Rearrangement of the conformation of Escherichia coli 5S RNA.

Several tRNA's from yeast'-' or from Escherichia coli" 6-9 can be trapped in a denatured biologically inactive form. In several instances, the denatured form is distinguishable from the native one by its chromatographic properties. Denatured yeast tRNA3LeU elutes in front of its native counterpart on Sephadex G-100 columns,4' 5 while denatured E. coli tRNATry is more strongly adsorbed on MAK columns than is the native form.8 In the course of a study on the properties of ribosomal 5S RNA from E. coli, two forms of this RNA with different chromatographic properties have also been observed. One of these forms cannot be reincorporated into the larger ribosomal subunit during reconstitution experiments starting with ribosomes stripped of 5S RNA and 50 per cent of their proteins by 2 M lithium chloride treatment. '0-'2 The form which has the same chromatographic properties as the native 5S RNA retains part of its ability to bind to the ribosomal subunit. Part of these results have been presented in a preliminary form in a report to the fourth meeting of the Federation of European Biochemical Societies.12 Materials and Methods. -Preparation of 5S RNA: 5S RNA was extracted from E. coli RNase ILocells and purified as previously described,'0 with only one modification. In order to prevent the degradation of RNA as much as possible at the time of extraction, frozen cells (25 gm) were mixed at 2-40C in a Turmix mixer with 25 ml of a Macaloid (American Tansul Co., Houston, Texas) suspension (6.25 gm in 500 ml of 0.05 M trisHCl, pH 7.6, 0.01 M MgCl2). Then 150 ml of a 90% (w/v) aqueous phenol solution and 125 ml of 0.01 M sodium acetate buffer, pH 5.0, 0.01 M magnesium acetate were added, and the suspension was mixed for 2 min at the maximum mixer speed. C'4.uracillabeled 5S RNA was prepared by the same procedure from cells grown on type-63 medium,'3 supplemented with 0.4% (w/v) glucose and 250 Mc/liter of CL-uracil (47.4 mc/ mmole, C.E.A., Saclay). Denaturation treatments: Urea treatment: 5S RNA was dissolved at the required concentration in 0.25 M tris-HCl buffer, pH 8.9, containing 7 M urea (freshly recrystallized) and 0.01 M EDTA. The solution was kept at 230C for 45 min and then quickly cooled in a salt-ice mixture. One-tenth volume of a cold 20% (w/v) sodium acetate solution adjusted at pH 5.0 was added and the RNA was precipitated by adding 2.5 vol of 95% ethanol, previously cooled at -10CC. Heat treatment: 5S RNA was dissolved at the required concentration in 0.15 M NaCl, 0.015 sodium citrate, pH 7.0. The solution was heated at 600C for 4 min and then quickly cooled by dilution in 29 vol of cold distilled water. The RNA was recovered by ethanol precipitation. Renaturation treatments: Renaturation in the presence of Mg++: 5S RNA was dissolved in 0.01 M tris-HCl, pH 6.9, 0.01 M MgCl2 at a concentration of about 160 ,ug/ml and heated at 600C for 5 min. It was then left to cool in air at room temperature. ' Renaturation in the absence of Mg++: The same procedure was applied to the RNA dissolved in 0.01 M tris-HCl, pH 6.9, 1 M NaCl, 0.01 M EDTA. Chromatographic procedures: Except when otherwise stated, chromatography on Sephadex Q-100 was performed on columns (1.5 cm X 90 cm) equilibrated at 2-40C