We demonstrate here, using RNA variants derived from tRNAAsp, that the minimalist aminoacylated structure able to interact efficiently with elongation factor Tu comprises a 10 base-pair helix linked to the 3'-terminal NCCA sequence. Shorter structures can interact with the elongation factor, but with significantly decreased affinity. Conserved features in the aminoacyl acceptor branch of tRNAs, such as base pair G53-C61 and the T-loop architecture, could be replaced respectively by the inverted base pair C53-G61 and by unusual anticodon loop or tetraloop sequences. Variants of whole tRNAAsp or of the 12 base-pair aspartate minihelix, with enlarged 13 base-pair long aminoacyl acceptor branches, as in selenocysteine-inserting tRNAs that are not recognized by elongation factor Tu, keep their binding ability to this factor. These functional results are well accounted for by the crystallographic structure of the Thermus thermophilus binary EF-Tu.GTP complex, which possesses a binding cleft accommodating the minimalist 10 base-pair domain of the tRNA aminoacyl acceptor branch.

The invariant threonine-62, which occurs in the effector region of all GTP/GDP-binding regulatory proteins, was substituted via site-directed mutagenesis by alanine and serine in the elongation factor Tu from Thermus thermophilus. The altered proteins were overproduced in Escherichia coli, purified and characterized. The EF-Tu T62S variant had similar properties with respect to thermostability, aminoacyl-tRNA binding, GTPase activity and in vitro translation as the wild-type EF-Tu. In contrast, EF-Tu T62A is severely impaired in its ability to sustain polypeptide synthesis and has only very low intrinsic and ribosome-induced GTPase activity. The affinity of aminoacyl-tRNA to the EF-Tu T62A.GTP complex is almost 40 times lower as compared to the native EF-Tu.GTP. These observations are in agreement with the tertiary structure of EF-Tu.GTP, in which threonine-62 is interacting with the Mg2+ ion, gamma-phosphate of GTP and a water molecule, which is presumably involved in the GTP hydrolysis.

A P-lactamase was purified 430-fold from the culture supernatant of Acinetobacter cafcoaceticus by ion exchange chromatography on CM-Sephadex and affinity chromatography on phenylboronic-acid-agarose. The purified enzyme was homogeneous as judged by SDS-PAGE, and was characterized with respect to molecular mass (38 and 41 kDa by gel filtration on Sephadex G-75 and SDS-PAGE, respectively), pH optimum (pH 7.0), temperature optimum (45 "C) and isoelectric point (9.3). The p-lactamase showed mainly cephalosporinase activity. It was inhibited by cloxacillin, carbenicillin, penicillanic acid sulphone (sulbactam) and aztreonam. It was not inhibited by clavulanic acid up to a concentration of 0.25mM. Neither EDTA nor p-chlormercuribenzoate, up to concentrations of 1 or 100 mM, respectively, affected activity. According to these characteristics, it is a typical CEP-N cephalosporinase.

An affinity electrophoresis system is described to allow determination of dissociation constants of lipopolysaccharide (LPS)-protein complexes. The LPS ligand is incorporated into polyacrylamide gels by addition to the polyacrylamide-N,N'-methylenebisacrylamide polymerization mixture. Quantitative evaluation revealed formation of immobile protein-ligand complexes. The method was applied both to R- and S-form LPS from Acinetobacter calcoaceticus. For a heat-modifiable outer membrane protein with Mr 18,000 from strain 69V the dissociation constant was determined to be 0.5 mM (EDTA-salt extracted R-LPS) and 0.3 mM (phenol-chloroform-petrolether extracted R-LPS). In comparison, for another A. calcoaceticus strain, CCM 5593, a higher dissociation constant of 1.0 mM (phenol-chloroform-petrolether extracted R-LPS) -indicative of lower affinity - was obtained. When S-LPS from A. calcoaceticus 69V was incorporated into the affinity gels, a dissociation constant of 0.02 mM was determined which indicates much stronger interactions than those exerted by R-LPS forms.

An undecagold cluster (Au,,) of molecular mass 6200Da was attached to the 3-(3-amino-3-carboxypropy1)uridine at position 47 of tRNAPhe from Escherichia coli. This modified tRNA can be enzymically aminoacylated with phenylalanine in the reaction catalyzed by phenylalanyl-tRNA synthetase. Au,,-labeled Phe-tRNAPh' forms a ternary complex with the elongation factor Tu . GTP and is active in poly(U)-dependent poly(phe) synthesis. The Au,, modification does not hinder the specific binding of tRNA to distinct ribosomal binding sites or the precise positioning of the aminoacyl and peptidyl residues in the peptidyltransferase center, and does not impair the translocation. The modified tRNA is suitable for the identification of ribosomal binding sites by scanning transmission electron microscopy and for crystallographic studies of the 70s ribosome at different states of the protein-elongation cycle.Protein biosynthesis is a complex process requiring a regulated action of many components of the translation system. Despite the rapid development of molecular biology, the determination of biological structures remains extremely important to study the mechanism of processes involved in gene expression. Among others, in the past decade much attention was focused on the elucidation of the three-dimensional structure of ribosomes. The present knowledge of the ribosomal structure and topology relies mostly on low-resolution studies using electron microscopy, neutron scattering and biochemical investigations including sequencing of ribosomal components, cross-linking and footprinting experiments. In the last years, crystallisation of ribosomal subunits and the complete 70s ribosomes have been reported (Wittmann et al., 1982; Mussig et al., 1989;von Bohlen et al., 1991 ;Ryazantsev et al., 1993), which may open the way for crystallographic structural analysis of this extremely complex ribonucleoprotein. Most succesful was, up to now, the crystallisation of 70s ribosomes from Thermus thermophilus diffracting up to 2.0 nm (Trakhanov et al., 1989) and the crystallisation of 70s ribosomes of the same species together with an average of 1.5 -1.8 equivalents Phe-tRNAPhe and a short mRNA chain composed of 35 * 5 uridine residues which diffract to higher than 1.5 nm resolution (Hansen et al., 1990).The enormous size of ribosomes with a molecular mass over 2.5-4.5 MDa, their complexity and instability are a hindrance to crystallisation and dictate the use of extremely heavy and dense groups for phasing (Weinstein et al., 1989). For this purpose, 'clusters', consisting of a core of several metal atoms linked together, such as an undecagold cluster (Au, ,), a tetrairidium cluster and tetrakis(acetoxymercuri)-methane, were used. Attachment of such clusters to specific sites of a tRNA and the following binding of this modified tRNA to the ribosomes will allow not only phasing but also localisation of tRNAs at the A, P and E sites of the ribosomal peptidyl transferase center, and in complexes of tRNA with enzymes and elongation factors. Hainfeld e...