Role of 5S RNA in the Functions of 50S Ribosomal Subunits

Abstract

ABSTRACT50S ribosomal subunits from Bacillus stearothermophilus can be reconstituted from their dissociated components, namely a 5S RNA-free protein fraction, a 5S RNA-free 23S ribosomal RNA fraction, and purified 5S RNA. The biological activity of reconstituted particles in polypeptide synthesis is dependent on the presence of 5S RNA. In the absence of 5S RNA, particles are produced that have greatly reduced activity in (a) polypeptide synthesis directed by synthetic, as well as natural, messenger RNA, (b) peptidyl transferase assay, (c Since 5S RNA was discovered as a component of the large ribosomal subunit (1), many studies have been done on its structure and biosynthesis. The primary sequence of 5S RNA from Escherichia coli was first elucidated by Brownlee, Sanger, and Barell (2). Yet, the function of 5S RNA has remained unknown. Several workers devised methods to remove 5S RNA from 50S subunits and found that removal of 5S RNA led to inactivation of 50S subunits (3-5). However, these workers were unable to restore activity to these inactive particles by adding 5S RNA. Thus, it was not possible to decide whether the inactivation was due to the removal of 5S RNA or to some structural disorganization of the particles caused by the treatment used.We have recently succeeded in reconstituting 50S ribosomal subunits from Bacillus stearothermophilus from their dissociated molecular components (6). Using this reconstitution system, we have now shown that 5S RNA is essential for the overall activity of 50S subunits in polypeptide synthesis. This paper describes information on the function of 5S RNA obtained in this system. MATERIALS AND METHODSThe following buffers are used: TMA-I; 0.01 M Tris HCl (pH 7.4)-0.01 M MgC12-0.03 M NH4Cl-6 mM 2-mercaptoethanol. TMA-II; Same as TMA-I, except 0.3 mM MgCl2. TRI; 0.03 M Tris * HC1 (pH 7.4)-0.02 M MgCl2-1 M KC1-6 mM 2-mercaptoethanol. TRO B. stearothermophilus 799 was grown at 610C to early-log phase in medium L (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 1 g of glucose per liter of H20). The pH is adjusted to 7.0 with NaOH. Cells were ground with two times their weight of alumina at 00C and extracted with five times the cell weight of TMA-I buffer containing 2 Ag/ml of RNase-free DNase. Alumina, unbroken cells, and cell debris were removed by centrifugation, first at 20,000 X g for 20 min, and then 78,000 X g for 30 min, to give the crude extract. Crude 70S ribosomes were obtained by centrifugation of the extract at 78,000 X g for 10 hr. The 70S ribosomes were then dialyzed against TMA-II overnight at 40C and the dissociated ribosomal subunits were separated and purified (7). All ribosomes were stored in TMA-I at -80'C.50S subunits (900 A260/ml in TMA-I) were treated with an equal volume of 8 M urea-4 M LiCl solution at 00C for 48 hr, and then centrifuged at low speed. The RNA pellet was washed once at 0C with a mixture (1:1) of TMA-I and 8 M urea-A M LiCl solution and then dissolved in water ("RNA 50" fraction). This fraction contains 23S RNA with one protein (...

Activities of B. stearothermophilus 50 S ribosomes reconstituted with prokaryotic and eukaryotic 5 S RNA

“…The following bacterial strains were grown in media, as cited by the references, to mid log phase before harvesting: E. coli Al9 [2] …”

“…Ribosomes from wheat germ were prepared as previously described [ 151, whilst Artemia salina ribosomes were a gift from J. Zimmermann. Poly-U directed polyphenylalanine synthesis was measured as previously published [2]. Yeast tRNAPhe was a generous gift from Dr. 0.…”

“…Reconstitution experiments have clearly demonstrated the importance of 5 S RNA as a structural component in the 50 S ribosomal subunit [ 1,2]. 50 S ribosomes reconstituted without 5 S RNA are void of several proteins and show greatly reduced biological activities.…”

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25

“…Its presence in the subunit is essential for ribosomal function (8,9), and it is situated near the interface region which is generally considered to be the main functional centre of the ribosome (10).…”

Structural analysis of 5S rRNA, 5S rRNA‐protein complexes and ribosomes employing RNase H and d(GTTCGG)

“…(c) It was also shown that TYCG can replace uncharged tRNAs at the ribosomal Asite to mediate the stringent-factor-dependent synthesis of pppGpp and ppGpp [37, 381. (d) Reconstitution experiments with B. stearothermopliilus [39] and E. coli [40] 50s ribosomal subunits lacking 5s rRNA revealed, among other deficiencies, a reduction in aminoacyl-tRNA binding to the ribosomal Asite.…”