Three tRNA binding sites on Escherichia coli ribosomes.

Abstract

The binding of N-acetyl-Phe-tRNArhe (an analogue of peptidyl-tRNA), Phe-tRNAPhe, and deacylated tRNAPhe to poly(U)-programmed tightly coupled 70S ribosomes was studied. The N-acetyl-Phe-tRNAPhe binding is governed by an exclusion principle: not more than one N-acetyl-Phe-tRNA hC can be bound per ribosome, although this peptidyl-tRNA analogue. can be present either at the aminoacyl-tRNA (A) site or the peptidyltRNA (P) site. Two Phe-tRNArhe molecules are accepted.by one ribosome in the presence of poly(U). This aminoacyl-tRNA binds enzymatically (in the presence of elongation factor Tu and GTP) and nonenzymatically tolthe A site and is then transferred to the P site, if that site is free. If this elongation factor G-independent movement is hampered, either by using an incubation temperature of OTC or by the addition of the translocation inhibitor vio- The ribosomal model comprising two tRNA binding sites (the A site for aminoacyl-tRNA and the P site for peptidyl-tRNA) is generally accepted for both pro-and eukaryotic ribosomes. This model is related to, and mainly caused by, the method usually chosen for the localization of aminoacyl-tRNA-i.e., the puromycin reaction (1 In all binding assays and at all tRNA concentrations, a blank (lacking 70S ribosomes) was determined and subtracted before calculating the binding data.Determination of Phenylalanine and Phe2. After binding [l4C]Phe-tRNAPheto the ribosomes, 100 ul of the reaction mixture was mixed with 12 Al of 1 M acetic acid to prevent further binding and peptidyltransferase activity, the ester bond was cleaved by addition of25 pA of1 M NaOH and incubation at 370C for 30 min, and the sample was acidified by the addition of 25 A.l of 1 M HC1. A total of 21,ul (seven 3-pA portions; -4000 cpm) of the final mixture was spotted on a cellulose sheet [20 X 20 cm, Polygram CEL 300, Machery and Nagel (Dfiren, Federal Republic ofGermany)] together with 9 ,ul each of 1.5 mM phenylalanine and saturated Phe2 ('2 mM; Sigma). After 8 hr (400 V in 2% pyridine acetate, pH 4.4, containing 15% acetone), phenylalanine and Phe2 were completely separated; Phe2 and phenylalanine migrated -10 and 7.5 cm, respectively, toward the cathode and were visualized with the ninhydrin reaction. The entire lane including the origin was cut in 1-cm-long pieces, and the radioactivity was determined. RESULTSBinding of Ac-Phe-tRNAPhe to Ribosomes. In the experiment described below, an Ac-[l4C]Phe-tRNAPhe preparation was used that contained -30% tRNAPhe. (.). The A and P site locations were tested by the puromycin reaction [reaction in the absence of EF-G indicates P site (e), reaction in the presence of EF-G indicates A site plus P site, and the difference is the A site (A)]. The Ac-Phe-tRNAPhe preparation contained -30% deacylated tRNAPh. Equivalent binding was obtained at 10 mM Mge+. With an Ac-Phe-tRNAP"' preparation containing < 10% deacylated tRNAPho, essentially the same binding curve was obtained, whereas the % A-site occupation was reduced.amo-unts of this Ac-Phe-tRNAPbe to poly(...

Structure-function relations in E. coli 16S RNA

“…For instance, while four tRNA-binding sites are proposed, only two or three are occupied at any one time. This agrees with the data of Rheinberger et al (1981) who found 2-2.5 tRNAs bound during translation. It also includes the R and D sites, which increase the fidelity of translation (Lake, 1981;Nierhaus et al, 1980).…”

“…The A and P sites are as usually proposed. The D (discharge) site corresponds to the E site of Rheinberger et al (1981). The name has been changed for acronymic reasons.…”

“…Ef: elongation factor. was provided by Nierhaus et al (1980) and Rheinberger et al (1981) on the basis of filter-binding studies. Velocity sedimentation, however, has yielded ambiguous results on this point (Schmitt et al, 1982;Grajevskaja et al, 1982).…”

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

“…Transfer RNAs (tRNAs) are the substrates used by ribosomes for synthesizing proteins+ To understand the mechanism of translation at the molecular level, it is essential to understand how tRNAs interact with the ribosome (Rheinberger et al+, 1981;Grajevskaja et al+, 1982;Kirillov et al+, 1983;Lill et al+, 1984)+ The anticodon arm of P-site tRNA interacts with mRNA in the vicinity of the cleft formed by the platform and the head of the 30S subunit (Lake, 1980;Gornicki et al+, 1984)+ The acceptor arms of A-site and P-site tRNAs interact with the peptidyl transferase center, which lies close to the base of the central protuberance of the 50S subunit (Ofengand, 1980;Olson et al+, 1982;Wower et al+, 1989)+ During peptide bond formation, the CCA-39 termini of A-site and P-site tRNAs must be proximal to allow peptidyl transfer, while their anticodons interact with adjacent codons on mRNA (Fairclough & Cantor, 1979a;Matzke et al+, 1980)+ Several models have been proposed for the arrangement of A-site and P-site tRNAs in the ribosome based on stereochemical considerations (Fuller & Hodgson, 1967;Woese, 1970;Rich, 1974;Sundaralingam et al+, 1975;Lake, 1977;Spirin & Lim, 1986;McDonald & Rein, 1987;Prabahakaran & Harvey, 1989;Nagano et al+, 1991;Easterwood et al+, 1994), fluorescence resonance energy transfer (FRET) (Johnson et al+, 1982;Paulsen et al+, 1983), crosslinking studies (Ofengand, 1980, Ofengand et al+, 1981Wower et al+, 1989Wower et al+, ,hotra et al+, 1998, and other methods (Spirin, 1983;Smith & Yarus, 1989;Wagenknecht et al+, 1989;Nierhaus et al+, 1998)+ Most of these models can be assigned to either the R (Rich, 1974) or S (Sundaralingam et al+, 1975) orientations+ In the R orientation, the T loop of A-site tRNA ...…”

tRNA binding to programmed ribosomes increases the ribosomal affinity for tuberactinomycin O

“…Increasing the ionic strength Maximal stimulation of the binding of tuberactinomycin to programmed ribosomes was achieved by the addition of a two-molar excess of tRNAPhe (375 pmol tRNAPhe, see table 1). At this molar ratio tRNAPhe binds exclusively to the ribosomal P-site [13]. The addition of more tRNA does not further increase the drug binding (table 1, cf.…”