Release factors differing in specificity for terminator codons.

Abstract

In a previous paper, initiator and terminator trinucleotides were shown to stimulate sequentially the binding of f-Met-tRNA to ribosomes and the release of free f-methionine from the ribosomal intermediate.1The formation of f-methio- nine was dependent upon a terminator codon, such as UGA, UAA, or UAG, and the release factor R discovered by Capecchi.2The separation of R into two components is described in this report.R1 cor- responds to the codons UAA and UAG; R2, to UAA and UGA.Methods.-R assay: The termination assay is described elsewhere.1Each reaction contained the following components in a final volume of 50 Al: 0.05 M Tris-acetate, pH 7.2; 0.03 M magnesium acetate; 0.05 M potassium acetate; 4-6 AMmoles of the (f ['H]- Met-tRNAf ..AUG.-.Ribosome) complex (4.0-6.7 A/Amoles of TCA-precipitable f ['H]- Met-tRNAf, 0.02 A2W0 unit was present/reaction); 0.96 A260 unit-of E. coli B ribosomes (washed with 0.5 M ammonium chloride); 0.17 mumole of AUG; and, where indicated, 7-7.7 mInmoles of terminator trinucleotide, and an R preparation.Reactions were incu- bated for 16 min at 30°unless otherwise stated; hence, the rate of f [3H]-methionine formation was determined in all experiments.The preparation of tRNAfMet (E. coli B), fractionated by benzoylated-DEAE-cellulose column chromatography,' was the gift of Dr. Michael Wilcox.The tRNAfmet was acylated with ['H]-methyl-methionine (3.1 c/mmole, Schwarz BioResearch Corp.) and then converted to f['H]-Met-tRNAfMet.lRibosomes were obtained from E. coli B as described by Lucas-Lenard and Lipmann,4 except that seven, rather than five, 0.5 M ammonium chloride washes were employed.R fractionation: Escherichia coli B supernatant and ribosome fractions, treated with DNase but not "preincubated," were prepared as described previously,5 except that cells were lysed with a French pressure cell at 18,000 psi, 0.005 M DTT replaced ,B-mercaptoethanol, and extracts were centrifuged for 5 hr at 137,000 X g.Release factors (prep.A, Table 1) were obtained as follows: R was precipitated by the addition of 116 gm of ammonium sulfate to 359 ml of the S-137 fraction; the pH was maintained at 7.8 by the addition of 0.5 N ammonium hydroxide.All steps were per- formed at 4°.The precipitate was collected by centrifugation at 30,000 X g for 15 min, dissolved in 139 ml of buffer A (0.05 M Tris-chloride, pH 8.0; 0.15 M potassium chloride; 0.001 M EDTA, adjusted to pH 7 with NaOH; and 0.003 M DTT) and dialyzed against