The localization of protein L19 on the surface of 50 S subunits of Escherichia coli aided by the use of mutants lacking protein L19

Three independently isolated mutants of Escherichia coli which apparently lacked protein L19 on their ribosomes, as judged by two-dimensional gels, were analyzed by a range of immunological tests to determine if the protein was indeed lacking. In two of the three, all the tests indicated that protein L19 was absent from both ribosome and supernatant. In the third, a drastically altered form of protein L19 was present on the ribosome. Electron micrographs of ribosomes obtained from the mutants were indistinguishable from those of wild type strains. The location of ribosomal protein L19 on the surface of the large subunit was determined. It was situated at the base of the 50 S particle facing the small subunit, on the side where the rod like appendage originates.     

Characterization of methylated neutral amino acids from Escherichia coli ribosomes.

The methylated neutral amino acids from both 30S and 50S ribosomal subunits of an Escherichia coli K strain were characterized. The 50S ribosomal subunit contains three methylated neutral amino acids: N-monomethylalanine, N-monomethylmethionine, and an as yet unidentified methylated amino acid found in protein L11. Both N-monomethylalanine and N-monomethylmethionine were found in protein L33. The amount of N-monomethylmethionine in this protein, however, is variable but not more than 0.25 molecules per protein. Thus protein L33 from this E. coli K strain has heterogeneity in its N-terminal amino acid and can start with either N-monomethylalanine or N-monomethylmethionine. The N-monomethylmethionine residue was not derived from the reduction of N-formylmethionine in the protein. The 30S ribosomal subunit contains only one methylated neutral amino acid: N-monomethylalanine.     

Identification of tyrosine residues that are susceptible to lactoperoxidase-catalyzed iodination on the surface of Escherichia coli 50s ribosomal subunits or 70s ribosomes

Further to our studies on the Escherichia coli 30S ribosomal subunit, the detailed surface topography of both 50S subunits and 70S ribosomes has been investigated by using iodination catalyzed by immobilized lactoperoxidase as the surface probe. In the 50S subunit, only proteins L2, L5, L10, and L11 were iodinated to a significant and reproducible extent. The targets of iodination were identified, after isolation of the individual iodinated proteins, and were as follows: in protein L2 (271 amino acids), tyrosine-102 and -160; in protein L5 (178 amino acids), tyrosine-142; in protein L10 (165 amino acids), tyrosine-132; in protein L11 (142 amino acids), tyrosine-7 and -61. In the 70S ribosome, only protein L5 was still iodinated to a significant extent from the 50S subunit, whereas in the 30S subunit the same spectrum of iodinated proteins was observed as that from iodinated isolated 30S subunits, with the exception that S21 was no longer present.     

Immunoblotting analysis of protein-protein crosslinks within the 50S ribosomal subunit of Escherichia coli. A study using dimethylsuberimidate as crosslinking reagent

50S ribosomal subunits of Escherichia coli have been crosslinked with the bifunctional imidoester dimethyl-suberimidate and the protein-protein crosslinks have been analyzed by immunoblotting, using antisera specific for the individual ribosomal proteins of the large ribosomal subunit. Crosslinked protein pairs which occurred in yields higher than 5% have been unambiguously identified. Thus 13 crosslinks have been identified, namely L1-L33, L5-L7/12, L6-L19, L7/12-L10, L7/12-L11, L9-L28, L10-L11, L13-L20, L16-L27, L17-L32, L18-L22, L19-L25 and L27-L33. These data, together with the results which we will be presenting elsewhere, contribute considerably to our knowledge of the protein topography of the 50S ribosomal proteins as determined by immunoelectron microscopy. We can now propose the approximate locations of ten proteins that have not previously been localized.     

Mapping proteins of the 50S subunit from Escherichia coli ribosomes

Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui.     

Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli

We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide. The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34. The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach. Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.     

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.     

The effect of magnesium starvation on the dissociation of ribosomal proteins from Escherichia coli K-12 ribosomes.

The effect of magnesium starvation upon the fate of individual ribosomal proteins was studied in Escherichia coli. During a 21 h incubation in the absence of Mg2+ the 30 S subunit was more susceptible to degradation, retaining an average 31.9% of its ribosomal proteins as compared to 40.0% for the 50 S subunit. An examination of those 50-S proteins dissociated to a lesser extent than the average value (L1, L2, L3, L7, L10, L13, L16, L17, L19, L21, L22, L23, and L29) revealed that, with the exception of L16, all were classified by Dohme and Nierhaus [5] as tightly bound. Of the ribosomal proteins dissocated during magnesium starvation only five were reincorporated (and these to a minimal degree) during recovery of cells in a medium containing Mg2+. These studies suggest that ribosomal proteins once released from the ribosome particles during magnesium starvation are not reutilized in the assembly of new subunits.     

Mutants of Escherichia coli lacking ribosomal protein L1

Two independently isolated mutants of Escherichia coli, RD19 and MV17-10, that appeared to lack protein L1 on their ribosomes, as determined by two-dimensional gels, were subjected to a battery of immunological tests to find if L1 was indeed lacking. The tests involved Ouchterlony double diffusion, modified immunoelectrophoresis, dimer formation on sucrose gradients, and affinity chromatography. By all these criteria, protein L1 was missing from the ribosome in these mutants. Nor was any L1 cross-reacting material detectable in the supernatant. There was, however, a specific two- to fivefold increase in concentrations of protein L11 in the supernatants of the mutants, which was evidence that protein L1 acts as a feedback inhibitor of expression of the operon coding for the genes for proteins L11 and L1.Electron micrographs of ribosomes obtained from these mutants were indistinguishable from those of wild-type strains. 50 S ribosomal subunits from mutants RD19 and MV17-10 were reconstituted with purified L1 from wild-type and investigated by immunoelectron microscopy. The three-dimensional location of ribosomal protein L1 on the surface of the large subunit was determined. L1 is located on the wider lateral protuberance of the 50 S subunit. The position of protein L1 in 50 S subunits reconstituted from mutants RD19 and MV17-10 was indistinguishable from the position in subunits from wild-type.     

Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

The ribosomes from four temperature-sensitive mutants of Escherichia coli have been examined for defects in cell-free protein synthesis. The mutants examined had alterations in ribosomal proteins S10, S15, or L22 (two strains). Ribosomes from each mutant showed a reduced activity in the translation of phage MS2 RNA at 44 degrees C and were more rapidly inactivated by heating at this temperature compared to control ribosomes. Ribosomal subunits from three of the mutants demonstrated a partial or complete inability to reassociate at 44 degrees C. 70-S ribosomes from two strains showed a reducton in messenger RNA binding. tRNA binding to the 30 S subunit was reduced in the strains with altered 30-S proteins and binding to the 50 S subunit was affected in the mutants with a change in 50 S protein L22. The relation between ribosomal protein structure and function in protein synthesis in these mutants is discussed.     

Differential Effects of Replacing Escherichia coli Ribosomal Protein L27 with Its Homologue from Aquifex aeolicus

The rpmA gene, which encodes 50S ribosomal subunit protein L27, was cloned from the extreme thermophile Aquifex aeolicus, and the protein was overexpressed and purified. Comparison of the A. aeolicus protein with its homologue from Escherichia coli by circular dichroism analysis and proton nuclear magnetic resonance spectroscopy showed that it readily adopts some structure in solution that is very stable, whereas the E. coli protein is unstructured under the same conditions. A mutant of E. coli that lacks L27 was found earlier to be impaired in the assembly and function of the 50S subunit; both defects could be corrected by expression of E. coli L27 from an extrachromosomal copy of the rpmA gene. When A. aeolicus L27 was expressed in the same mutant, an increase in the growth rate occurred and the "foreign" L27 protein was incorporated into E. coli ribosomes. However, the presence of A. aeolicus L27 did not promote 50S subunit assembly. Thus, while the A. aeolicus protein can apparently replace its E. coli homologue functionally in completed ribosomes, it does not assist in the assembly of E. coli ribosomes that otherwise lack L27. Possible explanations for this paradoxical behavior are discussed.     

Localization of two epitopes of protein L7/L12 to both the body and stalk of the large ribosomal subunit. Immune electron microscopy using monoclonal antibodies

Four molecules of ribosomal protein L7/L12 are found as two dimers on the Escherichia coli 50 S ribosomal subunit. Immune electron microscopy using monoclonal antibodies directed against two epitopes of protein L7/L12 has allowed placement of elements of each dimer. One monoclonal antibody, directed against a determinant in the COOH-terminal domain, allows localization of two identical determinants at or near the end of the subunit stalk. The same antibody was used to place two additional determinants at the periphery of stalkless subunits, in an area from which a stalk might be expected to project. A second antibody, directed against an epitope in the amino-terminal portion of L7/L12, caused loss of stalks from the 50 S subunits. The micrographs showed symmetrical oligometric complexes of the dissociated dimeric protein with bivalent antibody. Antibodies were also seen to bind to the body of stalkless subunits, in a region near the COOH-terminal sites. The results are explained by a model in which one dimer of protein L7/L12 exists in a folded conformation on the subunit body and the second dimer occurs in an extended conformation in the subunit stalk.     

Functional analysis of the ffh-trmD region of the Escherichia coli chromosome by using reverse genetics.

We have analyzed the essentiality or contribution to growth of each of four genes in the Escherichia coli trmD operon (rpsP, 21K, trmD, and rplS) and of the flanking genes ffh and 16K by a reverse genetic method. Mutant alleles were constructed in vitro on plasmids and transferred by recombination to the corresponding lambda phage clone (lambda 439) and from the phage clone to the E. coli chromosome. An ability to obtain recombinants only in cells carrying a complementing plasmid indicated that the mutated gene was essential, while an ability to obtain recombinants in plasmid-free cells indicated nonessentiality. In this way, Ffh, the E. coli homolog to the 54-kDa protein of the signal recognition particle of mammalian cells, and ribosomal proteins S16 and L19 were shown to be essential for viability. A deletion of the second gene, 21K, of the trmD operon reduced the growth rate of the cells fivefold, indicating that the wild-type 21-kDa protein is important for viability. A deletion-insertion in the same gene resulted in the accumulation of an assembly intermediate of the 50S ribosomal subunit, as a result of polar effects on the expression of a downstream gene, rplS, which encodes ribosomal protein L19. This finding suggests that L19, previously not considered to be an assembly protein, contributes to the assembly of the 50S ribosomal subunits. Strains deleted for the trmD gene, the third gene of the operon, encoding the tRNA (m1G37)methyltransferase (or TrmD) showed a severalfold reduced growth rate. Since such a strain grew much slower than a strain lacking the tRNA(m(1)G37) methyltransferase activity because of a point mutation, the TrmD protein might have a second function in the cell. Finally, a 16-kDa protein encoded by the gene located downstream of, and convergently transcribed to, the trmD operon was found to be nonessential and not to contribute to growth.     

Mapping the ribosomal RNA neighborhood of protein L11 by directed hydroxyl radical probing.

Ribosomal protein L11 is a highly conserved protein that has been implicated in binding of elongation factors to ribosomes and associated GTP hydrolysis. Here, we have analyzed the ribosomal RNA neighborhood of Escherichia coli L11 in 50 S subunits by directed hydroxyl radical probing from Fe(II) tethered to five engineered cysteine residues at positions 19, 84, 85, 92 and 116 via the linker 1-(p -bromoacetamidobenzyl)-EDTA. Correct assembly of the L11 derivatives was analyzed by incorporating the modified proteins into 50 S subunits isolated from an E. coli strain that lacks L11 and testing for previously characterized L11-dependent footprints in domain II of 23 S rRNA. Hydroxyl radicals were generated from Fe(II) tethered to L11 and sites of cleavage in the ribosomal RNA were detected by primer extension. Strong cleavages were detected within the previously described binding site of L11, in the 1100 region of 23 S rRNA. Moreover, Fe(II) tethered to position 19 in L11 targeted the backbone of the sarcin loop in domain VI while probing from position 92 cleaved the backbone around bases 900 and 2470 in domains II and V, respectively. Fe(II) tethered to positions 84, 85 and 92 also generated cleavages in 5 S rRNA around helix II. These data provide new information about the positions of specific features of 23 S rRNA and 5 S rRNA relative to protein L11 in the 50 S subunit and show that L11 is near highly conserved elements of the rRNA that have been implicated in binding of tRNA and elongation factors to the ribosome.     

Amino acid sequence of the ribosomal protein L21 of Escherichia coli.

The primary structure of protein L21 from the 50S subunit of Escherichia coli ribosomes has been completely determined by sequencing the peptides obtained by digestion of L21 with trypsin before and after modification of the arginine residues with 1,2-cyclohexanedione, Staphylococcus aureus protease, thermolysin, and pepsin. Automated Edman degradation using a liquid-phase sequenator was carried out on the intact protein as well as on a fragment arising from cleavage with cyanogen bromide. Protein L21 consists of a single polypeptide chain of 103 amino acids of molecular weight 11 565. An estimation of the secondary structure of protein L21 and a comparison with other E. coli ribosomal protein sequences are presented.     

Structure of LiCl core particles of 50 S ribosomal subunits from Escherichia coli by electron microscopy.

The structure of 50 S E. coli ribosomal subunits was studied by electron microscopy as these particles were gradually depleted of proteins by incubation with 0.5 to 6.0 m LiCl. Changes observed in the structure of the depleted subunits were correlated with the location of the deleted ribosomal proteins on the control 50 S particle. These changes were particularly striking in the "crown" region, the site of a considerable number of the proteins necessary for the biological activity of the 50 S subunit. Protein L 16, the first to be removed by the LiCl treatment, was found to be essential for the structural integrity of the large subunit through interactions with ribosomal proteins residing in the left-hand side crest and the interface. Based on electron microscopic evidence, a scheme was proposed for the structural changes accompanying the stepwise unfolding of the 50 S E. coli subunit by LiCl.     

Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts.

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.     

Probing the functional role and localization of Escherichia coli ribosomal protein L16 with a monoclonal antibody

A monoclonal antibody specific for Escherichia coli ribosomal protein L16 was prepared to test its effects on ribosome function and to locate L16 by immunoelectron microscopy. The antibody recognized L16 in 50 S subunits, but not in 70 S ribosomes. It inhibited association of ribosomal subunits at 10 mM Mg2+, but not at 15 mM Mg2+. Poly(U)-directed polyphenylalanine synthesis and peptidyltransferase activities were completely inhibited when the L16 antibody was bound to 50 S subunits at a molar ratio of 1. There was no inhibitory effect on the binding of elongation factors or on the associated GTPase activities. Fab fragments of the antibody gave the same result as the intact antibody. Chemical modification of the single histidine (His13) by diethyl pyrocarbonate destroyed antibody binding. Electron microscopy of negatively stained antibody subunit complexes showed antibody binding beside the central protuberance of the 50 S particle on the side away from the L7/L12 stalk and on or near the interface between the two subunits. This site of antibody binding is fully consistent with its biochemical effects that indicate that protein L16 is essential for the peptidyltransferase activity activity of protein biosynthesis and is at or near the subunit interface.     

Purification and properties of a ribosomal protein methylase from Eschericha coli Q13.

Ribosomal protein methylase has been purified from Escherichia coli strain Q13 using methyl-deficient 50S subunits as substrates. The purified enzyme (or enzyme complex) which is devoid of rRNA methylating activity is quite stable and has a pH optimum around 8.0. The Km for S-adenosyl-L-methionine is 3.2 muM. The molecular weight of the enzyme is 3.1 X 10(4); minor methylating activity was also detected for protein peaks with molecular weights of 1.7 X 10(4) and 5.6 X 10(4). Protein L11 is the major protein methylated by the purified enzyme. Product analysis revealed the presence of N epislon-trimethyllysine, a methylated neutral amino acid(s) previously observed in protein L11 and N epislon-monomethyllysine. Free ribosomal proteins were much better substrates for the methylation, indicating that methylation of 50S ribosomal proteins can occur before the complete assembly of the 50S ribosomal subunit.     

Tiamulin resistance mutations in Escherichia coli.

Forty "two-step" and 13 "three-step" tiamulin-resistant mutants of Escherichia coli PR11 were isolated and tested for alteration of ribosomal proteins. Mutants with altered ribosomal proteins S10, S19, L3, and L4 were detected. The S19, L3, and L4 mutants were studied in detail. The L3 and L4 mutations did not segregate from the resistance character in transductional crosses and therefore seem to be responsible for the resistance. Extracts of these mutants also exhibited an increased in vitro resistance to tiamulin in the polyuridylic acid and phage R17 RNA-dependent polypeptide synthesis systems, and it was demonstrated that this was a property of the 50S subunit. In the case of the S19 mutant, genetic analysis showed segregation between resistance and the S19 alteration and therefore indicated that mutation of a protein other than S19 was responsible for the resistance phenotype. The isolated ribosomes of the S19, L3, and L4 mutants bound radioactive tiamulin with a considerably reduced strength when compared with those of wild-type cells. The association constants were lower by factors ranging from approximately 20 to 200. When heated in the presence of ammonium chloride, these ribosomes partially regained their avidity for tiamulin.