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.     

Deletion of C-terminal residues of Escherichia coli ribosomal protein L10 causes the loss of binding of one L7/L12 dimer: ribosomes with one L7/L12 dimer are active

Escherichia coli ribosomal protein L10 binds the two L7/L12 dimers and thereby anchors them to the large ribosomal subunit. C-Terminal deletion variants (Delta10, Delta20, and Delta33 amino acids) of ribosomal protein L10 were constructed in order to define the binding sites for the two L7/L12 dimers and then to make and test ribosomal particles that contain only one of the two dimers. None of the deletions interfered with binding of L10 variants to ribosomal core particles. Deletion of 20 or 33 amino acids led to the inability of the proteins to bind both dimers of protein L7/L12. The L10 variant with deletion of 10 amino acids bound one L7/L12 dimer in solution and when reconstituted into ribosomes promoted the binding of only one L7/L12 dimer to the ribosome. The ribosomes that contained a single L7/L12 dimer were homogeneous by gel electrophoresis where they had a mobility between wild-type 50S subunits and cores completely lacking L7/L12. The single-dimer ribosomal particles supported elongation factor G dependent GTP hydrolysis and protein synthesis in vitro with the same activity as that of two-dimer particles. The results suggest that amino acids 145-154 in protein L10 determine the binding site ("internal-site") for one L7/L12 dimer (the one reported here), and residues 155-164 ("C-terminal-site") are involved in the interaction with the second L7/L12 dimer. Homogeneous ribosomal particles containing a single L7/L12 dimer in each of the distinct sites present an ideal system for studying the location, conformation, dynamics, and function of each of the dimers individually.     

Three binding sites for stalk protein dimers are generally present in ribosomes from archaeal organism

Ribosomes have a characteristic protuberance termed the stalk, which is indispensable for ribosomal function. The ribosomal stalk has long been believed to be a pentameric protein complex composed of two sets of protein dimers, L12-L12, bound to a single anchor protein, although ribosomes carrying three L12 dimers were recently discovered in a few thermophilic bacteria. Here we have characterized the stalk complex from Pyrococcus horikoshii, a thermophilic species of Archaea. This complex is known to be composed of proteins homologous to eukaryotic counterparts rather than bacterial ones. In truncation experiments of the C-terminal regions of the anchor protein Ph-P0, we surprisingly observed three Ph-L12 dimers bound to the C-terminal half of Ph-P0, and the binding site for the third dimer was unique to the archaeal homologs. The stoichiometry of the heptameric complex Ph-P0(Ph-L12)(2)(Ph-L12)(2)(Ph-L12)(2) was confirmed by mass spectrometry of the intact complex. In functional tests, ribosomes carrying a single Ph-L12 dimer had significant activity, but the addition of the second and third dimers increased the activity. A bioinformatics analysis revealed the evidence that ribosomes from all archaeal and also from many bacterial organisms may contain a heptameric complex at the stalk, whereas eukaryotic ribosomes seem to contain exclusively a pentameric stalk complex, thus modifying our view of the stalk structure significantly.     

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.     

Mechanism and rates of exchange of L7/L12 between ribosomes and the effects of binding EF-G

The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. On the basis of kinetic analysis, we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by ~27% and of dimers by ~47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.     

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.     

Purification of human mitochondrial ribosomal L7/L12 stalk proteins and reconstitution of functional hybrid ribosomes in Escherichia coli

Bacterial ribosomal L7/L12 stalk is formed by L10, L11, and multiple copies of L7/L12, which plays an essential role in recruiting initiation and elongation factors during translation. The homologs of these proteins, MRPL10, MRPL11, and MRPL12, are present in human mitochondrial ribosomes. To evaluate the role of MRPL10, MRPL11, and MRPL12 in translation, we over-expressed and purified components of the human mitochondrial L7/L12 stalk proteins in Escherichia coli. Here, we designed a construct to co-express MRPL10 and MRPL12 using a duet expression system to form a functional MRPL10-MRPL12 complex. The goal is to demonstrate the homology between the mitochondrial and bacterial L7/L12 stalk proteins and to reconstitute a hybrid ribosome to be used in structural and functional studies of the mitochondrial stalk.     

A point mutation in ribosomal protein L7/L12 reduces its ability to form a compact dimer structure and to assemble into the GTPase center

An Escherichia coli mutant, LL103, harboring a mutation (Ser15 to Phe) in ribosomal protein L7/L12 was isolated among revertants of a streptomycin-dependent strain. In the crystal structure of the L7/L12 dimer, residue 15 within the N-terminal domain contacts the C-terminal domain of the partner monomer. We tested effects of the mutation on molecular assembly by biochemical approaches. Gel electrophoretic analysis showed that the Phe15-L7/L12 variant had reduced ability in binding to L10, an effect enhanced in the presence of 0.05% of nonionic detergent. Mobility of Phe15-L7/L12 on gel containing the detergent was very low compared to the wild-type proteins, presumably because of an extended structural state of the mutant L7/L12. Ribosomes isolated from LL103 cells contained a reduced amount of L7/L12 and showed low levels (15-30% of wild-type ribosomes) of activities dependent on elongation factors and in translation of natural mRNA. The ribosomal activity was completely recovered by addition of an excess amount of Phe15-L7/L12 to the ribosomes, suggesting that the mutant L7/L12 exerts normal functions when bound on the ribosome. The interaction of Ser15 with the C-terminal domain of the partner molecule seems to contribute to formation of the compact dimer structure and its efficient assembly into the ribosomal GTPase center. We propose a model relating compact and elongated forms of L7/L12 dimers. Phe15-L7/L12 provides a new tool for studying the functional structure of the homodimer.     

The ribosomal domain of the bacterial release factors. The carboxyl-terminal domain of the dimer of Escherichia coli ribosomal protein L7/L12 located in the body of the ribosome is important for release factor interaction.

1. Polyclonal antibodies (pAb 1-73 and pAb 26-120) have been raised against both an N-terminal fragment of Escherichia coli ribosomal protein L7/L12 (amino acids 1-73), and a fragment lacking part of the N-terminal domain (amino acids 26-120). 2. Only pAb 26-120 inhibited release-factor-dependent in vitro termination functions on the ribosome. This antibody binds over the length of the stalk of the large subunit of the ribosome as determined by immune electron microscopy, thereby not distinguishing between the C-terminal domains of the two L7/L12 dimers, those in the stalk or those in the body of the subunit. 3. A monoclonal antibody against an epitope of the C-terminal two thirds of the protein (mAb 74-120), which binds both to the distal tip of the stalk as well as to a region at its base, reflecting the positions of the two dimers is strongly inhibitory of release factor function. 4. A monoclonal antibody against an epitope of the N-terminal fragment of L7/L12 (mAb 1-73), previously shown to remove the dimer of L7/L12 in the 50S subunit stalk but still bind to the body of the particle, partially inhibited release-factor-mediated events. 5. The mAb 74-120 inhibited in vitro termination with a similar profile when the stalk dimer of L7/L12 was removed with mAb 1-73, indicating that the body L7/L12 dimer, and in particular its C-terminal domains, are important for release factor/ribosome interaction. 6. The two release factors have subtle differences in their binding domains with respect to L7/L12.     

In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0.Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0.Ph-L12 complex and Ph-L11 could replace L10.L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.     

Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly

The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the prmA gene. The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of Escherichia coli prmA has no apparent phenotype. We have constructed a mutant of the extreme thermophile Thermus thermophilus in which the prmA gene has been disrupted with the htk gene encoding a heat-stable kanamycin adenyltransferase. This mutant shows no growth defects, indicating that T. thermophilus PrmA, like its E. coli homolog, is dispensable. Ribosomes prepared from this mutant contain unmethylated L11, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and are effective substrates for in vitro methylation by cloned and purified T. thermophilus PrmA. MALDI-TOF MS also revealed that T. thermophilus L11 contains a total of 12 methyl groups, in contrast to the 9 methyl groups found in E. coli L11. Finally, we found that, as with the E. coli methyltransferase, the ribosomal protein L11 dissociated from ribosomes is a more efficient substrate for in vitro methylation by PrmA than intact 70S ribosomes, suggesting that methylation in vivo occurs on free L11 prior to its incorporation into ribosomes.     

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.     

Structure of a Two-Domain N-Terminal Fragment of Ribosomal Protein L10 from Methanococcus jannaschii Reveals a Specific Piece of the Archaeal Ribosomal Stalk

Ribosomal stalk is involved in the formation of the so-called “GTPase-associated site” and plays a key role in the interaction of ribosome with translation factors and in the control of translation accuracy. The stalk is formed by two or three copies of the L7/L12 dimer bound to the C-terminal tail of protein L10. The N-terminal domain of L10 binds to a segment of domain II of 23S rRNA near the binding site for ribosomal protein L11. The structure of bacterial L10 in complex with three L7/L12 N-terminal dimers has been determined in the isolated state, and the structure of the first third of archaeal L10 bound to domain II of 23S rRNA has been solved within the Haloarcula marismortui 50S ribosomal subunit. A close structural similarity between the RNA-binding domain of archaeal L10 and the RNA-binding domain of bacterial L10 has been demonstrated. In this work, a long RNA-binding N-terminal fragment of L10 from Methanococcus jannaschii has been isolated and crystallized. The crystal structure of this fragment (which encompasses two-thirds of the protein) has been solved at 1.6 Å resolution. The model presented shows the structure of the RNA-binding domain and the structure of the adjacent domain that exist in archaeal L10 and eukaryotic P0 proteins only. Furthermore, our model incorporated into the structure of the H. marismortui 50S ribosomal subunit allows clarification of the structure of the archaeal ribosomal stalk base.     

Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elongation factor binding.

The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli ribosomes is assembled on the conserved region of 23 S rRNA termed the GTPase-associated domain. We replaced the L8 complex in E. coli 50 S subunits with the rat counterpart P protein complex consisting of P1, P2, and P0. The L8 complex was removed from the ribosome with 50% ethanol, 10 mM MgCl(2), 0.5 M NH(4)Cl, at 30 degrees C, and the rat P complex bound to the core particle. Binding of the P complex to the core was prevented by addition of RNA fragment covering the GTPase-associated domain of E. coli 23 S rRNA to which rat P complex bound strongly, suggesting a direct role of the RNA domain in this incorporation. The resultant hybrid ribosomes showed eukaryotic translocase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent, GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent activity was dependent upon the P complex binding and was inhibited by the antibiotic thiostrepton, a ligand for a portion of the GTPase-associated domain of prokaryotic ribosomes. This hybrid system clearly shows significance of binding of the P complex to the GTPase-associated RNA domain for interaction of EF-2 with the ribosome. The results also suggest that E. coli 23 S rRNA participates in the eukaryotic translocase-dependent GTPase activity in the hybrid system.     

Replacement of the L11 binding region within E.coli 23S ribosomal RNA with its homologue from yeast: in vivo and in vitro analysis of hybrid ribosomes altered in the GTPase centre.

Replacement of the protein L11 binding domain within Escherichia coli 23S ribosomal RNA (rRNA) by the equivalent region from yeast 26S rRNA appeared to have no effect on the growth rate of E.coli cells harbouring a plasmid carrying the mutated rrnB operon. The hybrid rRNA was correctly processed and assembled into ribosomes, which accumulated normally in polyribosomes. Of the total ribosomal population, < 25% contained wild-type, chromosomally encoded rRNA; the remainder were mutant. The hybrid ribosomes supported GTP hydrolysis dependent upon E.coli elongation factor G, although at a somewhat reduced rate compared with wild-type particles, and were sensitive to the antibiotic, thiostrepton, a potent inhibitor of ribosomal GTPase activity that binds to 23S rRNA within the L11 binding domain. That thiostrepton could indeed bind to the mutant ribosomes, although at a reduced level relative to that seen with wild-type ribosomes, was confirmed in a non-equilibrium assay. The rationale for the ability of the hybrid ribosomes to bind the antibiotic, given that yeast ribosomes do not, was provided when yeast rRNA was shown by equilibrium dialysis to bind thiostrepton only 10-fold less tightly than did E.coli rRNA. The extreme conservation of secondary, but not primary, structure in this region between E.coli and yeast rRNAs allows the hybrid ribosomes to function competently in protein synthesis and also preserves the interaction with thiostrepton.     

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.     

The C-terminal domain of Escherichia coli ribosomal protein L7/L12 can occupy a location near the factor-binding domain of the 50S subunit as shown by cross-linking with N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide.

All large ribosomal subunits contain two dimers composed of small acidic proteins that are involved in binding elongation factors during protein synthesis. The ribosomal location of the C-terminal globular domain of the Escherichia coli ribosomal acidic protein L7/L12 has been determined by protein cross-linking with a new heterobifunctional, reversible, photoactivatable reagent, N-[4-(p-azidosalicylamido)-butyl]-3-(2'-pyridyldithio)propionamide . Properties of this reagent are described. It was first radiolabeled with 125I and then attached through the formation of a disulfide bond to a unique cysteine of L7/L12, introduced by site-directed mutagenesis at residue 89. Intact 50S ribosomal subunits were reconstituted from L7/L12-depleted cores and the radiolabeled L7/L12Cys89. Irradiation of the reconstituted subunits resulted in photo-cross-linking between residue 89 and other ribosomal components. Reductive cleavage of the disulfide cross-link resulted in transfer of the 125I label from L7/L12Cys89 to the other cross-linked components. Two radiolabeled proteins were identified, L11 and L10. The location of both of these proteins is well established to be at the base of the L7/L12 stalk near the binding sites for the N-terminal domain of both L7/L12 dimers, and for elongation factors. The result indicates that L7/L12 can have a bent conformation bringing the C-terminal domain of at least one of the L7/L12 dimers at or near the factor-binding domain. The cross-linking method with radiolabeled N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide should be applicable for studies of other multicomponent complexes that can be reconstituted.     

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.     

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

The Escherichia coli ribosomal 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 A 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 NH(4)Cl 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 A 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.