Physical characteristics of 23 S rRNA from the 50 S ribosomal subunit of escherichia coli

Abstract

The in vitro reconstitution of the bacterial 30 S ribosomal subunits [l-2] and subsequently the 50 S subunits [3-51, has establishedthe fact that the information required for the assembly of the subunits is present in the structure of the proteins and/or RNAs. Previous work has also shown that the reconstitution process requires a rigid set of buffer conditions.Recently, Vasiliev and Zalite [6], using electron microscopy, concluded that the 23 S rRNA molecules are capable of acquiring a specific compact conformation which is morphologically close to intact 50 S subunits. However, their RNA was prepared in the presence of EtOH and spermidine, which have been shown to cause conformational changes on bacterial ribosomes [7,8]. Sieber et al. [9] also reported an electron microscopic analysis of the 23 S rRNA, which has an overall diameter larger than the dimensions reported by Vasiliev and Zalite 161, as well as the 50 S subunit [lo]. From these disparate results it is apparent that further physical studies are needed to clarify the structure of 23 S rRNA.We report here the physical characteristics of the 23 S rRNA under buffer conditions used for the formation of the first reconstitution intermediate, aswell as under low salt conditions normally used with hydrodynamic methods. We have compared these results with the hydrodynamic properties obtained previously on the first reconstitution intermediateOur results indicate that under reconstitution conditions, the conformation of the 23 S rRNA is considerably more extended than that found in the 50 S subunit, and that upon addition of protein the RNA structure does not appear to be appreciably affected. ElsevierlNorth-Holland Biomedical Press Materials and methods .l . Preparation of 50 S subunitsRibosomal subunits were prepared using previously described methods [12]. The 50 S and 30 S subunits were separated on a lo-30% exponential sucrose gradient formed in a Ti-15 Beckman zonal rotor spun at 32 000 rev./min for 14.5 h. The 50 S subunits were recovered from the sucrose fractions by precipitating with 2 volumes of ethanol after raising the Mg2+ and dithiothreitol concentration to 0.01 M and 0.001 M. The precipitate was pelleted by centrifugation at 20 000 X g for 30 min, dissolved and dialyzed at 4°C against 0.01 M Tris-HCl, pH 7.6,O.Ol M Mg(OAC)2, 0.06 M NH&l and 0.004 M fl-mercaptoethanol overnight. The purity of the ribosomal preparations was checked routinely by means of sedimentation velocity in the Beckman Model E analytical ultracentrifuge. 2. Preparation of 23 S rRNAThe 23 S and 5 S rRNAs were extracted from the 50 S subunits with phenol according to the method of Nierhaus et al. [5]. After the final extraction, the rRNA samples were precipitated with 2 volumes of EtOH, 20 mM Tris-HCl, pH 7.8, and pelleted at 10 000 revjmin for 30 min at 4°C in an HB-4 rotor. The pellets were dissolved in and dialyzed against 0.01 M Tris-HCI, pH 7.4,O.l M KC1 and 0.0015 M MgClz (Buffer I). The 23 S rRNA was separated from the 5 S rRNA by passing through a lo-30% exponentia...

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