The Identification of the Eukaryotic Ribosomal Proteins Homologous with Escherichia coli Proteins L7 and L12

Abstract

Antibodies were raised against eukaryotic (rat liver) and prokaryotic (E. coli) ribosomal particles and ribosomal proteins. The antisera were characterized and used to determine the identity of the eukaryotic proteins homologous to E. coli L7 and L12. The large subunit of rat liver ribosomes contains two acidic proteins, L40 and L41; they migrate during two-dimensional polyacrylamide gel electrophoresis in a way that mimics the behavior of L7 and L12. Rat liver L40 and L41 were found to be immunologically related to E. coli L7/L12, hence the proteins are likely to share some structural homology.The large subunits of Escherichia coli and other prokaryotic ribosomes contain a pair of acidic proteins which have been designated L7 and L12 from their positions on two-dimensional electropherograms (1). The proteins have been characterized and their primary sequences determined (2-7); L7 differs from L12 only in that its N-terminal serine is acetylated. There is information also on the function of the proteins, which seem essential for the binding of factors during initiation of protein synthesis and during the elongation and termination reactions (8)(9)(10)(11)(12)(13)(14).There are also two acidic proteins in rat liver 60S ribosomal subunits and they have electrophoretic properties similar to E. coli L7 and L12; the eukaryotic proteins have been designated L40 and L41 (15). We decided to attempt to determine if L7 and L12 had been conserved during evolution from prokaryotes to eukaryotes, and especially if E. coli L7 and L12 were homologous with rat liver L40 and L41. For this purpose antibodies raised against prokaryotic (E. coli) and eukaryotic (rat liver) ribosomal proteins were used. MATERIALS AND METHODSSeparation ofRibosomes and Ribosomal Particles. The following have been described before: the preparation of liver ribosomes (16) and ribosomal subunits (16, 17) from male SpragueDawley rats; the preparation of ribosomes (18) and ribosomal subunits (18, 19) from E. coli strain K12-A19 (20).Abbreviations: TP, total proteins-used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixture of all the proteins obtained from 80S ribosomes; A, antiserum or antibodies-A-TP40 is an antiserum (or antibody preparation) raised against a mixture of the proteins obtained from 40S ribosomal subunits; L7/L12, the structures of L7 and L12 are nearly identical (the N-terminal serine of L7 is acetylated), the notation, therefore, refers to a protein which is in either form; L40/ L41 is used in the same way to refer to two proteins of the large subunit of rat liver ribosomes although their primary sequence is not known.Separation of Ribosomal Protein. Proteins were extracted from ribosomes and ribosomal subunits with 67% acetic acid (16,21,22) or with 4 M urea-2 M LiCl (23). Individual E. coli ribosomal proteins were isolated and purified by carboxymethyl-cellulose chromatography and filtration through Sephadex G-100 (18, 24). Rat liver ribosomal proteins L40 and L41 were prepared by treating 2000 A2...

Acidic Phosphoproteins of the 60‐S Ribosomal Subunits from HeLa Cells

“…From the following common features: (a) migration to about the same position on two-dimensional polyacrylamide gel electrophoresis; (b) molecular weight; (c) characteristic faint staining by Coomassie blue, we conclude that the proteins L40a, L40b and L40c from HeLa 60-S ribosomal subunits correspond to acidic proteins EL7/EL12 of Artemia salina [6], L35/L36 of Saccharomyces cerevisiae [8] and probably to L40/L41 of rat liver [4,18]. It has been assumed that these proteins are homologous to the proteins L7/L12 of Escherichiu coli ribosomes [4,6,8,18].…”

“…It has been assumed that these proteins are homologous to the proteins L7/L12 of Escherichiu coli ribosomes [4,6,8,18].…”

“…These two proteins are engaged in several steps of peptide synthesis (for review, see [3]). Eukaryotic 60-S ribosomes possess a pair of acidic proteins of similar structure and function [4,5] and it has been suggested that their charge differences might also be due to a side-chain modification [6].…”

Peptidyl Transferase Center of Rat-Liver Ribosome Cores

“…However these authors do not identify them with L40/41, which according to them have a less acidic character and move behind the A1 -A4 series. In our case the release by 1 M ammonium chloride and ethanol, indicates that these proteins might be similar to L7/12 of E. coli ribosome and if so, equivalent to those originally named L40/41 [6].…”

“…Thus only proteins L7 and L12 are released when the treatment is performed at 0 "C, yielding particles inactive in elongation-factordependent functions [l 1. In addition higher temperatures (37 "C) almost totally remove proteins L10, L11 and partially proteins L1, L5, L8/9 and L25 [2-51. In the case of mammalian organisms the presence of proteins of similar characteristics to L7 and L12 has been detected in the ribosomes of rat liver by using immunological techniques [6]. However, no attempt has been reported for preparing protein-deficient particles derived from mammalian ribosomes using the ammonium chloride/ethanol method.…”