Studies on the RNA and protein binding sites of the E . coli ribosomal protein LIO

We have used modification of specific amino acid residues in the E. coli ribosomal protein LIO as a tool to study its interactions with another ribosomal protein, L7/L12, as well as with ribosomal core particles and with 23S RNA. The ribosome and RNA binding capability of LIO was found to be inhibited by modification of one more more of its arginine residues. This treatment does not affect the ability of LIO to bind four molecules of L7/L12 in a L7/L12-L10 complex. Our results support the view that LlO's role in promoting the L7/L12ribosome association is due primarily to its ability to bind to both 23S RNA and L7/L12 simultaneously. INTRODUCTION The E^ coli ribosomal proteins L7/L12 and LIO are near neighbors in the 50S ribosomal subunit (1). The interaction between these two proteins and the ribosome has been elucidated through studies using core particles depleted of either L7/L12 or of both L7/L12 and LIO (2-5) . These experiments demonstrated that LIO was required for the rebinding of L7VL12 to the ribosome. An L10-L7/ L12 interaction can also be detected when the two proteins have been removed from the ribosome by a simple ammonium chloride-ethanol extraction (6,7). The extracts contain a stable complex of four molecules of L7/L12 bound to one molecule of LIO (8,9). Complex formation can even be induced in vitro with mixtures containing purified L7/L12 and LIO (7,10,11). No other ribosomal components are necessary for the jji vitro association of these two proteins. The principal concern of the present experiments is to explore the relationship between the two observed binding functions of LIO: the capacity to associate with multiple copies of L7/L12 in the absence of ribosomes and the capacity to stimulate L7/L12 binding to ribosomes. It was found that LIO may interact with L7/L12 and with 23S RNA at the same time. The parallel expression of the two functions can be uncoupled after chemical modification of LIO, with a reagent specific for arginine. Here we report an analysis of the © Information Retrieval Limited 1 Falconberg Court London W1V5FG England 2637 Nucleic Acids Research functional effects of this treatment without attempting to give a full protein chemical description of the modified structure. Thus, modification of arginine residues in L10 abolishes its ability to associate with 23S RNA or with ribosomal core particles. The L10 so modified is still able to interact with L7/L12. Our data suggest that L10 is bifunctional, in the sense that its stimulates the ribosome binding of L7/L12 by virtue of its ability to interact simultaneously with both L7/L12 and 23S RNA. MATERIALS AND METHODS 50S ribosomal subunits were prepared from £. coli MRE 600 as described by Hardy et_ £l. (12). The ribosomes were extracted in an ammonium chloride-ethanol solution as previously described (2,13,14).This treatment yielded core particles and supernatants, which were used for preparing purified proteins (11). The pro3 14 teins were labelled _iri vitro by reductive methylation with H-HCHO and C-HCHO (15). 23S RNA was prepared by 3-fold phenol extraction followed by Sephadex G-100 chromatography and ethanol precipitation. Reconstitution of core particles with L10 and the L7/L12-L1O complex was done using the conditions of Schrier et al. (3). Here, the 100 pi aliquots contained 100 pmoles of core particles and 25-500 pmoles of the test protein. The reconstituted ribosomes were separated from non-bound protein by chromatography over Sepharose 6B columns equilibrated with the reconstitution buffer: 100 mM Tris HC1 pH 7.4, 20 mM Mg (Ac) , 100 mM NH^Cl and 14 mM B-mercaptoethanol. Fractions were collected and divided into two aliquots. One was used for determining the amount of ribosomes by measuring the absorbance at 260 nm. The other aliquot was us ting. 3 14 sed to estimate, the amount of Hor C-label by liquid scintillation counThe RNA-binding of L10, L7/L12 and L7/L12-L1O complex was assayed using the conditions of Dijk et^ _al. (16). Binding mixtures of 100-150 pi contained 70 pmoles 23S RNA and 30-400 pmoles test protein in 30 mM Tris-HCl pH 7.4, 20 mM MgCl., 350 mM KC1 and 6 mM g-mercaptoethanol. These mixtures were incubated 1 hr at 42 and then cooled on ice. The RNA-binding was analyzed • is described for the ribosome reconstitution. The conditions used for modifying L7/L12, L10, and the L7/L12-L10 complex with different reagents are summarized in Table 1. The reactivity of the residues under the conditions used for modification was studied using H-N125 ethylmaleimide to estimate the degree of modification of cysteines and I incorporation was used as a probe for tyrosine modification. In both cases