Preparation and properties of monomercurated tRNA

Abstract

Monomercurated tRNA has been prepared by exhaustive treatment of mercaptoethanol on polymercurated t RNA under non-denaturing conditions. The remained mercury atom is stable toward ultraviolet (254 nm) irradiation up to absorption of 100 quanta/nucleotide, but could be easily removed by mercaptoethanol under denaturing conditions. Monomercurated tRNA quantitatively binds to thiopropyl-Sepharose and can be completely desorbed by mercaptoethanol-containing solutions. Monomercurated tRNA is similar to the starting tRNA preparation in efficiency and specificity of aminoacylation and in factor-dependent polypeptide synthesis in vitro.One of the approaches to studying the functional topography of tRNA is determination of nucleoside residues involved in direct interactions with ribosomal proteins at different stages of translation. Here ultraviolet-induced tRNA-protein crosslinks could be used [l -51. For identification of proteins crosslinked to a definite tRNA molecule inside ribosomal complexes, as well as for location of cross-linked nucleoside residues in a tRNA sequence, it is necessary to isolate crosslinked nucleoproteins containing only this tRNA. This is possible if the tRNA molecule contains a specific affinity group, for instance, a mercury atom.Polynucleotides mercurated at C5 of pyrimidine bases are known to be firmly bound to sorbents containing an SH group [6 -81. Subsequent action of mercaptoethanol or similar compounds results in complete desorption of mercurated polynucleotides [6-81. Since ribosomal complexes contain several polynucleotides, a mercury atom in the respective tRNA must be introduced before complex formation. Therefore mercurated tRNAs should be functionally similar to the unmodified preparations and cause no side effects by the formation, ultraviolet irradiation and subsequent dissociation of complexes.Although mercuration of tRNA had been described earlier [9,10], the properties of mercurated tRNA mentioned above were not studied. In this paper we describe a method for preparation of monomercurated tRNA, which is able to bind reversibly to thiopropyl-Sepharose, but does not differ from the unmodified preparation in functional properties. MATERIALS AND METHODS MaterialsIndividual '4C-labelled amino acids (specific activity 25 -500 Ci/mol) were obtained from Amersham International; [203Hg](OA~), (specific activity 26 Ci/mol) from Isotop, USSR; individual tRNAPhe of Escherichia coli from Serva; elongation factors EF-Tu and EF-G from Biokhimreactiv, USSR: thiopropyl-Sepharose and DEAE-cellulose from Pharmacia; poly(U) and Fixion plates from Reanal, Hungary. Other reagents were of chemically pure grade.Labelled and unlabelled Hg(0Ac)z were prepared from [203Hg](N03)2 and HgClz ; total E. coli tRNA preparation was obtained according to [Ill; those deficient in tRNAPhe according to [12] ; total aminoacyl-tRNA synthetase according to [13]; 705 ribosomes of E. coli [activity in poly(U)-dependent binding of Phe-tRNAPhe = 80-90%] according to [14]; factor-dependent synthesis of poly(Phe) was ...

Direct tRNA-protein interactions in ribosomal complexes

“…70S ribosomes of E. coli MRE 600 (Biolar, Olaine, USSR) were used throughout this work. Monomercurated tRNAPhe was obtained according to (10) using tRNAPhe of E. coli (Boehringer, Mannheim). Aminoacylation of tRNAPhe with [3H]-phenylalanine (Amersham, England, specific radioactivity 137 Ci/mmole) and acetylation of Phe-tRNAPhe was carried out according to (11).…”

Nucleotide residues of tRNA, directly interacting with proteins within the complex of the 30 S subunit of E. coli ribosome with poly(U) and NAcPhe‐tRNAPhe

“…NAcPhe-tRNA Phe complexes, containing ordinary and monomercurated tRNA, are practically identical both in composition and properties and in the ratio of proteins cross-linked to tRNA under UV irradiation [8]. The use of monomercurated tRNA enables simple separation of tRNA (free or cross-linked to proteins) from other components of the complex after its irradiation and dissociation [8].…”

“…Their activity in poly(U)-dependent binding of NAcPhe-tRNA l'he was more than 90%. Monomer-curated tRNA was prepared according to [7] using tRNA phe, purchased from Boehringer (FRG). It was aminoacylated and acetylated as in [8].…”

“…Monomer-curated tRNA was prepared according to [7] using tRNA phe, purchased from Boehringer (FRG). It was aminoacylated and acetylated as in [8]. Formation of the ternary 30 S-poly(U).NAcPhe-tRNA Phe complex, its irradiation (254 nm), isolation of the mercurated NAcPbe-tRNA phe (both free and cross-linked to proteins) from the irradiated complex on thiopropyl-Sepharose and deacylation were carried out as described [8].…”

“…It was aminoacylated and acetylated as in [8]. Formation of the ternary 30 S-poly(U).NAcPhe-tRNA Phe complex, its irradiation (254 nm), isolation of the mercurated NAcPbe-tRNA phe (both free and cross-linked to proteins) from the irradiated complex on thiopropyl-Sepharose and deacylation were carried out as described [8]. The deacylated preparation was labelled at the 3' -end by using 1"4 phage-induced RNA ligase [9] and [32p]pCp, and subjected to electrophoresis in polyacrylamide gel [10% polyacrylamide, 0.5% bisacrylamide, 100 mM Tris-borate (pH 8.…”