Cores were prepared from 50S ribosomal subunits by incubation with 0.4 M LiCI/Mg++ (0.4c cores); 0.8c cores and corresponding SPO.4 0.8 split proteins were obtained from 0.4c cores. In the fragment reaction 0.4c cores were active, but 0.8c cores were not. Activity of the 0.8c cores could be restored by reconstitution with the SPO.4-0.8 fraction. The split proteins were separated by DEAE-cellulose chromatography and Sephadex gel filtration. The peptidyltransferase activity is correlated with the amount of protein Lii added to the 0.8c core under reconstitution conditions. Whether protein L11 displays the enzymatic activity itself or is part of the enzymatic center is discussed.Synthesis of peptide bonds, the primary function of ribosomes, does not require the entire complicated structure of the ribosome. The complexity is, however, required to ensure precision of translation during peptide-bond formation. The catalytic center of peptide-bond formation, referred to as peptidyltransferase, is located on the larger ribosomal subunit (1, 2). Peptide-bond formation can be tested in the "fragment reaction" (3), which uses 3'-fragments of a peptidyl-tRNA analogue [e.g., CACCA-(Ac [3H]Leu)], puromycin, 5OS subunits, and Mg++ and K+ ions. The last three bases (3'-end) of the tRNA in the donor site and the last two bases of the tRNA in the acceptor site seem to be crucial for peptidyltransferase activity (4). No factors and no GTP are involved in the reaction. Despite the abnormal requirement for 33% ethanol, this reaction is a model for peptide-bond formation in protein biosynthesis (4).Staehelin et al. (5) described the preparation of a series of 50S derived cores (a,3,-y) by isopycnic centrifugation in CsCl-Mg++ solutions. The (3-core showed only 20% activity in the poly(U) assay but it was fully active in the fragment assay; the y-core was not active in either system (5, 6); the split proteins, SP_, could restore the activity in the fragment assay of the 7y-core. We have prepared equivalent cores by LiCl treatment (7), which is more convenient for obtaining large amounts of cores and split proteins. In this paper, we show that the peptidyltransferase activity depends upon the presence of protein Li 1 in the core. MATERIALS AND METHODSPreparation and Analysis of LiCI Cores and Split Proteins.50S subunits were prepared as described (7) The supernatant containing the SPo.4_0.8 split proteins was dialyzed against core-buffer and lyophilized. By definition, the amount of equivalent units of split proteins was equal to the amount of A260 units of 0.8c cores. The dry protein-salt mixture was suspended in 5 ml of glass-distilled water. For removal of most of the RNA, one volume of 8 M urea in 4 M LiCl solution was added; after 24 hr at 00, the RNA was pelleted by centrifugation at 10,000 X g for 20 min. The supernatant was dialyzed against TM-buffer (pH 8.6) containing 6 M urea and applied to a DEAE-cellulose column (1.5 X 20 cm) that had been equilibrated with the same buffer. The column was washed with about 30 ml of...
The effect of 2-methoxy-5-nitrotropone on the ribosome has been studied. Treated particles are inactivated for polypeptide synthesis, aminoacyl-tRNA binding, and elongation-factor-dependent GTPase but catalize peptide bond formation. Treated particles (either 70-S ribosomes or ribosomal cores lacking proteins L7 and L12) recover their ability to hydrolyze GTP, but not to support polypeptide synthesis or aminoacyl-tRNA binding, upon the addition of these two untreated proteins. However, even in the absence of the added proteins the treated particles are able to carry out substantial GTP hydrolysis in the presence of 200/, methanol.Interaction of particles with antibiotics such as chloramphenicol and thiostrepton is not affected by treatment.When proteins from treated 50-5 subunits were analyzed by two-dimensional polyacrylamide gel electrophoresis, only proteins L3, and L15 were found not to be affected by the treatment. Proteins L1, L2, L4, L10, L13, Ll6, L17, L18, L22 and L24 showed spots of much less intensity than the controls and the other protein spots were not detected. A comparison of the resistant proteins with those present on CsC1-derived cores points to protein L15 as possibly involved in the peptidyl transferase centre of the ribosome.Chemical modification of ribosomal proteins is a method extensively used to study the relationship between structure and function in the ribosome. 2-Methoxy-5-nitrotropone, a specific reagent for the free primary amino groups of the amino acids, has been used as a tool to localize proteins in the 3 0 3 ribosomal subunit [l] and to carry out functional studies of the 50-5 subunit [2]. 50-S subunits treated with 2-methoxy-5-nitrotropone were inactive for polypeptide synthesis, the GTP hydrolysis which is dependent on elongation factor G (EF-G) being the only activity inactivated in the treated particles [2].It was therefore assumed that the inability of the treated ribosomes to synthesize proteins was due to an alteration in the translocation step of this process [2]. Since two proteins of the 50-S subunit, L7 and L12, are implicated in the EF-G-dependent functions of the ribosome [3-81, we have utilised intact ribosomes, 50-5 subunits and 50-S-derived particles deficient in L7 and L12 in order to decide whether the inactivation of ribosomes by 2-methoxy-5-nitrotropone is due to a specific alteration of these two proteins. I n this investigation we have studied the Abbreviations. EF-G, elongation factor G; EF-T, elongation factor T. effects of treatment with 2-methoxy-5-nitrotropone upon various activities of the ribosomal 50-5 subunit including functions dependent on elongation factors T and G (EF-T and EF-G). NATERIALS AND METHODS MaterialsEscherichia coli B or E. coli D-10 ribosomes were obtained as previously described [7] and subunits were prepared by sucrose gradient centrifugation in a zonal rotor [9]. Ribosomes and ribosome subunits were stored unfrozen a t -20 "C in a buffer containing 0.01 M Tris-HC1 pH 7.8, 0.01 M magnesium acetate, 0.15 M NH4C1, 0.001 M dith...
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