Undecagold cluster modified tRNA^Phe from Escherichia coli and its activity in the protein elongation cycle

Abstract

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...

Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation

“…The labeling reaction was as described (Plumbridge et al, 1980;Blechschmidt et al, 1994;Fei et al, 2008) with minor modifications, and typically was carried out in a 100-ml volume by incubating up to 18 nM (4.54 mg/ml) of tRNA Phe with 0.52 mM (4 mg/ml) of Cy3 NHS ester in 0.1 M sodium bicarbonate buffer, pH 8.3, at room temperature for 5 h, followed by an overnight incubation at 48C. The reaction was stopped by addition of 1/10 volume of 3 M NaOAc, pH 5.5.…”

“…Current methods for chemical modification of tRNA molecules with fluorescent dyes are based on presence in certain tRNAs at their elbow positions of high reactive unusual nucleotides, such as 4-thiouridine (s 4 U) at position 8 or 3-(3-amino-3-carboxypropyl)uridine (acp 3 U) at position 47. Such nucleotides can be modified with maleimide or NHS-linked fluorophores (Plumbridge et al, 1980;Janiak et al, 1990;Blechschmidt et al, 1994;Blanchard et al, 2004). After the labeling reaction is completed, excess dye can be removed by phenol extraction and ethanol precipitation, or by using gel filtration, or by a dialysis step (Walker and Fredrick, 2008), followed by separation of labeled from unlabeled tRNA using RP HPLC.…”

Monofunctionalized Gold Nanoparticles: Fabrication and Applications

“…Alternatively, monofunctionalized Au 11 clusters were obtained by stoichiometric ligand place-exchange with suitably functionalized phosphine ligands [72] and subsequent chromatography. [77] These facilitated synthetic approaches allowed reliable labeling of a multitude of biomolecules for TEM detection such as proteins, [13,78] antibodies [79] and RNA, [80][81][82] whereby, in the latter case, it was shown that the undecagold label did not interfere with the translation process in the ribosome due to its minuscule size [83,84] -in contrast to its Au 55 counterpart. [85] Recently, N-heterocyclic carbine-monofunctionalized undecagold was reported to have unprecedented thermal stability and catalytic activity in the electroreduction of CO 2 .…”

Preparation and high-resolution microscopy of gold cluster labeled nucleic acid conjugates and nanodevices

“…The labeled tRNA(Phe) no longer bound to the elongation factor (EF) Tu.GTP complex of Thermus thermophilus , which prevented is use as a probe to localize its binding sites. However, Blechschmidt et al (1993, 1994) reported an alternative approach: they used Phe-tRNA(Phe) containing 3-(3-amino-3-carboxypropyl) uridine at position 47, converted to a thiol derivative using 2-iminothiolane (Traut’s reagent). This was labeled with maleimido undecagold.…”