Febs Letters Volume 73, Number 1-2./Volume 74, Number 1-2. [antikvár]

Volume 73, number 1 IT-BS LETTERS January 1977 THE ROLE OF ESCHERICHIA COLI RIBOSOMAL PROTEINS L7 AND LI 2 IN PEPTIDE CHAIN PROPAGATION Bernard R. CLICK Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario MSG IL6, Canada Received 13 September 1976 Revised version received 2 December 1976 1. Introduction The ribosomal proteins L7 and LI 2 are involved in a number of factor-dependent partial reactions of initiation [1-3], elongation [4], and termination [5], which in each instance gives rise to the hydrolysis of GTP. It has been suggested that L7 and LI 2 might constitute a ubiquitous receptor site on the ribosome where the soluble factor-GTP complexes are bound [6—8]. A current model of ribosome functioning envisions a factor.GTP complex binding to the L7/L12 site, hydrolysis of the GTP, dissociation of the factor.GDP complex, and then binding of another factor-GTP complex to the L7/ LI 2 site [9]. Thus the L7/L12 site could be viewed as helping to keep the ribosome in phase, e.g., it could prevent the ribosome from simultaneously binding aminoacyl-tRNA and translocating. The involvement of L7/L12 in the partial reactions of proteins synthesis has been demonstrated in two different types of experiments. Ribosomes are either extracted with NH4a and ethanol to remove L7/L12 or are treated with antibodies against purified L7/L12 [9]. In both cases the ribosomes, after they are treated, are assayed in one of the factor-dependent partial reactions of protein synthesis. A notable exception to the apparent requirement of soluble factors for L7/L12 is the newly discovered elongation factor EF-P, which does not Abbreviations: poly(U), Polyuridylic acid. EE, Elongation factor. DTT, Dithiothreitol. North-Holland Publishing Company - A msterdam depend on exogenous GTP for its functioning [10-12]. We have examined the ability of ribosomes, from which L7 and LI2 have been completely removed, to catalyze the poly(U)-directed synthesis of polyphenylalanyl-tRNA, as a function of time. It was found that ribosomes, lacking both L7 and LI 2, are after an initial lag, able to support the synthesis of polyphenylalanyl-tRNA, suggesting that EF-Ty and EF-G do not have a stringent requirement for L7/L12 in order to function in peptide chain elongation. 2. Experimental Ribosomes without L7/L12 were prepared by a modification of the method of Hamel et al. [4]. E. coii Q13 ribosomes, 180 mg/ml, in a solution (buffer I) containing 0.5 M NH4a, 10 mM MgCl^, 10 mM Tris-HCl, pH 7.4, and 1 mM DTT were diluted to a final concentration of 4 mg/ml with a solution (buffer II) containing 1.0 M NH4CI, 2 mM MgQj, 10 mM imidazole-HCl, pH 7.4, and I niM DTT. Two separate batches of ice-cold ethanol (each being one half the volume of the diluted ribosomes) were added slowly with sUrring. Too rapid an addition of ethanol resulted in the incomplete removal of L7/L12. The precipitated ribosomes were pelleted by centrifugation at 30 000 X g for 15 min, suspended in buffer II, to a final ribosome concentration of 4 mg/ml and extracted a second time. The final ribosome pellet was dissolved in a 1