The proteins of donor tRNA‐binding site of Escherichia coli ribosomes

The effect of poly(U) on the arrangement of tRNAPhe in donor tRNA‐binding site of Escherichia coli ribosomes

“…In a previous paper [9] the labelling of both subunits by azido-tRNAPhe was reported in the presence of poly(U). These data were obtained using azidotRNAPhe with rather high (4 mol of reagent per mole of tRNAPhe) level of tRNA modification and a single ribosome preparation.…”

“…2,4-Dinitro-5-fluorophenylazide was prepared according to [8]. Aminoacylation of tRNAPhe was performed as in [9]. Poly(U) was from Reanal (Hungary).…”

“…Alkylation of tRNA with following attachment of arylazido groups was carried out as in [9]. The extent of tRNA modification was 1.8-4.0 mol of the reagent per mole of tRNA.…”

“…In the experiments on inhibition of noncovalent and covalent binding of azido-tRNA to ribosomes the excess (5-lOO-fold) of unmodified tRNA was added simultaneously with azido-tRNA. Irradiation of the complexes and separation of ribosomes into subunits with subsequent ethanol precipitation was carried out as in [9]. Ribosomal proteins (un-labelled and with cross-linked tRNA) were extracted from the ribosomal subunits by 2 M LiCl in 4 M urea at 4°C for 100 h according to [lo].…”

“…Gel electrophoresis of the probe and analysis of the labelled proteins were performed as in [ 111. Analytical separation of ribosomal subunits into rRNA and proteins was carried out as in [9] by centrifugation in sucrose density gradient in the presence of sodium dodecyl sulphate and EDTA.…”