A Series of Specific Ribonucleoprotein Fragments from the 30‐S Subparticle of Escherichia coli Ribosomes

Abstract

1. 30-5 ribosomal subparticles were hydrolysed with ribonuclease TI or pancreatic ribonuclease in the presence of 2 M urea. The RNA * protein fragments produced were separated on 501, polyacrylamide/0.5 ,Ilo agarose composite gels. Fractions from the composite gels were subjected to protein analysis on 17.5O/, periodate-soluble polyacrylamide gels run in the detergent sarkosyl, using the technique already published.2. Several RNA protein fragments were obtained by this procedure, each containing equimolar amounts of a few specific ribosomal proteins. Strict criteria for specificity of the proteins in each fragment were applied. Proteins 58 and 515 were found reproducibly in a small RNA * protein fragment, with only trace amounts of the remaining ribosomal proteins. Six proteins (57, S9, 510, 513, 514, and S19) constitute another group, and fragments containing three, four, five, or all six of these proteins were observed. Protein S20 was occasionally associated with this group, or with 56, 516 or 517, and possibly 521. The latter (including 520) were also found in association with S8 and 515.3. The individual proteins found in specific fragments are interpreted as being close neighbours in the 30-S particle. The fragment data can be incorporated into a re-arranged version of Nomura's "assembly map". I n our last paper [l] we described a method for the analysis of ribonucleoprotein (RNA * protein) fragments, which we obtained by mild nuclease digestion of Escherichia coli 30-5 ribosomes. The underlying objective of the work was that a precise analysis of small specific ribosomal fragments provides a direct method for examining the arrangement of proteins and RNA within the particle. I n this paper, we apply the technique to a number of RNA .protein fragments, produced under a variety of different hydrolysis conditions, and discuss the results in relation to Nomura's "assembly map" [2,31. MATERIALS AND METHODS Preparation of Ribosomes30-5 ribosomal subparticles were prepared from E. coli MRE 600 (obtained from MRE, Porton, U.K.) as described before [1,4], with the following modificaAbbreviations. RNA * protein, ribonucleoprotein; sarkosyl, N-lauryl sarcosine.Enzymes. T, ribonuclease (EC 2.7.7.26); pancreatic ribonuclease (EC 2.7.7.16).Definition. An A,,, unit is the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-em path length cell.tions. After grinding with alumina [a], the cell paste was extracted with 10 mM MgC1, rather than 0.1 mM, to maintain the ribosomes as 70-5 particles. These 70-5 particles were washed by spinning through 0.5 M NH4Cl, 10 mM MgCl,, 10 mM Tris-HC1 pH 7.6 (cf. [5]), and were then dissociated into subparticles by resuspending in 50mM KC1, 0.3 mM MgCl,, 10mM Tris-HC1 p H 7.6. The subparticles were separated in a zonal rotor as before [I], and the 30-5 ribosomes precipitated with ethanol. The precipitate was dissolved in 0.3 mM magnesium acetate, 10 mM Tris-HC1 pH 7.6, and dialysed against this buffer, or against 1 mM magnesium...

Random exchange of ribosomal proteins in EDTA sub‐particles

“…In order to determine whether some proteins remain specifically bound to the RNA in EDTA, the experiment of fig.2 was repeated (using a five-fold excess of unlabelled sub-particle in each case), and the radioactive proteins in each peak were analysed both on twodimensional gels [ 121 and Sarkosyl gels [7] using the methods we have described [ 131. Obviously, a 50s protein that remained specifically bound would not be found in the 30s peak (cf.…”

“…In particular, several authors have reported the isolation of specific ribo-nucleoprotein fragments from EDTA-treated [3-51 or de-salted [6] particles, which would suggest that the proteins remain specifically located on the RNA. However, in our own studies on specific fragments from E. coli ribosomes, we have been unable to obtain fragments from either the 30s or SOS sub-particles in the presence of EDTA which satisfy our rigorous criteria for specificity [7,8]. This has led us to suspect that in EDTA the proteins have lost their specific sites on the RNA, and in this paper we demonstrate that this is indeed the case, by examining the ability of the proteins to exchange between different ribosomal RNA's or sub-particles.…”

“…Ribosomal sub-particles from E. coli, either unlabelled or labelled with [ 14C] amino acids and [3H] uridine were prepared as described [7].…”

“…Sub-particles and RNA were mixed together at 0°C under various conditions (see text and legends to figures), and were then immediatelyseparated by electrophoresis on a 3% polyacrylamide/agarose gel slab, containing 20 mM KCI, 1 mM magnesium acetate, and 2.5 mM Tris buffer, as described [7]. Samples were run in adjacent 1 cm slots, and electrophoresis was continued until the Bromophenol Blue dye marker had run 6-7 cm.…”

The Use of Formaldehyde in RNA‐Protein Cross‐linking Studies with Ribosomal Subunits from Escherichia coli

“…Ammonium chloride-washed 30-S and 50-S E. coli ribosomal subunits were prepared exactly as described previously [22]. The subunits were either labelled with 14C-labelled amino acids and [3H]uridine to specific activities of 0.6 x lo6 counts min-' (mg protein)-' and 4 x lo6 counts min-' (mg RNA)-' respectively, using E. coli strain A19, or they were labelled with [32P]orthophosphate to a specific activity of approximately lo8 counts min-' (mg RNA)-' using strain MRE 600.…”