We have developed a simple method for measuring the missense substitution of amino acids at specified positions in proteins synthesized in vivo. We find that the frequency of cysteine substitution for the single arginine in Escherichia coli ribosomal protein L7/L12 is close to 10(‐3) for wild‐type bacteria, decreases to 4 x 10(‐4) in streptomycin‐resistant bacteria containing mutant S12 (rpsL), and is virtually unchanged in Ram bacteria containing mutant S4 (rpsD). We have also found that the frequency of the cysteine substitution for the single tryptophan in E. coli ribosomal protein S6 is 3‐4 x 10(‐3) for wild‐type bacteria, decreases to 6 x 10(‐4) in streptomycin‐resistant bacteria and is elevated to nearly 10(‐2) in Ram bacteria.

Incubation of electrophoretically pure samples of the Escherichia coli 50S ribosomal protein L7/L12 together with elongation factor Tu leads to the hydrolysis of GTP. Addition of elongation factor Ts stimulates this reaction. Elongation factor G cannot replace elongation factor Tu for the ribosome-free GTPase reaction dependent on L7/L12. The data suggest that elongation factor Tu and the protein L7/L12 interact directly at the ribosomal A site. The 50S ribosomal protein, L7/L12, is involved directly or indirectly in the-function of protein factors required for initiation, elongation, and termination of protein synthesis by the Escherichia colh system (reviewed in ref. 1). When the ternary complex consisting of elongation factor Tu (EF-Tu), aminoacyl-tRNA, and a noncleavable analogue of GTP is bound at the ribosomal A site, it can be shown by crosslinking experiments that EF-Tu and L7/L12 are near neighbors (2). It is, therefore, possible that a direct interaction between L7/L12 and EF-Tu is required for the expression of EF-Tu's function on the ribosome.One aspect of EF-Tu's function in protein synthesis is that it is involved in the hydrolysis of GTP and the production of GDP as well as inorganic phosphate (Pi) (see ref. 1). Furthermore, it is possible to trigger the hydrolysis of GTP by incubating EF-Tu with the antibiotic kirromycin in the absence of ribosomes, mRNA, and aminoacyl-tRNA (3, 4). Since a small molecule such as kirromycin can evoke this response, it seemed reasonable to expect that one or a few purified ribosomal components, which make up the EF-Tu binding domain, might do the same. We have shown that L7/L12 can replace the 50S subunit in an EF-Tu-dependent GTPase reaction in the presence of 30S ribosomal subunits, poly(U), and aminoacyl-tRNA.t Here we report that electrophoretically pure L7/L12 together with EF-Tu mediates the hydrolysis of GTP in the absence of all other components needed for protein synthesis. METHODSStandard Assay. All components tested for GTPase activity were studied in the following assay buffer: 20 mM Tris-HCl, pH 7.6/30 mM KCI/30 mM NH4Cl/10 Mg9l2/1 mM NaN3/1 mM dithiothreitol (Sigma)/O. 1 mM phenylmethylsulfonylfluoride (Sigma). Appropriate amounts of material were incubated in O.1-ml samples with 0.02 mM [y-32P]GTP (Amersham, specific activity 20-50 Ci/mol). After incubation for various times at 30' (30 min where not stated otherwise), the reaction was stopped by the addition of 0.1 ml of chilled 1 M HC104 in 2.5 mM KH2PO4. After 10 min on ice, 0.5 ml of a 6% suspension of activated charcoal in 1 M HCI was added; the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. mixture was agitated three times at 5-min intervals, and then cleared by centrifugation at 8000 X g for 2 min. An aliquot containing 0.5 ml of the clear supernatant was withdrawn; radioactivity was measured in a toluene-based sc...

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