On the nature of two ribosomal sites for specific sRNA binding.

Abstract

It is now well established that 30S ribosomal subunits alone can bind specific sRNA or aminoacyl sRNA in the presence of the corresponding messenger RNA. 1-4 Although 50S subunits alone cannot bind specific sRNA they stimulate the binding of specific sRNA about twofold when associated with 30S ribosomal subunits.5' 6 In this communication we present evidence supporting the notion that two sRNA molecules are bound to a 70S ribosome. These two sites are named site 1 (COOH side of the growing polypeptide chain) and site 2 (NH2 side of the growing polypeptide chain). Site 1 has about three times more affinity for sRNA than has site 2. Materials and Methods.-E. coli extract and other materials: Preparation of ribosomes, sRNA from E. coli B, and aminoacyl sRNA have been described in the preceding communications. 2, 5, 7 In some cases strain Q13 of E. coli was used. The ribosomes were washed 3 times and were free of the aminoacyl sRNA transfer factor. For preparation of E. coli soluble protein fraction, the Is-30 of Nirenberg and Matthaei8 was centrifuged for 90 min at 150,000 g and the supernatant fluid was dialyzed overnight against a buffer containing 0.01 M Tris-HCl (pH 7.8), 0.01 M magnesium acetate, 0.006 M P-mercaptoethanol, and 0.06 M KC1 (Buffer 1). This fraction was called S-150. Specific radioactivities of materials used in this paper were as follows: C'4-phenylalanine, 395 uc/,mole; H3-phenylalanine, 2800 ,uc/umole. Counting efficiency was 1.0 ' 1.5 X 106 cpm/uc and 1.0 1.8 X 105cpm/Ac for C14 and HI, respectively. When C14 and H3were counted simultaneously, the counting efficiency for C14 was 1.8 6.2 X 105 cpm/,uc. Reaction mixture for the binding of C14-phenylalanyl sRNA: A typical reaction mixture for phenylalanyl sRNA binding contained the following in umoles per 2.05 ml: 73.8 Tris-HCl (pH 7.1), 30.0 magnesium acetate, and 39.4 KC1. In addition, it contained 400 ,ug of poly U (ammonium salt) and C'4-phenylalanyl sRNA and 70S ribosomes. Incubation was carried out for 20 min at 22°C. In some cases bound CL4-phenylalanyl-sRNA was measured by the method of Nirenberg and Leder.9 Isolation of the ribosome C'4-phenylalanyl-sRNA-poly U complex and formation of polyphenylalanine: After the binding of C'4-phenylalanyl sRNA to 70S ribosomes was completed, the reaction mixture was centrifuged for 1.5 hr at 150,000 g. The pellet was suspended in 0.2 ml of Buffer 1. For polyphenylalanine synthesis the reaction mixture contained the following in uAmoles in a total volume of 1.20 ml: 56.2 Tris-HCl (pH 7.8), 23.0 magnesium acetate, 53.8 KC1, 4.8 (B-mercaptoethanol, 1.3 phosphoenolpyruvate (Na-salt), and 0.07 GTP. In addition, it contained 3 mg of S-150 protein, 34 ug of pyruvate kinase, 20 mg of a mixture of sRNA in which phenylalanine specific sRNA is aminoacylated with C2-phenylalanine, and 0.2 ml of the resuspended pellet described above. The mixture was incubated at 37'C for 15 min. NH2-terminal analysis of polyphenylalanine: At the end of the incubation period, an E. coli soluble protein fraction (Fraction A of ref. (7) was added to the reaction mixture to make the total protein content 5 mg. Immediately after the addition of the soluble protein, 0.2 ml of 0.1 M phenylalanine solution was added followed by 10% trichloracetic acid to a final concentration of 5%. The precipitate containing polyphenylalanyl sRNA was treated with hot trichloracetic acid, a mixture of ether-alcohol and with ether as described previously.'0 The precipitate was mixed with 1 ml of 5% NaHCO3 and 2.0 ml of 5% dinitrofluorobenzene (DNFB) in alcohol. The mixture was shaken at room temperature overnight and 50%0 trichloroacetic acid was added to a final concentration of 5%. The precipitate was collected and washed with ether to remove excess DNFB. The yellow precipitate was mixed with 1.5 ml of 6 N HCl and hydrolysis was carried out at 1151180 for 20 hr under a N2 atmosphere. After hydrolysis, the mixture was diluted with 3 ml of water and dinitrophenyl (DNP) phenylalanine was extracted with three 5 ml of ether. The ether