Robert R. Traut

Proposed Uniform Nomenclature for Mammalian Ribosomal Proteins

SummaryThe numbering systems for mammalian ribosomal proteins used in several laboratories have been correlated and a proposal for a standard system is presented.

Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3′‐ends of the tRNAs at the peptidyl‐transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl‐transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.

Identification of neighboring protein pairs in the Escherichia coli 30 S ribosomal subunit by crosslinking with methyl-4-mercaptobutyrimidate.

Abstract The 30 S ribosomal subunits of Escherichia coli were treated with methyl-4-mercaptobutyrimidate and oxidized to promote the formation of intermolecular disulfate bonds between neighboring proteins. Attention was focused on protein dimers, which were partially purified either by stepwise extraction of the 30 S particle with LiCl or by polyacrylamide/urea gel electrophoresis of the total crosslinked protein. Protein fractions were then analyzed by polyacrylamide/ sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the components of crosslinked protein pairs, indicated by molecular weight analysis, was accomplished by two-dimensional polyacrylamide/urea gel electrophoresis. The identification of 21 protein pairs is presented, 14 of which have not been reported previously.

Ribosomal proteins from rabbit reticulocytes: Number and molecular weights of proteins from ribosomal subunits

Abstract The ribosomal proteins from 40 S and 60 S subunits of rabbit reticulocytes were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots stained with Coomassie brilliant blue were cut out and the proteins were extracted. The material extracted from each spot was mixed with proteins of known molecular weight and then analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Both the total number and the molecular weights of each of the proteins were determined by these procedures. Thirty-two proteins were identified in the 40 S subunits; their molecular weights ranged from 8000 to 39,000 (average mol. wt = 25,000). Thirty-nine proteins were identified in the 60 S subunit; their molecular weights ranged from 9000 to 58,000 (average mol. wt = 31,000). The sum of the molecular weights of the individual proteins from each subunit is in agreement with previous estimations, derived from physico-chemical measurements of the total protein in mammalian ribosomal subunits. The molecular weight distribution obtained for the isolated proteins was nearly identical to that derived from spectrophotometric analysis of polyacrylamide-sodium dodecyl sulfate gels of the total protein mixtures from each subunit stained with Coomassie brilliant blue. The results are consistent with the hypothesis that reticulocyte ribosomes contain one copy of most of their protein constituents.

[43] Cross-linking of ribosomes using 2-iminothiolane (methyl 4-mercaptobutyrimidate) and identification of cross-linked proteins by diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis☆

Publisher Summary This chapter discusses the cross linking of ribosomes and identification of cross-linked proteins by gel electrophoresis. The presence of a readily cleavable bond in the cross-linking reagent permits reversal of the cross-linking reaction and regeneration of monomeric components from isolated cross-linked complexes, thus facilitating their identification. Methods are described in the chapter that employ reversible cross-linking and analysis of a complex mixture of cross-linked products. The reagent 2-iminothiolane, formerly called methyl 4-mercaptobutyrimidate reacts with lysine amino groups in the intact ribosomal subunit to form amidine derivatives containing sulfhydryl groups. The cross-linked subunits retain the capacity to reassociate to form 70 S ribosomes and retain up to 50% of their activity in polyphenylalanine synthesis. Methods for the separation and identification of cross-linked dimers are also described. Of particular general applicability is the technique of diagonal polyacrylamide/sodium dodecyl sulfate (SDS) gel electrophoresis. It is a two-dimensional electrophoretic separation, utilizing the size dependence of the mobility of proteins in SDS to distinguish cross-linked from monomeric proteins. The first electrophoresis is performed under nonreducing conditions, and the second under reducing conditions. This results in a pattern in which non-cross-linked proteins fall on a diagonal line and cross-linked proteins fall beneath the diagonal.

Cross-linking of initiation factor IF3 to proteins of the Escherichia coli 30 S ribosomal subunit☆

Abstract Complexes of 30 S subunits and [14C]IF3 were allowed to react with the protein cross-linking reagents, N,N′-p-phenylenedimaleimide or dimethylsuberimidate. Non-cross-linked IF3 was removed from the complex by centrifugation in a buffer containing a high salt concentration, and the total protein was extracted from the pelleted particles. The mixture of cross-linked products was analyzed by radioimmunodiffusion with antisera prepared against all of the individual 30 S ribosomal proteins. Radioactivity was found in the precipitin bands formed with antisera against ribosomal proteins S1, S11, S12, S13, S19 and S21. The results show that IF3 was present in covalent cross-linked complexes containing those 30 S ribosomal proteins and imply that they comprise or are near the binding site for initiation factor IF3.

Identification by diagonal gel electrophoresis of nine neighboring protein Pairs in the Escherichia coli 30 S ribosome crosslinked with methyl-4-mercaptobutyrimidate

The 30 S ribosomal subunits of Escherichia coli were allowed to react with methyl-4-mercaptobutyrimidate under conditions in which amidine linkages were formed between lysine e-amino groups in ribosomal proteins and the imidate group of the bifunctional reagent. Cross-linking between sulfhydryl groups close enough to form intermolecular disulfide bonds was promoted by mild oxidation of the modified ribosomes. Crosslinked protein-protein dimers were separated by diagonal electrophoresis in polyacrylamide/dodecyl sulfate gels. Disulfide linked dimers were first separated by electrophoresis in the oxidized form in one dimension, then cleaved by mild reduction to regenerate their monomeric components prior to electrophoresis in the second dimension. Preliminary identification of the paired proteins was obtained from the molecular weights of the monomeric and crosslinked species. Final identification of the proteins in each dimer was established by electrophoresis of the monomeric proteins in a two-dimensional polyacrylamide gel electrophoresis system containing urea. The following nine dimers, five of which have not been reported previously, were identified: S2–S3, S4–S6, S4–S8, S4–S9, S4–S12, S5–S8, S5–S9, S7–S8 and S7–S9.

Cryo-electron microscopic localization of protein L7/L12 within the Escherichia coli 70 S ribosome by difference mapping and Nanogold labeling.

The Escherichia coliribosomal protein L7/L12 is central to the translocation step of translation, and it is known to be flexible under some conditions. The assignment of electron density to L7/L12 was not possible in the recent 2.4 Å resolution x-ray crystallographic structure (Ban, N., Nissen, P., Hansen, J., Moore, P. B., and Steitz, T. A. (2000)Science 289, 905–920). We have localized the two dimers of L7/L12 within the structure of the 70 S ribosome using two reconstitution approaches together with cryo-electron microscopy and single particle reconstruction. First, the structures were determined for ribosomal cores from which protein L7/L12 had been removed by treatment with NH4Cl and ethanol and for reconstituted ribosomes in which purified L7/L12 had been restored to core particles. Difference mapping revealed that the reconstituted ribosomes had additional density within the L7/L12 shoulder next to protein L11. Second, ribosomes were reconstituted using an L7/L12 variant in which a single cysteine at position 89 in the C-terminal domain was modified with Nanogold (Nanoprobes, Inc.), a 14 Å gold derivative. The reconstruction from cryo-electron microscopy images and difference mapping placed the gold at four interfacial positions. The finding of multiple sites for the C-terminal domain of L7/L12 suggests that the conformation of this protein may change during the steps of elongation and translocation.

A new two-dimensional gel electrophoresis system for the analysis of complex protein mixtures: application to the ribosome of E. coli.

A new method for two-dimensional polyacrylamide gel electrophoresis of proteins is described. The method, illustrated here by its application for the analysis of ribosomal proteins of E. coli, has a high resolving power. The proteins S15 and S16 can be resolved either following alkylation or under reducing conditions. This was not possible with urea gel systems previously employed. The method should be advantageous in the identification of the components of dimers formed with the reagent methyl 4-mercaptobutyrimidate. An additional advantage of the new method is that both dimensions are run at an acidic pH. For ribosomal proteins it is therefore unnecessary to either polymerize the protein sample in the middle of the first dimension disc gel or to electrophorese two samples with opposite polarity.

Cross-linking of Selected Residues in the N- and C-terminal Domains of Escherichia coli Protein L7/L12 to Other Ribosomal Proteins and the Effect of Elongation Factor Tu

Five different variants of protein L7/L12, each with a single cysteine substitution at a selected site, were produced, modified with125I-N-[4-(p-azidosalicylamido)-butyl]-3-(2′-pyridyldithio)propionamide, a radiolabeled, sulfhydryl-specific, heterobifunctional, cleavable photocross-linking reagent that transfers radiolabel to the target molecule upon reduction of the disulfide bond. The proteins were reconstituted with core particles depleted of wild type L7/L12 to yield 70 S ribosomes. Cross-linked molecules were identified and quantified by the radiolabel. No cross-linking of RNA was detected. Two sites in the dimeric N-terminal domain, Cys-12 and Cys-33, cross-linked strongly to L10 and in lower yield to L11 but to no other proteins. The three sites in the globular C-terminal domain all cross-linked strongly to L11 and, in lower yield, to L10. Weaker cross-linking to 50 S proteins L2 and L5 occurred from all three C-terminal domain locations. The 30 S ribosomal proteins S2, S3, S7, S14, S18 were also cross-linked from all three of these sites. Binding of the ternary complex [14C]Phe-tRNA·elongation factor Tu·guanyl-5′-yl imidodiphosphate) but not [14C]Phe-tRNA·elongation factor Tu·GDP·kirromycin increased labeling of L2, L5, and all of the 30 S proteins. These results imply the flexibility of L7/L12 and the transient proximity of three surfaces of the C-terminal domain with the base of the stalk, the peptidyl transferase domain, and the head of the 30 S subunit.