as cleavage modes in modified heteroduplexes. A mechanism of enzymatic action is proposed.

Amino Acid Sequence↗

PubMed2004

Hydrogen loss from nucleobase nitrogens upon electron attachment to isolated DNA and RNA nucleotide anions.

Electron transfer to isolated nucleotide monoanions in collisions with Na vapor induces hydrogen loss from nitrogen of the transient nucleobase anion. The cross section for this process is linearly correlated with the number of N-H hydrogens and is highest for guanine. The process is much faster than microseconds since only dehydrogenated dianions survived for mass spectrometric detection. The lifetime of the adenosine 5(')-monophospate dianions was measured to be 0.2 ms in an electrostatic ion storage ring but also a longer-lived component with a lifetime of at least 10 ms was identified. Implications of dissociation along the N-H coordinate for a nucleotide in DNA are briefly discussed in terms of Watson-Crick base pairs.

Anions↗

PubMed1999

Application of oligonucleoside methylphosphonates in the studies on phosphodiester hydrolysis by Serratia endonuclease.

The endonuclease from Serratia marcescens is a non-specific enzyme that cleaves single and double stranded RNA and DNA. It accepts a phosphorylated pentanucleotide as a minimal substrate which is cleaved in the presence of Mg2+ at the second phosphodiester linkage. The present study is aimed at understanding the role of electrostatic and hydrogen bond interactions in phosphodiester hydrolysis. Towards this objective, six pentadeoxyadenylates with single stereoregular methylphosphonate substitution within this minimal substrate (2a-4b) were synthesized following a protocol described here. These modified oligonucleotides were used as substrates for the Serratia nuclease. The enzyme interaction studies revealed that the enzyme failed to hydrolyze any of the methylphosphonate analogues suggesting the importance of negative charge and/or hydrogen bond acceptors in binding and cleavage of its substrate. Based on these results and available site-directed mutagenesis as well as structural data, a model for nucleic acid binding by Serratia nuclease is proposed.

Binding Sites↗

PubMed1979

Assembly of vesicular stomatitis virus nucleocapsids in vivo: a kinetic analysis.

Pulse-chase labeling and cell fractionation were used to examine the pathways taken by the three nucleocapsid polypeptide species of vesicular stomatitis virus into nucleocapsids and then into virions. An improved method of polyacrylamide gel electrophoresis resolved nucleocapsid polypeptides N and NS from cellular actin, facilitating accurate quantitation of the viral polypeptides. Contrary to previous belief, the rate of NS synthesis was found to be a constant fraction of total virus protein synthesis throughout infection, indicating a consistent mechanism of virus protein synthesis regulation. In the kinetic studies, each polypeptide species displayed the following characteristic behavior. (i) Structural polypeptide N was the only species that entered a metabolically active soluble pool before assembly into nucleocapsids. The size of this pool increased with time after infection, causing an increasing delay in the appearance of pulse-labeled N molecules in nucleocapsids. (ii) Throughout infection, the entire complement of L molecules entered nucleocapsids immediately after their synthesis, without diversion through a soluble pool. (iii) Although 75% of newly synthesized molecules of the transcriptase-associated protein NS entered a soluble pool, they never emerged from the compartment. At all times after infection, about 25% of the NS molecules bypassed the soluble pool and entered nucleocapsids directly after their synthesis, as if in concert with L. These results indicate that VSV nucleocapsid assembly in vivo is a stepwise process, comprising an initial condensation of N with the viral RNA, followed by attachment of L and NS, analogous to the stepwise assembly of Sendai virus nucleocapsids. (D. W. Kingsbury, C.-H. Hsu, and K. G. Murti. Virology 91:86-94, 1978). About half of the intracellular nucleocapsids were recovered in a form that sedimented at anomalously low centrifugal forces, reflecting an association with large cellular organelles. This attachment was mediated mainly by electrostatic forces, since these "bound" nucleocapsids were released by elevated salt concentrations. The kinetic behavior of nucleocapsid polypeptides was the same in both fractions, providing no evidence for a division of nucleocapsid functions between cellular compartments.

Actins↗

PubMed1999

Conformational analysis of single-base bulges in A-form DNA and RNA using a hierarchical approach and energetic evaluation with a continuum solvent model.

The analysis and prediction of non-canonical structural motifs in RNA is of great importance for an understanding of the function and design of RNA structures. A hierarchical method has been employed to generate a large variety of sterically possible conformations for a single-base adenine bulge structure in A -form DNA and RNA. A systematic conformational search was performed on the isolated bulge motif and neighboring nucleotides under the constraint to fit into a continuous helical structure. These substructures were recombined with double-stranded DNA or RNA. Energy minimization resulted in more than 300 distinct bulge conformations. Energetic evaluation using a solvation model based on the finite-difference Poisson-Boltzmann method identified three basic classes of low-energy structures. The three classes correspond to conformations with the bulge base stacked between flanking nucleotides (I), location of the bulge base in the minor groove (II) and conformations with a continuous stacking of the flanking helices and a looped out bulge base (III). For the looped out class, two subtypes (IIIa and IIIb) with different backbone geometries at the bulge site could be distinguished. The conformation of lowest calculated energy was a class I structure with backbone torsion angles close to those in standard A -form RNA. Conformations very close to the extra-helical looped out bulge structure determined by X-ray crystallography were also among the low-energy structures. In addition, topologies observed in other experimentally determined bulge structures have been found among low-energy conformers. The implicit solvent model was further tested by comparing an uridine and adenine bulge flanked by guanine:cytosine base-pairs, respectively. In agreement with the experimental observation, a looped out form was found as the energetically most favorable form for the uridine bulge and a stacked conformation in case of the adenine bulge. The inclusion of solvation effects especially electrostatic reaction field contributions turned out to be critically important in order to select realistic low-energy bulge structures from a large number of sterically possible conformations. The results indicate that the approach might be useful to model the three-dimensional structure of non-canonical motifs embedded in double-stranded RNA, in particular, to restrict the number of possible conformations to a manageable number of conformers with energies below a certain threshold.

DNA↗

PubMed1997

Solution structure of a metal-binding site in the major groove of RNA complexed with cobalt (III) hexammine.

BACKGROUND: Solvated metal ions are critical for the proper folding and function of RNA. Despite the importance of these ions, the details of specific metal ion-RNA interactions are poorly understood. The crystal structure of a group I intron ribozyme domain characterized several metal-binding sites in the RNA with osmium (III) hexammine bound in the major groove. A corresponding method for locating and characterizing metal-binding sites of RNA in solution is of obvious interest. NMR should be ideal for localizing metal hexammine ions bound to the RNA because of the large concentration of protons around the metal center. RESULTS: We have solved the solution structure of the P5b stem loop from a group I intron ribozyme bound to a cobalt (III) hexammine ion. The location of the ion is precisely determined by intermolecular nuclear Overhausser effect cross-peaks between the cobalt (III) hexammine protons and both exchangeable and non-exchangeable RNA protons in the major groove. The binding site consists of tandem G-U base pairs in a sequence of four consecutive G residues ending in a GAAA tetraloop, as originally identified in the crystal structure. The edges of the bases in the major groove present an electrostatically negative face and a variety of hydrogen-bond acceptors for the cobalt (III) hexammine ion. The metal ion ligand is bound near the guanosine nucleotides of the adjacent G-U base pairs, where it makes hydrogen bonds with the N7 and carbonyl groups of both guanines. The carbonyl groups of the uracil residues add to the negative surface of the binding pocket, but do not form hydrogen bonds with the hexammine. Additional hydrogen bonds form with other guanine residues of the GGGG sequence. The structure of the binding site does not change significantly on binding the cobalt (III) hexammine. The structure of the complex in solution is very similar to the structure in the crystal. CONCLUSIONS: The structure presents a picture of how tandem G-U base pairs bind and position metal ions within the RNA major groove. The binding site is performed in the absence of metal ions, and presents a negative pocket in the major groove with a variety of hydrogen-bond acceptors. Because G-U base pairs are such a common motif in RNA sequences, it is possible that this RNA-metal ion interaction is critical in forming large complex RNA structures such as those found in the ribosome and self-splicing introns. This structure was determined using cobalt (III) hexammine as an analog for hexahydrated magnesium, a technique that may be applicable to other RNA sequences. Metal hexammines may prove to be useful general probes for locating RNA metal ion binding sites in solution.

Base Sequence↗

PubMed1999

The folding of centromeric DNA strands into intercalated structures: a physicochemical and computational study.

We have carried out a physicochemical and computational analysis on the stability of the intercalated structures formed by cytosine-rich DNA strands. In the computational study, the electrostatic energy components have been calculated using a Poisson-Boltzmann model, and the non-polar energy components have been computed with a van der Waals function and/or a term dependent on the solvent-accessible surface area of the molecules. The results have been compared with those obtained for Watson-Crick duplexes and with thermodynamic data derived from UV experiments. We have found that intercalated DNA is mainly stabilized by very favorable electrostatic interactions between hydrogen-bonded protonated and neutral cytosines, and by non-polar forces including the hydrophobic effect and enhanced van der Waals contacts. Cytosine protonation electrostatically promotes the association of DNA strands into a tetrameric structure. The electrostatic interactions between stacked C.C+ pairs are strongly attenuated by the reaction field of the solvent, and are modulated by a complex interplay of geometric and protonation factors. The forces stabilizing intercalated DNA must offset an entropic penalty due to the uptake of protons for cytosine protonation, at neutral pH, and also the electrostatic contribution to the solvation free energy. The latter energy component is less favorable for protonated DNA due to the partial neutralization of the negative charge of the molecule, and probably affects other protonated DNA and RNA structures such as C+-containing triplexes.

Centromere↗

PubMed2005

Study on the interactions between anti-HIV-1 active compounds with trans-activation response RNA by affinity capillary electrophoresis.

The study on the interactions between two anti-human immunodeficiency virus type 1 (anti-HIV-1) active compounds with trans-activation response (TAR) RNA by affinity capillary electrophoresis (ACE) with UV absorbance detection is presented. The results showed that the novel active molecules could interact with TAR RNA and inhibit the reproduce process of HIV-1. The binding constants were estimated by the change of migration time of the analytes through the change of concentrations of TAR RNA in the buffer solution. The yielded binding constants of 8.87 x 10(3)M(-1) for active compound C(3) and 8.42 x 10(3)M(-1) for MC(3) at 20.0 degrees C, 0.626 x 10(3)M(-1) and 0.644 x 10(3)M(-1) at 37.0 degrees C, respectively. The thermodynamic parameters Delta H and DeltaS were obtained and shown that both hydrophobic and electrostatic interaction played roles in the binding processes. The results showed that the presented method was an easy and simple method to evaluate the interaction of small molecules with some bioactive materials.

Anti-HIV Agents↗

PubMed2006

Biological relevance of a stable biochemical interaction between the tombusvirus-encoded P19 and short interfering RNAs.

The Tomato bushy stunt virus (TBSV)-encoded p19 protein (P19) is widely used as a robust tool to suppress RNA interference (RNAi) in various model organisms. P19 dimers appropriate 21-nucleotide (nt) duplex short interfering RNAs (siRNAs) generated by Dicer presumably to prevent programming of the RNA-induced silencing complex (RISC). In the context of virus infection, this model predicts that P19 mutants compromised for siRNA binding cannot prevent RISC-mediated degradation of TBSV RNA and thus reduce viral pathogenicity. To test this, we used P19/43 (R-->W), which is less pathogenic than wild-type P19 (wtP19), and P19/75-78 (RR-->GG), with pathogenicity properties (i.e., viral spread and symptom induction) comparable to those of a P19-null mutant. We demonstrate that P19/43 still suppresses RNAi-mediated viral RNA degradation in infected Nicotiana benthamiana, while P19/75-78 is unable to prevent this clearance of viral RNA, leading to an irreversible recovery phenotype. Gel filtration and immunoprecipitation assays show that at the onset of the infection, wtP19, P19/43, and P19/75-78 readily accumulate, and they form dimers. The wtP19 is stably associated with duplex approximately 21-nt TBSV siRNAs, while P19/75-78 does not bind these molecules, and the electrostatic interaction of P19/43 with siRNAs is perturbed for approximately 21-nt duplexes but not for longer siRNAs. This is the first clear demonstration of a direct correlation between a novel structurally orchestrated siRNA binding of an RNAi suppressor and its roles in viral pathogenesis. The findings should be particularly valuable for the RNAi field in general because the P19 mutants enable precise determination of siRNA appropriation effects.

Chromatography, Gel↗

PubMed1999

Cell cycle-dependent distribution and specific inhibitory effect of vectorized antisense oligonucleotides in cell culture.

Factors limiting the use of antisense phosphodiester oligodeoxynucleotides (ODNs) as therapeutic agents are inefficient cellular uptake and intracellular transport to RNA target. To overcome these obstacles, ODN carriers have been developed, but the intracellular fate of ODNs is controversial and strongly depends on the means of vectorization. Polyamidoamine dendrimers are non-linear polycationic cascade polymers that are able to bind ODNs electrostatically. These complexes have been demonstrated to protect phosphodiester ODNs from nuclease degradation and also to increase their cellular uptake and pharmacological effectiveness. We studied the intracellular distribution of a fluorescein isothiocyanate-labeled ODN vectorized by a dendrimer vector and found that intracellular ODN distribution was dependent on the phase of the cell cycle, with a nuclear localization predominantly in the G2/M phase. In addition, in order to evaluate the relevance of ODN vectors in enhancing the inhibition of the targeted genes' expression, we developed a rapid screening system which measures the transient expression of two reporter genes, one used as target, the other as control and vice versa. This system was validated through investigating the effect of the dendrimer vector on ODN biological activity. Antisense sequence-specific inhibition of more than 70% of one reporter gene was obtained with a chimeric ODN containing four phosphorothioate groups, two at each end.

3T3 Cells↗

PubMed1995

Interaction of methylene blue with transfer RNA--a spectroscopic study.

The binding of methylene blue (MB) with tRNA was studied using absorption, fluorescence and circular dichroic spectroscopy. In the spectral titration of MB with tRNA, hypochromism was observed in the absorption maximum of the dye in the visible region till P/D = 4 and thereafter the intensity increased with a red shift at P/D > 9, indicating electrostatic and intercalative binding at low and high P/D ratios, respectively. Analysis of absorption data, following Schwarz's procedure, showed that the electrostatic binding is cooperative in nature (cooperatively parameter q = 50) with a binding constant K = 7.77 x 10(3) M-1. A non-linear Scatchard plot was observed for the intercalative binding (at P/D > 4), probably due to a difference in the spectral characteristics of the dye intercalated between the base pairs and that between the bases in the single stranded domains. Quenching of fluorescence was observed for both the binding processes. In the circular dichroism spectra of tRNA-MB complexes at high P/D (approximately 30), nonconservative positive ICD bands were seen at 620 and 680 nm while at low P/D (approximately 2), two conservative negative CD bands at 300 and 660 nm and two bisignate bands with cross overs at 565 and 605 nm were observed. The short wavelength component of the bisignate band at 565 nm is negative while that of the 605-nm band is positive, indicating that the former arises from left handed and the latter from the right handed helical disposition of dye molecules along the tRNA backbone. The changes in the CD spectrum of tRNA on dye binding could be due to a conformational change of the nucleic acid or a negative CD being induced at that region.

Circular Dichroism↗

PubMed1981

Bound Ribosomes of Pea Chloroplast Thylakoid Membranes: Location and Release in Vitro by High Salt, Puromycin, and RNase.

The mode of attachment of 70S ribosomes to thylakoid membranes from pea leaves was studied by determining the proportion of the bound RNA which was released by various incubation conditions. The results supported a model in which several classes of bound ribosomes could be distinguished: (a) very tightly bound, not released by any conditions yet tested (20% of the total); (b) monomeric ribosomes attached by electrostatic interaction with the membranes (30 to 40% of the total) and released by high salt; and (c) polysomes, with some of the ribosomes attached by a combination of electrostatic interactions and insertion of the nascent polypeptide chain into the membrane. These required a combination of puromycin and high salt for release. Other ("hanging") ribosomes of the polysomes were inferred to be attached through mRNA but not actually attached to the membranes directly; they could be released by RNase under low salt conditions, as well as by puromycin plus high salt.To obtain these results, chloroplasts had to be prepared in media containing 0.2 molar Tris at pH 8.5. Using Tricine buffers at pH 7.5 yielded thylakoid membranes whose ribosomes were removed almost completely by high salt alone; these showed no response to puromycin. However, pH 7.5 had to be used in all cases for ribosome dissociation in high salt media, as the ribosome structure appeared to be degraded by high salt at pH 8.5, and release then occurred without the need for puromycin.The kinetics of ribosome release by high salt showed a rapid initial phase with a half-life of 20 seconds. The extent of release by high salt was very dependent on the temperature of the incubation. Plotting the data according to the Arrhenius interpretation shows a significant break at about 15 C, with apparent activation energy of 20 kilocalories per mole below that temperature and 5 kilocalories per mole above that temperature. This result suggests that membrane fluidity might be an important factor permitting release of ribosomes under high salt conditions.Electron microscope pictures of the washed thylakoids showed polysomes closely associated with the outer membranes of grana stacks, and with the stroma lamellae. Following digitonin treatment of the membranes and centrifugation, fractions enriched in Photosystem I and presumed stroma lamellae were also enriched in bound RNA.

Journal Article↗

PubMed2005

Single-molecule measurements of the persistence length of double-stranded RNA.

Over the past few years, it has become increasingly apparent that double-stranded RNA (dsRNA) plays a far greater role in the life cycle of a cell than previously expected. Numerous proteins, including helicases, polymerases, and nucleases interact specifically with the double helix of dsRNA. To understand the detailed nature of these dsRNA-protein interactions, the (bio)chemical, electrostatic, and mechanical properties of dsRNA need to be fully characterized. We present measurements of the persistence length of dsRNA using two different single-molecule techniques: magnetic tweezers and atomic force microscopy. We deduce a mean persistence length for long dsRNA molecules of 63.8 +/- 0.7 nm from force-extension measurements with the magnetic tweezers. We present atomic force microscopy images of dsRNA and demonstrate a new method for analyzing these, which yields an independent, yet consistent value of 62 +/- 2 nm for the persistence length. The introduction of these single-molecule techniques for dsRNA analysis opens the way for real-time, quantitative analysis of dsRNA-protein interactions.

Biophysics↗

PubMed2004

Role of an active site guanine in hairpin ribozyme catalysis probed by exogenous nucleobase rescue.

The hairpin ribozyme is a small catalytic RNA with reversible phosphodiester cleavage activity. Biochemical and structural studies exclude a requirement for divalent metal cation cofactors and implicate one active site nucleobase in particular, G8, in the catalytic mechanism. Our previous work demonstrated that the cleavage activity that is lost when G8 is replaced by an abasic residue is restored when certain nucleobases are provided in solution. The specificity and pH dependence of exogenous nucleobase rescue were consistent with several models of the rescue mechanism, including general acid base catalysis, electrostatic stabilization of negative charge in the transition state or a requirement for protonation to facilitate exogenous nucleobase binding. Detailed analyses of exogenous nucleobase rescue for both cleavage and ligation reactions now allow us to refine models of the rescue mechanism. Activity increased with increasing pH for both unmodified ribozyme reactions and unrescued reactions of abasic variants lacking G8. This similarity in pH dependence argues against a role for G8 as a general base catalyst, because G8 deprotonation could not be responsible for the pH-dependent transition in the abasic variant. Exogenous nucleobase rescue of both cleavage and ligation activity increased with decreasing pH, arguing against a role for rescuing nucleobases in general acid catalysis, because a nucleobase that contributes general acid catalysis in the cleavage pathway should provide general base catalysis in ligation. Analysis of the concentration dependence of cytosine rescue at high and low pH demonstrated that protonation promotes catalysis within the nucleobase-bound ribozyme complex but does not stabilize nucleobase binding in the ground state. These results support an electrostatic stabilization mechanism in which exogenous nucleobase binding counters negative charge that develops in the transition state.

Base Sequence↗

PubMed2004

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.

Acylation↗