In this study codon usage bias of all experimentally known genes of Lactococcus lactis has been analyzed. Since Lactococcus lactis is an AT rich organism, it is expected to occur A and/or T at the third position of codons and detailed analysis of overall codon usage data indicates that A and/or T ending codons are predominant in this organism. However, multivariate statistical analyses based both on codon count and on relative synonymous codon usage (RSCU) detect a large number of genes, which are supposed to be highly expressed are clustered at one end of the first major axis, while majority of the putatively lowly expressed genes are clustered at the other end of the first major axis. It was observed that in the highly expressed genes C and T ending codons are significantly higher than the lowly expressed genes and also it was observed that C ending codons are predominant in the duets of highly expressed genes, whereas the T endings codons are abundant in the quartets. Abundance of C and T ending codons in the highly expressed genes suggest that, besides, compositional biases, translational selection are also operating in shaping the codon usage variation among the genes in this organism as observed in other compositionally skewed organisms. The second major axis generated by correspondence analysis on simple codon counts differentiates the genes into two distinct groups according to their hydrophobicity values, but the same analysis computed with relative synonymous codon usage values could not discriminate the genes according to the hydropathy values. This suggests that amino acid composition exerts constraints on codon usage in this organism. On the other hand the second major axis produced by correspondence analysis on RSCU values differentiates the genes into two groups according to the synonymous codon usage for cysteine residues (rarest amino acids in this organism), which is nothing but a artifactual effect induced by the RSCU values. Other factors such as length of the genes and the positions of the genes in the leading and lagging strand of replication have practically no influence in the codon usage variation among the genes in this organism.

Interaction between Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and its substrates have been studied by fluorescence quenching. In the absence of other substrates, glutamine, tRNAG'" and ATP bind with dissociation constants of 460, 0.22 and 180 pM, respectively. The presence of other substrates has either no effect or, at best a weak effect, on binding of ligands. Attempts to isolate enzyme-bound aminoacyl adenylate did not succeed. Binding of the phosphodiester, 5'-(methy1)adenosine monophosphate (MeAMP), to GlnRS was studied by fluorescence quenching and radioactive-ligand binding. tRNA also only has a weak effect on phosphodiester binding.Selectively pyrene-labeled GlnRS was used to obtain shape and size information for free GlnRS. A comparison with the GlnRS shape in the GlnRS/tRNAG1" crystal structure indicates that no major change in shape and size occurs upon tRNAG'" binding to GlnRS. S,S'-Bis(8-anilino-l-naphthalene sulfonate) (bis-ANS), a non-covalent fluorescent probe, was also used to probe for conformational changes in GlnRS. This probe also indicated that no major conformational change occurs upon tRNAG'" binding.We conclude that lack of tRNA-independent pyrophosphate-exchange activity in this enzyme is not a result of either lack of glutamine or ATP binding in the absence of tRNA, or formation of aminoacyl adenylate and slow release of pyrophosphate. A conformational change is implied upon tRNA binding, which promotes pyrophosphate exchange. Fluorescence studies indicate that this conformational change must be limited and local in nature.

The urea-induced unfolding of glutaminyl-tRNA synthetase, a multidomain protein, has been studied by equilibrium and kinetic methods, using chemical modification, fluorescence, and CD spectroscopy. The far-UV CD, fluorescence, and sulfhydryl reactivity clearly demonstrated the existence of a stable intermediate state at around 2 M urea. The intermediate showed higher binding of 1-anilino-8-naphthalenesulfonic acid. Furthermore, near-UV CD study of the intermediate showed significantly disrupted tertiary structure with only a small change in the secondary structure, which is a characteristic of molten globule states. The activation energies (delta G++) calculated from unfolding kinetics monitored by CD and fluorescence suggest that the intermediate state may be separated from the native and the unfolded state by high activation energy barriers.

Glutaminyl-tRNA synthetase from Escherichia coli is a member of a subgroup of aminoacyl-tRNA synthetases that do not catalyze ATP-PPi exchange in the absence of the cognate tRNA. Such behavior suggests conformational changes upon substrate binding. Two different fluorescent probes, pyrenylmaleimide and acrylodan, were used to specifically label a nonessential sulfhydryl group of GlnRS. Conformational changes induced by substrates were studied using glutaminyl-tRNA synthetase labeled with these two environment-sensitive probes. ATP was shown to cause a significant conformational change that alters the mode of binding to tRNA(Gln) to GlnRS. The alteration of the salt sensitivity pattern of tRNA(Gln) binding to GlnRS by ATP supports this. Binding of tRNA(Gln) causes a conformational change that may be different in nature for the ATP/GlnRS complex and free GlnRS. Hydrodynamic parameters deduced from fluorescence polarization studies and the use of a noncovalent probe indicate that the ATP-induced conformational change may not be global in character.

Conformational changes that occur upon substrate binding are known to play crucial roles in the recognition and specific aminoacylation of cognate tRNA by glutaminyl-tRNA synthetase. In a previous study we had shown that glutaminyltRNA synthetase labeled selectively in a nonessential sulfhydryl residue by an environment sensitive probe, acrylodan, monitors many of the conformational changes that occur upon substrate binding. In this article we have shown that the conformational change that occurs upon tRNAG'" binding to glnRS/ATP complex is absent in a noncognate tRNA tRNAG1"-glnRS/ATP complex. CD spectroscopy indicates that this cognate tRNAG1"-induced conformational change may involve only a small change in secondary structure. The Van? Hoff plot of cognate and noncognate tRNA binding in the presence of ATP is similar, suggesting similar modes of interaction. It was concluded that the cognate tRNA induces a local conformational change in the synthetase that may be one of the critical elements that causes enhanced aminoacylation of the cognate tRNA over the noncognate ones.Keywords: conformational change; discrimination; fluorescence; synthetase: tRNA Translation of nucleotide sequences in mRNAs to amino acid sequences in proteins is characterized by high degree of accuracy. This level of accuracy is primarily determined at the level of aminoacylation of tRNAs by aminoacyl-tRNA synthetases (Schimme1 & Soll, 1979;Schimmel, 1987;Carter, 1993). The correct aminoacylation of all the tRNAs involves positive recognition of the cognate tRNAs (McClain, 1993) and negative discrimination of the noncognate tRNAs (Schmitt et al., 1993). Recognition of cognate tRNAs has been studied intensively, and structural elements that are responsible for recognition have now been mapped for many systems (Normanly & Abelson, 1989;Mechulam et al., 1995). Very little is known, however, about the factors that are responsible for negative discrimination.Even in the case of recognition of cognate tRNAs where the identity elements have been mapped, it is not clear exactly how this elements influence enzymatic properties and translates recognition into catalytic competence. Due to pioneering work by Soll and co-workers, glutaminyl-tRNA synthetase has emerged as one Reprint requests to: Siddhartha Roy, Department of Biophysics, Bose Institute, P 1/12. C.I.T. Scheme VI1 M, Calcutta 700054, India; e-mail: siddarth@boseinst.ernet.in.Abbreviations: GlnRS, glutaminyl-tRNA synthetase; acrylodan, 6-acryloyl-2-dimethyl aminonaphthalene; CD, circular dichroism: tempol, (4-hydroxy-2,2,6,6-tetramethylpiperidine-I -0xyl): GluRS, glutamyl-tRNA synthetase.of the best systems to study the recognition and the consequent catalytic activation process. Jahn et al. (1991) and Ibba et al. (1996) have shown that the correct recognition of anti-codon bases increases kc,, of the enzyme glutaminyl-tRNA synthetase of Escherichia coli significantly. Because the anti-codon binding pocket is approximately 35 A away from the active site, a protein conformational ...