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Research has been conducted on the structure of the specificity domain of the Bacillus subtilis RNase P RNA. The experiments have suggested that this structure is composed of a core, a functional unit of this domain, and a buttress module which provides the structural stability to the core module.

Results indicate that structure of the human tRNA(sup)Lys3UUU anticodon stem coupled with the modified amino acid t(sup)6A(sub)37 facilitates ribosome binding. Data indicate that the modified nucleoside influences anticodon loop conformation, which is mediated by t(sup)6A.

Results point out that it is critical for human tRNA(sup)LysUUU species to undergo anticodon domain modification for its effective binding to AAA-programmed ribosomes and wobbling to AAG codons.

A study of mitochondria from Caenorhabditis elegans which not only has the G:U pair preserved but also proximal nucleotides that inhibits charging by a nonmitochondrial enzyme is presented. Mitochondrial alanyl-tRNA synthetase from C. elegans has been cloned and its microhelix oligonucleotides have been assessed as substrates for aminoacylation. Results show that G:U recognition continues even with an acceptor helix that does not recognize nonmitochondrial systems. The mitochondrial enzyme is similar to other alanine enzymes including the well-studied Escherichia coli protein.

Kinetic and thermodynamic analyses were performed to determine the role of the protein component of Bacillus subtilis RNase P in catalytic reactions of B. subtilis precursor tRNA(super Asp) scission. The functional characteristics of ribonuclease P (RNase P) holoenzyme were also correlated to RNase P RNA in 10 millimole magnesium chloride and 100 millimole ammonium chloride. The protein component of RNase P was found to assist recognition of pre-tRNA by supporting the interaction between the enzyme and the 5'-precursor component of the substrate.

Research was conducted to study the various features associated with the molecular recognition of tRNA by pseudouridine 55 synthase and compare them with those involved in the relationship between tRNA and (m5U54)-methyltransferase. Results showed that the elimination of base pairs in the structure eliminated any form of pseudouridine 55 synthase activity. They also suggested that tRNA mutants located at the base of nucleotide 59 functioned as substrates for the synthase activity.

Research was conducted to investigate the mechanisms of pseudouridine synthases, an enzyme which transforms uridine to pseudouridine in a number of positions. Plasmid pLX1 was turned into BL21(DE3) cells while reaction mixtures were incubated in a buffer. Results showed that the target uracil remained bounded to the enzyme until product formation. They also indicated the importance of fluorouracil in covalent complex formation via 39 target positions.

The importance of the intrinsic ATPase in 80S ribosomes of higher eukaryotes was determined through identification of four binding sites in liver ribosomes of rabbits. Results have shown that the rabbit liver ribosomes contain only three tRNA binding sites A, P and E. The presence of ATP weakened the tight binding of the deacetylated tRNA at the E site. The E site interacted with deacetylated tRNA tightly blocking the binding of aminoacyl-tRNA to the A site.

The role of RNA transfer protein structures as a requirement in modifying enzyme binding, catalysis, and recognition was analyzed through inhibitory studies of structural variants of yeast tRNAasp and Escherichia coli tRNAleu. Results have shown that sequence G36pG37 is an essential identity element particularly in relation to substrate yields of heterologous tRNA species.

The role of RNA tertiary structure in transfer RNA recognition as well as the formation of an RNA tertiary base pair were investigated through identifying the structural determinant of G15*G48. Results have shown that the dependence of the N2*N3 pairing of G15*G48 on A13*A22 indicated the ability to manipulate a G*G base pair through another RNA structural element. The correlation between G15*G48 and A13*A22 indicated a control of an RNA tertiary base pair to modify the function of the RNA.

The influence of the zinc-containing C-terminal region on the binding of isoleucyl-tRNA synthetase was investigated. Gel mobility shift assay was utilized to study the complex formation between the zinc peptide and tRNA. Results have shown that the binding of peptide Zn2+-53C to isoleucyl-transfer RNA synthetase (tRNAIle) was nonspecific and that the peptide binding was zinc-dependent. The same kind of C-terminal when combined with other parts of synthetase can form a specific RNA binding pocket.

The melting behavior of several synthetic polynucleotides and a mixture of natural tRNAs was studied by monitoring the changes in the whole UV absorbance spectrum at several pH values. The second-order absorbance data were analyzed with a soft-modeling multivariate curve resolution procedure that allows the determination of the number of different species or conformations present along the melting experiment and the calculation of the melting profile and the pure spectrum for each chemical species or conformation. The melting temperature, [T.sub.m], for each thermal transition can be calculated from the melting profiles, and structural information on the different species or conformations can be obtained from their pure spectra. The multistranded species formed at certain pH conditions show several sharp thermal transitions related to the loss of the initial highly ordered structure. For these transitions, the mixture of species obtained in the denaturing process can be resolved when several data matrices, each giving additional information, are analyzed simultaneously with the mathematical procedure proposed.

The biochemical structure of Thermus thermophilus histidyl-transfer RNA (tRNA) synthetase (HisRSTT)-histidine complex was analyzed by multiple isomorphous replacement (MIR). MIR analysis of the HisRSTT-histidine complex indicated the presence of a crystal structure that was highly homologous to Escherichia coli HisRS complex. The enzyme was also composed of an N-terminal catalytic domain, a 90-residue C-terminal alpha/beta domain and a HisRS-specific alpha-helical domain.

The thiol located at position 443 of Escherichia coli prolyl-tRNA synthetase (ProRS) is not essential for catalysis, but is likely to be in a buried region that forms the prolyl-adenylate substrate binding pocket. This is indicated by results of the chemical modification and site-directed mutagenesis of the single cysteine located at position 443 in motif 3 of Escherichia coli ProRS. It was found that chemical modification of C443 hampers the ability of the enzyme to form the activated aminoacyl-adenylate, which is required for tRNA aminoacylation. Also, the aminoacyl-adenylate binding site is in close proximity to C443 of motif 3.

The thermodynamic effects of coaxial stacking of helixes with GA and CC mismatches were analyzed by utilizing model systems with four- or five-nucleotide overhangs on a hairpin stem. The coaxial stacking of helices stabilizes GA mismatches and CC mismatches during helix interfaces. Furthermore, energetic preference for 5' or 3' break in the phosphate backbone adjacent to the GA mismatch was not detected in the mismatch interface systems.

A revised model was developed for the polypeptide chain elongation cycle in which a discriminatory role is ascribed to the conformational differences of cognate and near-cognate transfer ribonucleic acids (tRNA). The model for protein elongation begins with the post-translocation of the peptidyl-tRNA ternary complex to the ribosomal P site. The post-translocation process initiates the release of aa-tRNA and EF-Tu.guanine diphosphate (GDP) due to the hydrolysis of guanine triphosphate.

High affinity binding of trivalent arsenicals to arginyl aminoacyl-tRNA transferase can occur by a thiol-independent mechanism. The presence of vicinal thiol groups is indicated by the reversible enzyme inhibition by phenylarsenoxides. The characterization of all mutant enzymes is carried out and the ability to bind a radioiodinated phenylarsenoxide derivative is retained. The blocking of the binding of the radiolabeled phenylarsenoxide due to the reaction of the enzyme with the bifunctional reagent indicates the binding of the two reagents at a single common site.

The structural conformation of tyrosyl-tRNA synthase, a 370 amino acid polypeptide from a eukaryotic pathogen, can attain transversion in a critical acceptor helix base pair which is responsible for species selective microhelix aminoacylation. The enzyme contains class I aminoacyl-tRNA synthetase and aligns with prokaryotic tyrosyl-tRNA synthetase in the class-defining active site region indicating that species selective microhelix can be used as species-specific inhibitor of tRNA synthetase.

To analyse the function of C-terminal domain, one of the domain of class I tRNA synthetase, in specific RNA binding, the anticodon binding of Escherichia coli methionyl tRNA synthetase was fused to maltose binding protein. This experiment revealed that the fusion protein and the isolated domain are stable and has native-like structural characteristics. Both the fusion protein and isolated domain specifically bind to a small RNA hairpin oligonucleotide.

A nanosecond of molecular dynamics (MD) simulation on the solvated anticodon loop of tRNA(super Asp) is analyzed, directing to address the ramifications of the detected differences in ten 100 ps molecular dynamics trajectories produced by modifying initial protocol of the systems. The resulting divergent trajectories differ in structural and dynamical characteristics. No relation exists between the modifications made in the system and the properties of the trajectories. The multiple MD recognized the protocol stability.

X-ray analysis of cross-transplantation and cross-aminoacylation between Escherichia coli class I glutaminyl- and class II aspartyl-tRNA synthetases reveals that both tRNAs share three identity nucleotides, G73, G10 and V35. Transplantation experiments reveal the feasibility of transplantation between Class I and class II systems and that conversion of aspartate to glutamine needs more sequence changes than conversion of glutamine to aspartate. Transplantation studies identify a new identity nucleotide C38 in yeast tRNA (Asp) for aspartylation.

Analysis of the crystal structure of unchanged tRNA(Gln), complexed with glutaminyl-tRNA synthetase from Escherichia coli and ATP, reveals the importance of pseudouridinylation for the stabilization of tRNA through water-regulated connection of the changed bases. The absence of water molecules, which cross-link the N5 of pseudo-uridines to their 5'-phosphate, is the notable change in unmodified tRNA.

A comparison of the intermediates of selenocysteine tRNA's precursor reveals that selenium affects tRNA (ser)(sec) stability and methylation. Selenocysteine also affects the methylation of the mcm U residue's ribose portion. The precusor tRNA used in the comparison lacks nucleoside modification. The microinjection method incorporates either one of the tRNAs into Xenopus oocytes.

Tyrosyl-tRNA synthetase from Bacillus stearothermophilus exhibits reactivity to tyrosine and ATP. Conversion of lysine 233 to alanine reduces the reactivity of the active subunit to ATP under saturated and unsaturated conditions. This reduction in substrate affinity causes a noncooperative enzyme to cooperate.

Pre-steady state kinetic analysis along with site-directed mutagenesis helps examine the function of threonine 234 in catalysis of tyrosyl adenylate formation by tryosyl-tRNA synthetase from Bacillus stearothermophilus. The transition state is stabilized by threonine 234 during the development of tyrosyl adenylate in tyrosyl-tRNA synthetase. Double mutant free energy cycles facilitate the examination of the function of coupling between T234 and K230.

A powerful evolutionary association is revealed by the basic geometries of two members of a class I enzyme, glutamyl-tRNA synthetase (GluRS) and glutaminyl-tRNA synthetase (GlnRs) of Escherichia coli, whose zinc constitution is measured. No zinc is seen in GlnRS eliminated to cause a structural modification in the enzyme and reduce the aminoacylation metabolism. The process of evolution of aminoacyl-tRNA synthetases may cause a frequent increase and fall in the zinc-binding domains. This is revealed by the close association between the two enzymes in the evolutionary stage.

Glutaminyl-tRNA synthetase (GlnRS) reacts with tRNA(Gln) in various ways, depending on the important conformational modification in GlnRS stimulated by ATP. Proteins reveal conformational modifications when stimulated by ligands. The molecular levels reveal the precise properties of the modifications which are associated with particular happenings in the working system. Allosteric influences are observed in hemoglobin and aspartate transcarbamylase due to the modifications. Hexolunase, which represents the active conformation, reveals the change in the protein due to the modifications.

A coupled transcription system was developed from Escherichia coli to examine the application of synthetic tRNA for in vitro protein engineering. T7 RNA polymerase was used to synthesize DNA sequences in vitro, clone a DNA plasmid and transcribe in vitro. The sequences resembled the natural tRNA Ala/UGC gene from Escherichia coli which had varying anticodons. The result of the change of CUA from UGC alanine anticodon corresponded with the UAG stop codon, while the rare AGG arginine codon corresponded to the CCU.

The first efficient synthesis of 5-taurinomethyluridine [T m.sup.5]U and 5-taurinomethyl-2-thiouridine [T m.sup.5][s.sup.2]U and their physicochemical properties are described. The synthesis of modified ribonucleosides involved the reaction of 5-substituted uridine derivatives with a tetrabutylammonium salt of taurine that is highly reactive and well-soluble in common organic solvents.

The measurement results of several RNA hairpin in NaCl and KCl concentration carried out by using optical tweezers is reported. The results suggest that the most stable secondary structure of RNA molecules might vary depending on the monovalent cation concentration.

The crystal structure of the ternary complex of heterotetrameric phenylalanyl-tRNA synthetase (PheRS) from Thermus thermophilus with cognate [tRNA.sup.Phe] and a nonhydrolyzable phenylalanyl adenylate analogue (PheOH-AMP) was determined. It reveals conformational changes in [tRNA.sup.Phe] induced by the PheOH-AMP binding and suggests that the idiosyncratic feature of PheRS, which aminoacylates the 2'-OH group of the terminal ribose is dictated by the system specific topology of the CCA end-binding site.

An attempt is made to investigate the structural properties of human mitochondrial tRNA (super Leu(UUR) along with a focus on domains within this molecule that appear unstable. Further, a series of tRNAs featuring mutations within the D and anticodon stems were prepared using nuclease probing.

A method based on 1H-1H NOE interactions is used to identify the binding site of spermine on tRNA. The carbon-bound methylene protons next to the amino groups are used to follow the interaction with tRNA. These protons resonate between 2.8-3.1 ppm where they overlap with the tRNA sugar resonances. This overlap is resolved by using spermines enriched with 13C in the carbons bonded to the -NH2+- and NH3+ groups. Results show that spermine interacts at the corner of the L-shaped tRNA molecule where the T_C and D loops intersect. These results are useful in the further understanding of the function of polyamines in cell growth and carcinogenesis.

An interresidually cyclized uridylyl(3'-5')uridine is synthesized. It has a bent formation similar to the U-turn in tRNA. Enzymatic assay, UV, 1H, 13C and 31P NMR studies characterize the compound. This cyclic dimer is not digested by snake venom phosphodiesterase and calf spleen phosphodiesterase due to its highly restricted base conformation. The cyclic structure is confirmed by the digestion of this dimer by nuclease P1 and HPLC analysis.

The nucleotide sequences of two tRNA(super Leu) species that have been isolated from Escherichia coli A19 have been studied with a post-labeling method. The two leucine isoacceptors tRNA(sub 4)(super Leu) and tRNA(sub 5)(super Leu) are responsible for reading the UUR codons in an in vitro protein synthesis. Results reveal that the first positions of the anticodon of both tRNA(super Leu) species have conformationally rigid modified pyrimidines. The modifications suggest that the tRNAs do not recognize non-cognate phenylalanine codons.