Ribosomal protein L7/L12 cross-links to proteins in separate regions of the 50 S ribosomal subunit of Escherichia coli

The 50 S ribosomal subunits from Escherichia coli were modified by reaction with 2-iminothiolane under conditions in which 65 sulfhydryl groups, about 2/protein, were added per subunit. Earlier work showed that protein L7/L12 was modified more extensively than the average but that nearly all 50 S proteins contained sulfhydryl groups. Mild oxidation led to the formation of disulfide protein-protein cross-links. These were fractionated by urea gel electrophoresis and then analyzed by diagonal gel electrophoresis. Cross-linked complexes containing two, three, and possibly four copies of L7/L12 were evident. Cross-links between L7/L12 and other ribosomal proteins were also formed. These proteins were identified as L5, L6, L10, L11, and, in lower yield, L9, L14, and L17. The yields of cross-links to L5, L6, L10, and L11 were comparable to the most abundant cross-links formed. Similar experiments were performed with 70 S ribosomes. Protein L7/L12 in 70 S ribosomes was cross-linked to proteins L6, L10, and L11. The strong L7/L12-L5 cross-link found in 50 S subunits was absent in 70 S ribosomes. No cross-links between 30 S proteins and L7/L12 were observed.     

Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: separation and identification of contact sequences of neighboring proteins after CNBr fragmentation

Mammalian ribosomal proteins were cross-linked in situ with the primarily cysteine-selective heterobifunctional reagents N-succinimidyl 2-(4-hydroxy-2-maleimidophenylazo)benzoate (reagent A, maximum range approx. 8 A) and N-succinimidyl 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoate (reagent B, maximum range approx. 12 A). With reagent B the secondarily attached (N-aryolated) protein becomes labelled specifically at the receptor amino group (lysine). The cross-linked proteins were fragmented with CNBr in attempts to isolate and identify sequences involved in the next-neighbor contacts. Two experimental schemes were adopted. Heavy complexes containing the large protein L4 cross-linked to protein L14 and/or L18 were isolated and treated with CNBr. The split products were submitted to diagonal electrophoresis for separation and identification of the two pairs of contact fragments. Proteins cross-linked with the radiolabelled reagent B were submitted to diagonal electrophoresis. The labelled receptor proteins were excised and treated with CNBr. Fragments carrying the contact sequences were separated by gradient gel electrophoresis and identified by autoradiography. By use of these methods CNBr fragments were isolated containing one or the dual contact sites of the following binary protein complexes: L4-L14, L4-L18, L4-L13a/L18a, L6'-L23, L6-L29, L7-L29, L14-L13a, L21-L18a, and L27-L30 (asterisks indicate the labelled receptor proteins). By varying the site of labelling of the heterobifunctional reagents and the methods of protein fragmentation a complete analysis of the contact sequences of these proteins should be possible.     

Determination of the disulfide structure of sillucin, a highly knotted, cysteine-rich peptide, by cyanylation/cleavage mass mapping

The disulfide structure of sillucin, a highly knotted, cysteine-rich, antimicrobial peptide, isolated from Rhizomucor pusillus, has been determined to be Cys2--Cys7, Cys12--Cys24, Cys13--Cys30, and Cys14--Cys21 by disulfide mass mapping based on partial reduction and CN-induced cleavage enabled by cyanylation. The denatured 30-residue peptide was subjected to partial reduction by tris(2-carboxyethyl)phosphine hydrochloride at pH 3 to produce a mixture of partially reduced sillucin species; the nascent sulfhydryl groups were immediately cyanylated by 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate. The cyanylated species, separated and collected during reversed phase high-performance liquid chromatography, were treated with aqueous ammonia, which cleaved the peptide chain on the N-terminal side of cyanylated cysteine residues. The CN-induced cleavage mixture was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry before and after complete reduction of residual disulfide bonds in partially reduced and cyanylated species to mass map the truncated peptides to the sequence. Because the masses of the CN-induced cleavage fragments of both singly and doubly reduced and cyanylated sillucin are related to the linkages of the disulfide bonds in the original molecule, the presence of certain truncated peptide(s) can be used to positively identify the linkage of a specific disulfide bond or exclude the presence of other possible linkages.     

Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome--protein kinase systems from rat cerebral cortex.

Ribosomal protein phosphorylation was investigated in isolated ribosomal subunits and polyribosomes from rat cerebral cortex in the presence of [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Ribosomal proteins that were most readily phosphorylated in isolated cerebral ribosomal subunits included proteins S2, S3a, S6 and S10 of the 40 S subunit and proteins L6, L13, L14, L19 and L29 of the 60 S subunit. These proteins were also phosphorylated in cellular preparations of rat cerebral cortex in situ or in vitro [Roberts & Ashby (1978) J. Biol. Chem. 253, 288-296; Roberts & Morelos (1979) Biochem. J. 184, 233-244]. However, several additional ribosomal proteins were phosphorylated when isolated 40 S or 60 S subunits were separately incubated in the reconstituted system. Analogous results were obtained with an equimolar mixture of cerebral 40 S and 60 S subunits under comparable conditions. In contrast, extensive exposure of purified cerebral polyribosomes to the catalytic subunit resulted in phosphorylation of only those ribosomal proteins of the 40 S subunit that were most highly labelled after the administration of [32P]Pi in vivo: proteins S2, S6 and S10. Ribosomal proteins of 60 S subunits that were readily phosphorylated in isolated cerebral polyribosomes included proteins L6, L13 and L29. These results indicate that polyribosome formation markedly decreases the number of ribosomal protein sites available for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. Moreover, the findings suggest that, of the ribosomal protein phosphorylations observed in rat cerebral cortex in vivo, proteins S2, S6, S10, L6, L13 and L29 can be phosphorylated in polyribosomes, whereas proteins S3a, S5, L14 and L19 may become phosphorylated only in free ribosomal subunits.     

Synthesis and application of two reagents for the introduction of sulfhydryl groups into proteins

Two reagents are described which can be used for the introduction of sulfhydryl groups into proteins. Mercaptopropionylhydrazide modifies specifically periodate-oxidized N termini of proteins, provided that the N-terminal residue is serine or threonine. 3-(Phenyldithio)propionimidate introduces a disulfide bond at lysine residues of proteins. Reduction converts the disulfide into a sulfhydryl group. The imidate compound was found to react with a high specificity with only one lysine residue of ribosomal protein L7/L12.     

A novel methodology for assignment of disulfide bond pairings in proteins.

A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, beta-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.     

Characterization and primary structure of proteins L28, L33 and L34 from Bacillus stearothermophilus ribosomes.

The complete amino acid sequences of 3 proteins from the 50S subunit of Bacillus stearothermophilus ribosomes were determined by N-terminal sequence analysis and by sequencing of overlapping fragments obtained from enzymatic digestions and chemical cleavages. The proteins BstL28, BstL33 and BstL34, named according to the equivalent proteins in Escherichia coli ribosomes, consist of 60, 49, and 44 amino acid residues and have calculated molecular masses of 6811.0, 5908.6, and 5253.9 Da, respectively. They are highly basic with a content of positively charged residues ranging between 29% for L33 and 45% for L34. The 3 proteins were positioned in the 2-dimensional map of B stearothermophilus 50S ribosomal proteins. The electrophoretic mobilities confirm sizes and net charges deduced from the sequences.     

YmL9, a nucleus-encoded mitochondrial ribosomal protein of yeast, is homologous to L3 ribosomal proteins from all natural kingdoms and photosynthetic organelles.

The nuclear gene for mitochondrial ribosomal protein YmL9 (MRP-L9) of yeast has been cloned and sequenced. The deduced amino acid sequence characterizes YmL9 as a basic (net charge + 30) protein of 27.5 kDa with a putative signal peptide for mitochondrial import of 19 amino acid residues. The intact MRP-L9 gene is essential for mitochondrial function and is located on chromosome XV or VII. YmL9 shows significant sequence similarities to Escherichia coli ribosomal protein L3 and related proteins from various organisms of all three natural kingdoms as well as photosynthetic organelles (cyanelles). The observed structural conservation is located mostly in the C-terminal half and is independent of the intracellular location of the corresponding genes [Graack, H.-R., Grohmann, L. & Kitakawa, M. (1990) Biol. Chem. Hoppe Seyler 371, 787-788]. YmL9 shows the highest degree of sequence similarity to its eubacterial and cyanelle homologues and is less related to the archaebacterial or eukaryotic cytoplasmic ribosomal proteins. Due to their high sequence similarity to the YmL9 protein two mammalian cytoplasmic ribosomal proteins [MRL3 human and rat; Ou, J.-H., Yen, T. S. B., Wang, Y.-F., Kam, W. K. & Rutter, W. J. (1987) Nucleic Acids Res. 15, 8919-8934] are postulated to be true nucleus-encoded mitochondrial ribosomal proteins.     

A mutant from Escherichia coli which lacks ribosomal proteins S17 and L29 used to localize these two proteins on the ribosomal surface

A mutant of Escherichia coli has been isolated which lacked ribosomal proteins S17 and L29, as judged by two-dimensional gel electrophoresis. A battery of immunological tests was used to confirm this result. Ribosomes of this mutant were used as a control for the localization of proteins S17 and L29 on the surface of the ribosomal subunits of E. coli. Protein S17 has been localized on the 30S subunit body, 3-5 nm away from the lower pole, while protein L29 is located at the back of the 50S particle on the opposite side to the interface.     

Disulfide interaction in situ between two neighbouring proteins in mammalian 60-S ribosomal subunits. Isolation of the contact region of the larger protein.

A disulfide complex is formed in situ under gentle conditions between two neighbouring proteins in the 60-S subunits of mammalian ribosomes. The proteins have been identified as L 4 and L 29. The complex is easily isolated from whole ribosomes, and can be utilized for preparing the two proteins in a very pure state for further characterization. Chymotryptic cleavage of the complex or the isolated larger protein (L 4) in the presence of SDS produces two unequal fragments of this protein in nearly quantitative yield. The smaller fragment (approx. 12 000 daltons) contains the contact sequence. Only this fragment of protein L 4 is labelled when rat liver ribosomes are incuabted with iodo[14C]acetate under conditions of complex formation. Protein L 29 is resistant to chymotrypsin in the presence of sodium dodecyl sulfate.     

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.     

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes

Protein constituents at the subunit interface of rat liver ribosomes were analysed by cross-linking with the bifunctional reagent, diepoxybutane (distance between reactive groups 4 A). Isolated 40S and 60S subunits were labelled with 125I and recombined with unlabelled complementary subunits. The two kinds of selectively labelled 80S ribosomes were treated with diepoxybutane at low concentration. Radioactive ribosomal proteins covalently attached to the rRNA of the unlabelled complementary subparticles were isolated by repeated gradient centrifugation. The RNA-bound, labelled proteins were identified by two-dimensional gel electrophoresis. The experiments showed that proteins S2, S3, S4, S6, S7, S13, and S14 in the small subunit of rat liver ribosomes are located at the ribosomal interface in close proximity to 28S rRNA. Similarly, proteins L3, L6, L7, and L8 were found at the the interface of the large ribosomal subunit in the close vicinity of 18S rRNA.     

5S rRNA-recognition module of CTC family proteins and its evolution

The effects of amino acid replacements in the RNA-binding sites of homologous ribosomal proteins TL5 and L25 (members of the CTC family) on ability of these proteins to form stable complexes with ribosomal 5S RNA were studied. It was shown that even three simultaneous replacements of non-conserved amino acid residues by alanine in the RNA-binding site of TL5 did not result in noticeable decrease in stability of the TL5-5S rRNA complex. However, any replacement among five conserved residues in the RNA-binding site of TL5, as well as of L25 resulted in serious destabilization or complete impossibility of complex formation. These five residues form an RNA-recognition module in TL5 and L25. These residues are strictly conserved in proteins of the CTC family. However, there are several cases of natural replacements of these residues in TL5 and L25 homologs in Bacilli and Cyanobacteria, which are accompanied by certain changes in the CTC-binding site of 5S rRNAs of the corresponding organisms. CTC proteins and specific fragments of 5S rRNA of Enterococcus faecalis and Nostoc sp. were isolated, and their ability to form specific complexes was tested. It was found that these proteins formed specific complexes only with 5S rRNA of the same organism. This is an example of coevolution of the structures of two interacting macromolecules.     

The complex between ribosomal proteins and aminoacyl-tRNA: the interactions and hydrolytic activities are not confined to the proteins L2 and L16 of Escherichia coli ribosomes

The capacity of some Escherichia coli (E. coli) ribosomal proteins to bind to tRNA and to hydrolyse their aminoacylated derivatives has been analysed. The following results were obtained: (1) The basic proteins L2, L16 and L33 and S20 bound f[3H]Met-tRNA to a similar extent as the total proteins from 30 S (TP30) or 50 S (TP50) when tested by nitrocellulose filtration, in contrast to the more acidic proteins L7/L12 and S8. (2) The proteins of the peptidyltransferase centre, L2 and L16, showed no distinct specificity, binding various charged tRNAs from E. coli and Saccharomyces cerevisiae (S. cerevisiae). (3) A number of isolated ribosomal proteins hydrolysed aminoacyl-tRNA as assessed by trichloroacetic acid precipitation, in contrast to the TP30 and TP50. (4) The loss of radiolabel from Ac[14C]Phe-tRNA and from [14C]tRNA in the presence of these proteins could not be prevented by RNasin, a ribonuclease inhibitor, whereas that mediated by a sample of non-RNase-free bovine serum albumin was inhibited. (5) When double-labelled, Ac[3H]Phe-[14C]tRNA was incubated with L2 both radiolabels were lost, indicating that this potential candidate for a peptidyltransferase enzyme does not specifically cleave the ester bond between the aminoacyl residue and the tRNA.     

The complete primary structure of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus

The amino acid sequences of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus have been completely determined. This has been achieved by sequence analyses of peptides derived from enzymatic digestions of the proteins with trypsin, chymotrypsin, pepsin, Staphylococcus aureus protease, and Armillaria mellea protease as well as by chemical cleavage with hydroxylamine and cyanogen bromide. Based on the primary structures of the six proteins, their secondary structures were predicted using four different computer prediction programs. A comparison of the amino acid sequences of the studied proteins from B. stearothermophilus with the homologous proteins from Escherichia coli revealed that in four proteins (L1, L15, L24 and L29) between 40-50% of the residue in the sequences are identical, whereas this value is significantly higher (69%) for L14 and lower (28%) for L23. The distribution of those amino acid residues which are identical in the corresponding proteins from the two bacteria is not random along the protein chain: some regions are highly conserved whereas others are not. This finding indicates that the regions which are conserved during evolution are important for the spatial structure and/or function of the protein.     

Methylation of ribosomal proteins in Bacillus subtilis

We measured the methylation of ribosomal proteins from the 30S and 50S subunits of Bacillus subtilis after growing the cells in the presence of [1-14C]methionine and [methyl-3H]methionine. Two-dimensional polyacrylamide gel electrophoretic analysis revealed a preferential methylation of the 50S ribosomal proteins. Proteins L11 and L16, and possibly L9, L10, L18, and L20, were methylated. On the other hand, only two possibly methylated proteins were found on the 30S subunit. A comparison of these results with those for Escherichia coli suggests a common methylation pattern for the bacterial ribosomal proteins.     

Studies on proteins of animal ribosomes XXIV. Localisation of proteins in ribosomal subunits of rat liver studied by chemical substitution with p-nitrophenyl acetate and methyl acetimidate

The reaction of [³H]p-nitrophenyl acetate (NPA) or [¹⁴C]methyl acetimidate (MAI) with amino groups of ribosomal proteins from the rate has been studied.A comparison has been made between the reactivity of the proteins in situ in the ribosomal subunit with that of isolated protein mixtures.In the small subunit reactivity compared with the protein mixture was only 10–65% in the case of NPA but 45 to more than 100% in the case of MAI.In the large subunit reactivity to MAI was 10–60% that of the isolated protein mixture. This suggests that the large subunit has a denser structure than the small one.In agreement with earlier experiments with iodoacetamide the proteins S2, 5, 7, 8, 10 and 13 of the small subunit and L15, 17, 20, 24, 25, 27, 29, 33, 34, 35 and 38 in the large subunit are quite accessible while proteins S9, 14, 19, 20, 24, 25, 27, 29 and 30 of the small subunit and L1, 7, 8, 10, 11, 19, 28, 31 and 32 of the large one are relatively inaccessible.     

Nucleotide sequence and linkage map position of the genes for ribosomal proteins L14 and S8 in the maize chloroplast genome

The nucleotide sequence of a 1287-base-pair segment of the maize (Zea mays) chloroplast DNA, encoding chloroplast ribosomal proteins L14, S8 and the C-terminal part of L16, has been determined using the dideoxy-chain-termination method. These data from a monocot plant are compared to the corresponding data from a dicot and a lower plant and from two bacteria. The deduced amino acid sequence of maize chloroplast L14 shows 80%, 81%, 51% and 52% and that of S8 shows 75%, 58%, 39% and 38% sequence identity, respectively, to the corresponding sequences of Nicotiana tabacum, Marchantia polymorpha, Bacillus stearothermophilus and Escherichia coli. The starting map coordinates of rpL14 and rpS8 in the physical map of the maize chloroplast DNA [Larrinua, I. M., Muskavitch, K. M. T., Gubbins, E. J. and Bogorad, L. (1983) Plant Mol. Biol. 2, 129-140] are 31.330 and 31.841. The gene order is rpL16-spacer-rpL14-spacer-rpS8. Shine-Dalgarno sequences (GGA and AGGAGG) and computer-derived stem-loop structures of dyad symmetry are present in the spacers and the 3' downstream region of rpS8, respectively, but a chloroplast promoter-like sequence could not be detected suggesting that the latter might be located further upstream in this ribosomal protein gene cluster in maize chloroplast DNA.     

Covalent binding of 4-nitrobenzyl mercaptan S-sulfate to the sulfhydryl groups of hepatic cytosolic proteins and bovine serum albumin with mixed disulfide bond formation

4-Nitrobenzyl [35S]mercaptan S-sulfonic acid ([35S]NBM S-sulfate), a new type of reactive metabolite of the thiol [35S]NBM in rat liver cytosol fortified with 3'-phosphoadenosine 5'-phosphosulfate, bound rapidly and covalently at pH 7.4 and 37 degrees C to the sulfhydryl groups of rat liver cytosolic proteins with formation of disulfide bonds. From the radioactive proteins was isolated and identified the sole amino acid adduct, S-([35S]NBM)cysteine, after their acid hydrolysis under the anaerobic conditions. Bovine serum albumin (BSA), a model protein with a single SH group, also reacted readily with radioactive NBM S-sulfate to form a disulfide bond in stoichiometric manner. S-([35S]NBM)-cysteine was also isolated and identified as the sole amino acid adduct from the well-washed, radioactive BSA after the same anaerobic acid hydrolysis. A normal hepatic level of GSH not only retarded the BSA-NBM adduct formation completely, but also detached the radioactivity from BSA by the reduction of the disulfide bond with formation of [35S]NBM and its disulfide. Of twenty-one amino acids examined at pH 7.4 and 37 degrees C, only cysteine reacted with NBM S-sulfate and afforded S-(NBM)cysteine with concomitant formations of S-sulfocysteine, cystine, NBM, and its disulfide.