The binding site of ribosomal protein L10 in eubacteria and archaebacteria is conserved: reconstitution of chimeric 50S subunits.

It has been shown by electron microscopy that the selective removal of the stalk from 50S ribosomal subunits of two representative archaebacteria, namely Methanococcus vaniellii and Sulfolobus solfataricus, is accompanied by loss of the archaebacterial L10 and L12 proteins. The stalk was reformed if archaebacterial core particles were reconstituted with their corresponding split proteins. Next, structurally intact chimeric 50S subunits have been reconstituted in vitro by addition of Escherichia coli ribosomal proteins L10 and L7/L12 to 50S core particles from M vaniellii or S solfataricus, respectively. In the reverse experiment, using core particles from E coli and split proteins from M vaniellii, stalk-bearing 50S particles were also obtained. Analysis of the reconstituted 50S subunits by immunoblotting revealed that E coli L10 was incorporated into archaebacterial core particles in both presence or absence of E coli L7/L12. In contrast, incorporation of E coli L7/L12 into archaebacterial cores was only possible in the presence of E coli L10. Our results suggest that in archaebacteria - as in E coli - the stalk is formed by archaebacterial L12 proteins that bind to the ribosome via L10. The structural equivalence of eubacterial and archaebacterial L10 and L12 proteins has thus for the first time been established. The chimeric reconstitution experiments provide evidence that the domain of protein L10 that interacts with the ribosomal particle is highly conserved between eubacteria and archaebacteria.     

Occurrence in the archaebacterium Sulfolobus solfataricus of a ribosomal protein complex corresponding to Escherichia coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)2.P0

Two-dimensional electrophoresis of total protein from 50 S ribosomal subunits of the archaebacterium Sulfolobus solfataricus demonstrated a complex between two proteins that was stable in 6 M urea, but dissociable in detergent or below pH 5.5. The proteins, numbered L1 and L10 according to their electrophoretic mobilities, corresponded to Escherichia coli ribosomal proteins L10 and L7/L12, respectively. The members of the complex were therefore designated Sso L10e and Sso L12e. Sso L12e had other properties in common with E. coli L7/L12: low molecular weight, relative acidity, selective release from the ribosome by high salt/ethanol, and dimeric structure. The Sso L12e.Sso L10e complex was isolated by gel filtration of total 50 S proteins in 4 M urea. The stoichiometry of the components was approximately four copies of Sso L12e to one copy of Sso L10e. The occurrence in an archaebacterium of a complex of acidic ribosomal proteins similar to E. coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)/.P0 strongly supports the concept that this element of quaternary structure is a major conserved feature of the ribosome and reaffirms its importance in the translocation step of protein synthesis.     

Oligomeric state and mode of self-association of Thermotoga maritima ribosomal stalk protein L12 in solution

The "stalk" of the prokaryotic 50S ribosomal subunit is comprised of four copies of the protein L7/L12. In Escherichia coli, L7/L12 is a dimeric protein at micromolar concentrations, which is able to undergo rapid subunit exchange. A recent structural study indicated a tetrameric arrangement of the L12 proteins isolated from Thermotoga maritima, in which the proteins engaged in two different dimerization modes. In one mode, the two monomers of L12 form a tight symmetric and parallel dimer held together by a four-helix bundle, which encompasses the hinge region between the N- and C-terminal domains. In the other mode, the two monomers bind through their N-terminal region in an antiparallel configuration, in which one monomer comprises an alpha-helical hinge and the other monomer adopts an elongated shape with an unfolded hinge region. Presently, it is unclear which dimer contact prevails in solution and on the ribosome. Using cysteine mutants of T. maritima labeled with fluorescent probes, we investigated the mode of interactions between L12 subunits. Data from Forster resonance energy transfer experiments support a dimerization of L12 in solution, in which two monomers bind through their N-terminal region in an antiparallel configuration. We also demonstrate that the rate of subunit exchange in T. maritima L12 is significantly slower at 25 degrees C than that in the E. coli system. The exchange rate increases with increasing temperature and approaches the one observed for the E. coli system at 50 degrees C. Possible factors responsible for this difference are discussed.     

The binding site for ribosomal protein complex L8 within 23 s ribosomal RNA of Escherichia coli

The ribosomal protein complex L8 of Escherichia coli consists of two dimers of protein L7/L12 and one monomer of protein L10. This pentameric complex and ribosomal protein L11 bind in mutually cooperative fashion to 23 S rRNA and protect specific fragments of the latter from digestion with ribonuclease T1. Oligonucleotides protected either by the L8 complex alone or by the complex plus protein L11 were isolated from such digests and shown to rebind specifically to these proteins. They were also subjected to nucleotide sequence analysis. The longest oligonucleotide, protected by the L8 complex alone, consisted of residues 1028-1124 of 23 S rRNA and included all the other RNA fragments produced in this study. Previously, protein L11 had been shown to protect residues 1052-1112 of 23 S rRNA. It is concluded that the binding sites for the L8 protein complex and for protein L11 are immediately adjacent within 23 S rRNA of E. coli.     

Evidence for the ability of L10 ribosomal proteins of Salmonella typhimurium and Klebsiella pneumoniae to regulate rplJL gene expression in Escherichia coli

Genes rplJ, coding for ribosomal protein L10 of Salmonella typhimurium and Klebsiella pneumoniae, have been cloned on pUC plasmid. The resultant multicopy recombinant plasmids were detrimental for the growth of normal JM101 E. coli host cells and harmless for the mutant JF3029 host. This negative effect is the evidence for the ability of heterologous L10 proteins to regulate expression of rplJL genes in E. coli. Nucleotide sequence was determined completely for S. typhimurium rplJL' DNA portion and partially for rplJL' genes of K. pneumoniae. According to the nucleotide sequence data obtained three amino acid substitutions differ L10 proteins of S. typhimurium and E. coli and the long range, providing for the coupled translations of L10 and L7/L12 cistrons in E. coli mRNA is also valid for S. typhimurium and K. pneumoniae.     

Examination of protein sequence homologies: I. Eleven Escherichia coli L7/L12-type ribosomal "A" protein sequences from eubacteria and chloroplast

Seven complete and four partial sequences of Escherichia coli L7/L12-type ribosomal "A" proteins obtained from various bacteria (E. coli, Bacillus subtilis, Micrococcus lysodeikticus, Rhodopseudomonas spheroides, Desulfovibrio vulgaris, Streptomyces griseus, Bacillus stearothermophilus, Clostridium pasteurianum, Arthrobacter glacialis, and Vibrio costicola) and spinach chloroplast have been reexamined using a computer program that searches for homologous tertiary structures. Comparison matrices for the sequences show that they match the sequence of E. coli L7 (EL7) if one assumes the insertion or deletion of certain residues at sites corresponding to residues 1, 38, 49, and 92 of EL7. That two additional insertion points are found only in the spinach chloroplast protein suggests that the chloroplast protein probably diverged from the bacterial forms. Further phylogenetic relationships among these 11 prokaryote-type "A" proteins are discussed with respect to average correlation coefficients computed, taking into account the existence of the gaps.     

The phosphorylation of ribosomal proteins L14 and S3 in Krebs II ascites cells.

Krebs II ascites cells incubated in Earle's saline (lacking glucose and amino acids) contain ribosomes with proteins S6 and Lgamma phosphorylated, as do ascites cells grown in the peritonea of mice or hamster fibroblasts grown in Eagle's medium. When ascites cells were incubated in Eagle's medium (containing glucose and amino acids) there was extensive glycolysis, producing very acidic conditions, and ribosomal proteins S3 and L14 became phosphorylated whereas Lgamma became dephosphorylated. This altered pattern of phosphorylation could not be produced merely by incubating ascites cells in Earle's saline at a decreased pH, but a rather similar pattern was produced when Earle's saline was supplemented with amino acids (but with glucose still omitted). These results suggest that depriving ascites cells of glucose may induce the synthesis of a protein (or proteins), necessary for alteration of the pattern of phosphorylation of the ribosomal proteins.     

An archaebacterial gene from Methanococcus vannielii encoding a protein homologous to the ribosomal protein L10 family

An open reading frame upstream of the Methanococcus vannielii L12 gene has been detected. The beginning of this open reading frame agrees with the N-terminal region of a protein (MvaL10) which has been isolated from the 50 S ribosomal subunit of M. vannielii and sequenced. The length of this gene is 1008 nucleotides, coding for 336 amino acids. Excellent sequence similarities were found to the L10-like ribosomal proteins from Halobacterium halobium and man. The N-terminal part of the MvaL10 protein shows significant sequence similarities to the E. coli L10 protein. MvaL10 is more than twice as long as E. coli L10 but is of length similar to those of the homologous halobacterial and human proteins. Interestingly, the C-terminal region of MvaL10 shows exceptionally high similarity to the C-terminal sequence of the MvaL12 protein. This is not the case for the E. coli proteins but was also observed for the human, Halobacterium and Sulfolobus proteins.     

The proximity of the C-terminal domain of Escherichia coli ribosomal protein L7/L12 to L10 determined by cysteine site-directed mutagenesis and protein-protein cross-linking.

Oligonucleotide-directed mutagenesis was used to produce a serine 89 to cysteine 89 substitution in the C-terminal globular domain of Escherichia coli ribosomal protein L7/L12. Cys-89 represented the only cysteine residue in the protein. L7/L12Cys89 was overproduced in E. coli and purified. An allele replacement strain was also constructed. Growth of this strain was indistinguishable from that of wild type. Ribosomes from the allele replacement strain were used to determine the location of the C-terminal domains of L7/L12 by disulfide cross-linking. A new homobifunctional cysteine-specific cross-linking reagent, 1,4-di[3'-(2'-pyridyldithio)-propionamido]butane, and diagonal gel electrophoresis were used to identify ribosomal proteins cross-linked to L7/L12Cys89. A cross-link between L7/L12 and the single cysteine in L10 was found, in addition to L7/L12 dimers. The L7/L12Cys89-L10 cross-link locates the C-terminal domain of at least one L7/L12 dimer on the body of the large subunit and supports our previous model (Olson, H. M., Sommer, A., Tewari, D. S., Traut, R. R., and Glitz, D. G. (1986) J. Biol. Chem. 261, 6924-6932) that depicts one of the two dimers of L7/L12 on the surface of the body of the 50 S subunit in a bent conformation with the C-terminal domain in close proximity to the N-terminal domain at the base of the stalk.     

Overexpression of the methanococcal ribosomal protein L12 in Escherichia coli and its incorporation into halobacterial 50 S subunits yielding active ribosomes

The gene for the ribosomal L12 protein from the archaebacterium Methanococcus vannielii was cloned into the expression vector pKK223-3. The protein was overexpressed and remained stable in Escherichia coli XL1 cells. Purification yielded a protein with the same amino acid composition and sequence as in Methanococcus but it was acetylated at the N terminus as in the case with the homologous protein of E. coli. The in vivo incorporation of the overexpressed protein into the E. coli ribosomes was not observed. The overexpressed M. vannielii protein MvaL12e was incorporated into halobacterial ribosomes, thereby displacing the corresponding halobacterial L12 protein. Intact 70 S ribosomes were reconstituted from halobacterial 50 S subunits carrying the MvaL12e protein. These ribosomes were as active as native halobacterial ribosomes in a poly(U) assay. On the other hand, our attempts to incorporate L12 proteins from Bacillus stearothermophilus and E. coli into halobacterial ribosomes were not successful. These results support the conclusion which is based on primary sequence and predicted secondary structure comparisons that there exist two distinct L12 protein families, namely the eubacterial L12 protein family and the eukaryotic/archaebacterial L12 protein family.     

Engineering and characterization of the ribosomal L10.L12 stalk complex. A structural element responsible for high turnover of the elongation factor G-dependent GTPase

The ribosomal stalk protein L12 is essential for events dependent on the GTP-binding translation factors. It has been recently shown that ribosomes from Thermus thermophilus contain a heptameric complex L10.(L12)2.(L12)2.(L12)2, rather than the conventional pentameric complex L10.(L12)2.(L12)2. Here we describe the reconstitution of the heptameric complex from purified L10 and L12 and the characterization of its role in elongation factor G-dependent GTPase activity using a hybrid system with Escherichia coli ribosomes. The T. thermophilus heptameric complex resulted in a 2.5-fold higher activity than the E. coli pentameric complex. The structural element of the T. thermophilus complex responsible for the higher activity was investigated using a chimeric L10 protein (Ec-Tt-L10), in which the C-terminal L12-binding site in E. coli L10 was replaced with the same region from T. thermophilus, and two chimeric L12 proteins: Ec-Tt-L12, in which the E. coli N-terminal domain was fused with the T. thermophilus C-terminal domain, and Tt.Ec-L12, in which the T. thermophilus N-terminal domain was fused with the E. coli C-terminal domain. High GTPase turnover was observed with the pentameric chimeric complex formed from E. coli L10 and Ec-Tt-L12 but not with the heptameric complex formed from Ec-Tt-L10 and Tt.Ec-L12. This suggested that the C-terminal region of T. thermophilus L12, rather than the heptameric nature of the complex, was responsible for the high GTPase turnover. Further analyses with other chimeric L12 proteins identified helix alpha6 as the region most likely to contain the responsible element.     

Escherichia coli ribosomal protein L10 is rapidly degraded when synthesized in excess of ribosomal protein L7/L12.

In Escherichia coli the genes encoding ribosomal proteins L10 and L7/12, rplJ and rplL, respectively, are cotranscribed and subject to translational coupling. Synthesis of both proteins is coordinately regulated at the translational level by binding of L10 or a complex of L10 and L7/L12 to a single target in the mRNA leader region upstream of rplJ. Unexpectedly, small deletions that inactivated the ribosome-binding site of the rplL gene carried on multicopy plasmids exerted a negative effect on expression of the upstream rplJ gene. This effect could be overcome by overproduction of L7/L12 in trans from another plasmid. This apparent stimulation resulted from stabilization of the overproduced L10 protein by L7/L12, presumably because free L10, in contrast to L10 complexed with L7/L12, is subject to rapid proteolytic decay. The contribution of this decay mechanism to the regulation of the rplJL operon is evaluated.     

Protein topography of Sulfolobus solfataricus ribosomes by cross-linking with 2-iminothiolane. Sso L12e, Sso L10e, and Sso L11e are neighbors

Large ribosomal subunits from Sulfolobus solfataricus were cross-linked with 2-iminothiolane in order to investigate the arrangement of proteins in the region containing the multicopy acidic protein Sso L12e, the protein homologous to Escherichia coli L7/L12. Proteins from cross-linked 50 S subunits were extracted and fractionated by chromatography on CM-cellulose. Fractions containing Sso L12e were analyzed by "diagonal" (two-dimensional reducing/nonreducing) dodecyl sulfate polyacrylamide gel electrophoresis. Sso L12e appeared in cross-linked homodimers and also in cross-linked complexes that contained Sso L10e, the protein equivalent to E. coli L10. In addition, Sso L12e was found in cross-links to L4, L6a, L26, and L29. N-terminal sequences obtained for L6a and L26 showed them to have significant homologies to E. coli proteins L11 and L23, respectively. The results indicate the presence in this archaebacterial ribosome of Sso L12e dimers and their location near Sso L10e and Sso L11e. The Sso L12e-L29 (Sso L23e) cross-link suggests proximity between components of the factor-binding and peptidyltransferase domains, since E. coli L23 is a protein affinity-labeled by puromycin. The (Sso L12e)4-Sso L10 pentameric complex, identified previously from studies in solution, appears to represent correctly the arrangement of these proteins in the ribosome. The occurrence in the archaebacterial ribosome of this unique structural element, similar to those shown previously in eubacteria and eukaryotes, reinforces the concept that the protein quaternary structure of the ribosomal factor-binding domain is highly conserved.     

Structure and evolution of the L11, L1, L10, and L12 equivalent ribosomal proteins in eubacteria, archaebacteria, and eucaryotes

The genes corresponding to the L11, L1, L10, and L12 equivalent ribosomal proteins (L11e, L1e, L10e, and L12e) of Escherichia coli have been cloned and sequenced from two widely divergent species of archaebacteria, Halobacterium cutirubrum and Sulfolobus solfataricus, and the L10 and four different L12 genes have been cloned and sequenced from the eucaryote Saccharomyces cerevisiae. Alignments between the deduced amino acid sequences of these proteins and to other available homologous proteins of eubacteria and eucaryotes have been made. The data suggest that the archaebacteria are a distinct coherent phylogenetic group. Alignment of the proline-rich L11e proteins reveals that the N-terminal region, believed to be responsible for interaction with release factor 1, is the most highly conserved region and that there is specific conservation of most of the proline residues, which may be important in maintaining the highly elongated structure of the molecule. Although L11 is the most highly methylated protein in the E. coli ribosome, the sites of methylation are not conserved in the archaebacterial L11e proteins. The L1e proteins of eubacteria and archaebacteria show two regions of very high similarity near the center and the carboxy termini of the proteins. The L10e proteins of all kingdoms are colinear and contain approximately three fourths of an L12e protein fused to their carboxy terminus, although much of this fusion has been lost in the truncated eubacterial protein. The archaebacterial and eucaryotic L12e proteins are colinear, whereas the eubacterial protein has suffered a rearrangement through what appear to be gene fusion events. Within the L12e derived region of the L10e proteins there exists a repeated module of 26 amino acids, present in two copies in eucaryotes, three in archaebacteria, and one in eubacteria. This modular sequence is apparently also present in the L12e proteins of all kingdoms and may play a role in L12e dimerization, L10e-L12e complex formation, and the function of the L10e-L12e complex in translation.     

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.     

Structural basis for the function of the ribosomal L7/12 stalk in factor binding and GTPase activation

The L7/12 stalk of the large subunit of bacterial ribosomes encompasses protein L10 and multiple copies of L7/12. We present crystal structures of Thermotoga maritima L10 in complex with three L7/12 N-terminal-domain dimers, refine the structure of an archaeal L10E N-terminal domain on the 50S subunit, and identify these elements in cryo-electron-microscopic reconstructions of Escherichia coli ribosomes. The mobile C-terminal helix alpha8 of L10 carries three L7/12 dimers in T. maritima and two in E. coli, in concordance with the different length of helix alpha8 of L10 in these organisms. The stalk is organized into three elements (stalk base, L10 helix alpha8-L7/12 N-terminal-domain complex, and L7/12 C-terminal domains) linked by flexible connections. Highly mobile L7/12 C-terminal domains promote recruitment of translation factors to the ribosome and stimulate GTP hydrolysis by the ribosome bound factors through stabilization of their active GTPase conformation.     

Studies on the RNA and protein binding sites of the E. coli ribosomal protein L10.

We have used modification of specific amino acid residues in the E. coli ribosomal protein L10 as a tool to study its interactions with another ribosomal protein, L7/L12, as well as with ribosomal core particles and with 23S RNA. The ribosome and RNA binding capability of L10 was found to be inhibited by modification of one more of its arginine residues. This treatment does not affect the ability of L10 to bind four molecules of L7/L12 in a L7/L12-L10 complex. Our results support the view that L10's role in promoting the L7/L12-ribosome association is due primarily to its ability to bind to both 23S RNA and L7/L12 simultaneously.