Mechanisms of molecular recognition of tRNAs by aminoacyl-tRNA synthetases.

Interactions of Escherichia coli isoleucyl- and glutamyl-tRNA synthetases and their cognate tRNAs were analyzed by phosphate-alkylation mapping with N-nitroso-N-ethylurea and/or by 1H-NMR analysis. When E. coli tRNA(Ile) was bound with isoleucyl-tRNA synthetase, many of the phosphate groups in the anticodon loop and stem and in the D-stem were protected from alkylation. This result is consistent with that of analysis of imino proton resonances due to the secondary and tertiary base pairs. These analyses also suggested that the L-shaped tertiary structure of tRNA(Ile) is distorted upon complex formation with IleRS because of disruption of some tertiary base pairs. In the case of E. coli tRNA(Glu), several phosphate groups in the D-stem and the variable loop were significantly protected by the cognate synthetase. These results indicate that the two tRNAs, unlike other tRNAs studied so far, have some of the "identity determinants" in the D-stem and/or in the anticodon stem.     

Organization of ribosomal RNA genes in Mycoplasma capricolum

Summary DNA segments carrying rRNA genes of Mycoplasma capricolum have been cloned and characterized by restriction endonuclease mapping, DNA-RNA hybridization and nucleotide sequencing. The M. capricolum genome has two sets of rRNA gene clusters, where the arrangement is in the order of (5)16S-23S-5S(3). The spacer region between 16S and 23S rDNA is extremely rich in AT and does not carry any tRNA genes. Present address: Division of Hematology and Immunology of Internal Medicine, Kanazawa Medical University, Uchinada-Cho, Kahoku-Gun Ishikawa Pref. 920-02, Japan     

Key residues on microtubule responsible for activation of kinesin ATPase

Microtubule (MT) binding accelerates the rate of ATP hydrolysis in kinesin. To understand the underlying mechanism, using charged‐to‐alanine mutational analysis, we identified two independent sites in tubulin, which are critical for kinesin motility, namely, a cluster of negatively charged residues spanning the helix 11–12 (H11–12) loop and H12 of α‐tubulin, and the negatively charged residues in H12 of β‐tubulin. Mutation in the α‐tubulin‐binding site results in a deceleration of ATP hydrolysis (k_cat), whereas mutation in the β‐tubulin‐binding site lowers the affinity for MTs (K_0.5MT). The residue E415 in α‐tubulin seems to be important for coupling MT binding and ATPase activation, because the mutation at this site results in a drastic reduction in the overall rate of ATP hydrolysis, largely due to a deceleration in the reaction of ADP release. Our results suggest that kinesin binding at a region containing α‐E415 could transmit a signal to the kinesin nucleotide pocket, triggering its conformational change and leading to the release of ADP.     

Involvement of a Calcium-Dependent Protein Kinase in Hypoosmotic Turgor Regulation in a Brackish Water Characeae Lamprothamnium succinctum

Calcium ion is a key messenger in turgor regulation of internodalcells of Lamprothamnium succinctum in response to hypoosmotictreatment. An increase in the concentration of cytosolic freecalcium ion ([Ca²⁺]_c) is prerequisite for the turgor regulation[Okazaki and Tazawa (1990) J. Membr. Biol. 114: 189], We examinedwhether or not a calcium-dependent protein kinase (CDPK) isinvolved in the Ca²⁺-mediated turgor regulation of Lamprothamniumcells. A 53-kDa CDPK which phosphorylated preferentially histoneH1 but poorly myelin basic protein or casein, was detected inthe cell extract of Lamprothamnium by an in-gel protein kinaseassay. This protein kinase was detected by Western blottingand was immunoprecipitated using an anti-Dunaliella tertiolectaCDPK antibody which can neutralize the Dunaliella CDPK activity[Yuasa et al. (1995) Plant Cell Physiol. 36: 699]. The 53-kDaCDPK was partially purified from Lamprothamnium and its activitywas shown to be inhibited by the antibody and K-252a, a proteinkinase inhibitor. Microinjection of the antibody into the cytosblof Lamprothamnium cells inhibited the decrease in turgor pressurein response to hypoosmotic treatment. However, a transient increasein [Ca²⁺]_c, which was suggested by a transient reduction ofthe velocity of cytoplasmic streaming, was induced in antibody-injectedcells after hypoosmotic treatment. Turgor regulation upon hypoosmotictreatment was inhibited when the cells were treated with K-252a.These results imply that CDPK of Lamprothamnium functions ata down-stream position of Ca²⁺-mobilization in processing turgorregulation in response to hypoosmotic treatment. ² These authors contributed equally to the work.