Structure of Free Thermus flavus 5 S rRNA at 1.3 nm Resolution from Synchrotron X-ray Solution Scattering

Abstract

The shape of free Thermus flavus 5 S rRNA in solution at 1.3 nm resolution is restored from synchrotron x-ray scattering data using an ab initio simulated annealing algorithm. The free 5 S rRNA is a bent elongated molecule displaying a compact central region and two projecting arms, similar to those of the tRNA. Ribosomal 5 S rRNA, an essential component of the ribosome, is approximately 120 nucleotides long. The ribosomal particles lacking the 5 S rRNA have a strongly reduced activity in protein synthesis (1-3), in particular, reduced peptidyl transferase activity. Because of its functional importance and the fact that the 5 S rRNA interacts specifically with several ribosomal proteins (4), it is of great interest to know the threedimensional structure or a reliable shape of this RNA molecule. Nearly 1000 different prokaryotic and eukaryotic 5 S rRNA sequences have been determined so far. The predicted secondary structure (5) is shown in Fig. 1. The size of the 5 S rRNA limits the possibility of its three-dimensional structure determination by nuclear magnetic resonance (NMR). Therefore, in the past, we tried to crystallize several 5 S rRNA species (6, 7). The crystals of an isolated 5 S rRNA suitable for the x-ray analysis were obtained particularly from the thermophilic bacterium Thermus flavus. These crystals, however, diffract only to about 0.75 nm resolution and are extremely sensitive to radiation, even under cryogenic conditions. The intrinsic flexibility of the whole 5 S rRNA molecule and small differences in the primary structure seem to influence significantly the quality of the crystals. The T. flavus 5 S rRNA was then divided into five domains, A through E, and these domains were chemically synthesized and crystallized for x-ray analysis (8, 9). In parallel, we began to analyze the shape of the entire 5 S rRNA in solution using small angle scattering, an established method of monitoring the low resolution structure of biological macromolecules (10). Recently, new approaches have been developed to ab initio restore low resolution structure from the scattering data (11, 12), and these were successfully applied to study proteins and ribosomes (13-15). In the present paper, an ab initio low resolution model of the free 5 S rRNA in solution is derived from synchrotron radiation small angle x-ray scattering (SAXS) 1 data. The atomic models of the 5 S rRNA fragments are tentatively positioned inside the low resolution shape. The model obtained may be further used for crystallographic molecular replacement studies, as well as for the analysis of the complex formation with binding proteins in solution. EXPERIMENTAL PROCEDURESIsolation and Purification-The Escherichia coli cells from strain MRE600 were grown at 37°C in a Luria broth medium. The 70 S ribosomes were isolated by previously described extraction and centrifugation steps (2). The intact ribosomes were dissociated into their 30 and 50 S subunits at magnesium concentrations below 5 mM (1). The 5 S rRNA was separated from the large ribosomal subunit b...

Structural similarity of E. coli 5S rRNA in solution and within the ribosome

“…17 Similar conclusions can be drawn from a comparison of the general size and shape of a free 5S rRNA molecule with the results of a study on the 5S rRNA molecule bound with the ribosome. 8 Reliable information on the effect of the interactions with the ribosomal proteins on the structure of the 5S rRNA molecule will be obtained from the x-ray dif-FIGURE 1 A general model of the secondary structure of procaryotic 5S rRNA based on crystal structure of H. marismortui 5S rRNA within ribosome. 3 fraction study of the free 5S rRNA and its complexes with proteins out of the ribosome.…”

“…It could not be solved on the basis of the x-ray diffraction data because of insufficient quality of the crystals grown, or on the basis of NMR study because of its size (molecular mass of approximately 40 kDa). 8 However, data for the spatial structure of fragments of 5S rRNA had been collected. Analysis of the experimental data in combination with the results of computer simulations has led to the first three-dimensional structural model of the ribosomal 5S rRNA from X. laevis 9 published in PDB.…”

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

“…Although the tertiary structure of 5S rRNA has been determined for the free molecule (Funari et al 2000;Lorenz et al 2000) and its isolated domains (Betzel et al 1994;Xiong and Sundaralingam 2000;Huber et al 2001), and as a part of a ribosomal complex (Ban et al 2000;Harms et al 2001;Spahn et al 2001;Yusupov et al 2001), its precise function in protein synthesis is not fully understood. Biochemical studies with ribosomes from Escherichia coli led to the hypothesis that 5S rRNA acts to facilitate communication between the different functional centers, helping to coordinate the multiple events catalyzed by the ribosome (Bogdanov et al 1995;Dokudovskaya et al 1996), and this view was further supported by a later study of yeast ribosomes (Smith et al 2001).…”

Modelling of bacteriophage capsids and free nucleic acids

“…7. The ab initio approaches clearly establish the bent structure of RNA and an uneven surface, similar to the DAMMIN models found for rRNA (Funari et al, 2000) Anhydrous and hydrated three-dimensional models of capsids, together with their central slabs. The 23 220 AA residues (grey) of the anhydrous model (A) were constructed on the basis of 1FRS.…”