Binding of 16 S rRNA to

Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20" C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S127~ST3^~S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA Interactions 1n spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, Sll, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. Ihis set includes 4 chloroplast-synthesized proteins: S6, STl, SI5 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are Included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit. INTRODUCTION Chloroplast ribosomes present the peculiarity that they are of prokaryotic type (1,2,3) but that many of their proteins are encoded in the nucleus and are synthesized in the cytoplasm. Therefore the assembly of all the chloroplast ribosomal components raises fundamental problems. One approach consists 1n the study of RNA-protein interactions. A great amount of work has been done on the protein-RNA interactions In Escherichia coli ribosomes (4,5,6,7,8). Concerning the 30S ribosomal subunit it has been shown by Nomura's group and it is generally admitted, that 7 ribosomal proteins (S4, S7, S8, S13, S15, SI7, S20) are independently and specifically bound to the 16S rRNA (4). Cooperative interactions between ribosomal proteins and 16S rRNA have also been found (9). These results provide the basis for the assembly of the 30S ribosomal subunits 1n E. coli. Several methods were used to determine which proteins bind to the 16S rRNA (10). These last years, a new technique for the study of protein-nucleic acid interactions have been developed, using blots of proteins on © IRL Press Limited, Oxford, England. 7293 Nucleic Acids Research ni t rocel lu lose sheets (11). This technique is a t t ract ive since one blot containing e lectrophoret ical ly separated ribosomal proteins can be used to determine interact ions with rRNA and after washing, can be re-used with d i f ferent conditions of binding. The prote in-b lo t t ing method is par t i cu la r ly useful for the study of RNA-protein interact ions in the case of chloroplast ribosomes because the preparation of a l l the isolated chloroplast ribosomal proteins, not yet achieved at the present time and needed by other methods, can be avoided. In the present study, the pro te in-b lo t t ing method was assayed with E_̂ co l i 30S ribosomal components and results are compared with known protein-16S rRNA interact ions. Binding of chloroplast 30S ribosomal proteins with chloroplast 16S rRNA was also determined, giving for the f i r s t time an insight on the protein-RNA interact ions 1n the chloroplast 30S ribosomal subunit. I t is shown that chloroplast 16S rRNA strongly binds to 7 chloroplast 30S ribosomal proteins in str ingent condit ions, and that 3 out of these 7 proteins are found in the LiCl washed 30S ribosomal subunit (core pa r t i c l e ) . MATERIALS AND METHODS Preparation of_ ribosomal subunits Chloroplasts were isolated from spinach leaves ( Spinacia oleracera, L., var. G&ant d'hiver) purchased from a local market. Ribosomal subunits were isolated as previously described (3) , but 10 yg/ml of heparin were added to the osmotically disrupt ing medium. Escherichia Col 1 (K12 strain) were grown in the LB (Luria-Bertrani) medium. The bacteria were col lected in the exponential phase of growth, washed with a medium containing 10 mM Tr1s-HCl, pH 7.6, 1 mM Mg acetate, 5 mM d i th io th re i t o l and stocked at -20° C. Frozen bacteria were ground 10 min in presence of carborundum (s i l ic ium carbure) in a medium containing 10 mM Tris HC1, pH 7.6, 1 mM Mg acetate, 5 mM d1th1othre1tol. After the disrupt ion of the bacter ia, carborundum and ce l l debris were removed by a 20 m1n centr i fugat ion at 30,000 g. The pel lets were reextracted in the same manner. Supernatants containing the 70S ribosomes were pooled and layered on 10-432 sucrose gradients containing 1 mM Mg for the obtention of the ribosomal subunits. Treatment of chloroplast 30S nbosomal subunits by_ LiCl About 10 A26Q units of chloroplast 30S ribosomal subunits were treated with 1M or 2M of LiCl in the conditions described by Homann and Nierhaus (12). Core part ic les were pel leted at 220,000 g for 3 hrs at 4° C. Proteins contained 1n the supernatant were precipi tated by 10% TCA at room temperature and were prepared for electrophoresis as indicated below.