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.     

Characterization of the region on protein L7/L12 involved in binding to ribosomal particles

Tryptic digestion of reductively methylated protein L7/L12 yields a large tryptic fragment, which comprises amino acids 1-59. At the most, two molecules of this fragment can bind to a 50-S ribosomal particle, deprived of protein L7/L12. Besides, binding of each single 1-59 fragment competes with binding of one dimeric L7/L12 molecule. Molecular weight studies on the fragment reveal a monomeric structure. Digestion of the 1-59 fragment with carboxypeptidase Y leads to the formation of a 1-55 fragment. The binding characteristics of the latter fragment are similar to those of the 1-59 fragment. The results suggest that a monomeric stretch of L7/L12, comprising the first 55 amino acids, is sufficient for attaching L7/L12 to the ribosome.     

Structural investigations of the highly flexible recombinant ribosomal protein L12 from Thermotoga maritima

Ribosomal protein L7/L12, the only multicopy component of the ribosome, is involved in translation factor binding and in the ribosomal GTPase center. The gene for L7/L12 from Thermotoga maritima was cloned and the protein expressed at high levels in Escherichia coli. Purification of L7/L12 was achieved under non-denaturing conditions via heat treatment and two chromatographic steps. Circular dichroism melting profiles were monitored at 222 nm, showing the melting temperature of the protein at pH 7.5 around 110 degrees C, compared to approximately 60 degrees C for the highly homologous Escherichia coli protein. The unfolding was reversible and renaturation closely followed the path of the thermal melting. Dynamic light scattering, gel filtration chromatography, and crosslinking experiments suggested that under physiological buffer conditions Thermotoga maritima L7/L12 exists as a tetramer. The protein was crystallized under two conditions, yielding an orthorhombic (C222(1)) and a cubic (12(1)3) space group with an estimated two and three to four L7/L12 molecules per asymmetric unit, respectively. The crystals contained the full-length protein, and cryogenic buffers were developed which improved the mosaic spreads and the resolution limits. For the structure solution isoleucine was mutated to methionine at two separate positions, the mutant forms expressed as selenomethionine variants and crystallized.     

Delta Lys120, a mutation which destabilizes the ribosome-binding domain of ribosomal protein L7/L12

Five-residue-long deletions centered on Ala63, Ala75, and Glu118 of ribosomal protein L7/L12 gave low mutant yields (5% or less) when the mutant genes were cloned in phage M13mp18 and controlled by the L10 promotor. Deletions of Glu118-Lys120 or Lys120 (the COOH-terminus of L7/L12) gave higher mutant yields, up to 50% with L7/L12 delta Lys120. L7/L12 delta Lys120 was not preferentially found in the S100 and not preferentially removed by LiCl washing, but was preferentially extracted from 70S ribosomes in the presence of 28-35% ethanol in 0.25-0.5 M NH4Cl. It follows that delta Lys120 destabilizes the ribosome-binding domain of ribosomal protein L7/L12 in an ethanol-containing solvent, which raises the question whether Lys120 is part of the ribosome-binding domain of L7/L12 during some step of protein synthesis or whether it is essential to preserve the conformation of the physiological ribosome-binding domain under structurally stressful conditions.     

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.     

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.     

Structure of the C-terminal domain of the ribosomal protein L7/L12 from Escherichia coli at 1.7 A

The structure of a C-terminal fragment of the ribosomal protein L7/L12 from Escherichia coli has been refined using crystallographic data to 1.7 A resolution. The R-value is 17.4%. Six residues at the N terminus are too disordered in the structure to be localized. These residues are probably part of a hinge in the complete L7/L12 molecule. The possibility that a 2-fold crystallographic axis is a molecular 2-fold axis is discussed. A patch of invariant residues on the surface of the dimer is probably involved in functional interactions with elongation factors.     

Studies on the binding of the ribosomal protein complex L7/12-L10 and protein L11 to the 5''-one third of 23S RNA: a functional centre of the 50S subunit.

The RNA binding sites of the protein complex of L7/12 dimers and L10, and of protein L11, occur within the 5'-one third of 23S RNA. Binding of the L7/12-L10 protein complex to the 23S RNA is stimulated by protein L11 and vice-versa. This is the second example to be established of mutual stimulation of RNA binding by two ribosomal proteins or protein complexes, and suggests that this may be an important principle governing ribosomal protein-RNA assembly. When the L7/12-L10 complex is bound to the RNA, L10 becomes strongly resistant to trypsin. Since the L7/12 dimer does not bind specifically to the 23S RNA, this suggests that L10 constitutes a major RNA binding site of the protein complex. Only one of the L7/12 dimers is bound strongly in the (L7/12-L10)-23S RNA complex; the other can dissociate with no concurrent loss of L10.     

RNA secondary structure and translation inhibition: analysis of mutants in the rplJ leader

We have carried out measurements of the stable binding of the ribosomal protein (r-protein) complex L10-L7/L12 to mutant forms of the mRNA leader of the rplJ operon of Escherichia coli. One of the point mutations, base 1548, which lies within the L10-L7/L12-protected region, almost completely abolishes in vitro formation of a stable complex of L10-L7/L12 with rplJ mRNA leader, and a second point mutation, base 1634, strongly reduces it. These observations constitute strong support for the proposition that L10-L7/L12 binds to the rplJ leader in bringing about translational feedback. To account for the action of these and other mutations, and to explain the mechanism of translation feedback inhibition, we suggest a secondary structure model involving alternate forms of the rplJ mRNA leader.     

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.     

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.     

Dimer state of protein L7/L12 and EF-G-dependent reactions of ribosomes.

A number of different monomer and dimer derivatives of protein L7/L12 has been studied in EF-G-dependent reactions on the ribosome. It has been shown that only dimer derivatives of protein L7/L12 are able to interact with the ribosome. This means that it is the dimer forms of protein L7/L12 that are present in the functionally active ribosome. It is likely that the N-terminal sequence of protein L7/L12 is responsible for dimerization of the protein in solution and at the same time contributes mainly to the interaction of the protein L7/L12 dimer with the ribosome. The results obtained suggest that there are four copies of protein L7/L12 in the translating ribosome.     

From structure and dynamics of protein L7/L12 to molecular switching in ribosome

Based on the (1)H-(15)N NMR spectroscopy data, the three-dimensional structure and internal dynamic properties of ribosomal protein L7 from Escherichia coli were derived. The structure of L7 dimer in solution can be described as a set of three distinct domains, tumbling rather independently and linked via flexible hinge regions. The dimeric N-terminal domain (residues 1-32) consists of two antiparallel alpha-alpha-hairpins forming a symmetrical four-helical bundle, whereas the two identical C-terminal domains (residues 52-120) adopt a compact alpha/beta-fold. There is an indirect evidence of the existence of transitory helical structures at least in the first part (residues 33-43) of the hinge region. Combining structural data for the ribosomal protein L7/L12 from NMR spectroscopy and x-ray crystallography, it was suggested that its hinge region acts as a molecular switch, initiating "ratchet-like" motions of the L7/L12 stalk with respect to the ribosomal surface in response to elongation factor binding and GTP hydrolysis. This hypothesis allows an explanation of events observed during the translation cycle and provides useful insights into the role of protein L7/L12 in the functioning of the ribosome.     

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.     

Genomic organization of the human ubiquitin-conjugating enzyme gene, UBE2L6 on chromosome 11q12

The human UBE2L6 gene encodes UbcH8(Kumar), a ubiquitin-conjugating enzyme (E2) highly simliar in primary structure to UbcH7 which is encoded by UBE2L3. Like UBC4 and UBC5 in yeast, these proteins demonstrate functional redundancy. Herein we report the intron/exon structure of UBE2L6. Comparison of the genomic organization of UBE2L6 with UBE2L3 demonstrates that these genes remain highly conserved at the genomic as well as at the protein level. We also describe the chromosomal localization of UBE2L6, which maps to chromosome 11q12.     

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.     

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.     

Ribosomal protein L7/L12 has a helix-turn-helix motif similar to that found in DNA-binding regulatory proteins.

Inspection of the structure of the C-terminal domain of ribosomal protein L7/L12 (1) reveals a helix-turn-helix motif similar to the one found in many DNA-binding regulatory proteins (2-5). The 19 alpha-carbon atoms of the L7/L12 alpha-helices superimpose on the DNA binding helices of CAP and cro with root-mean-square distances between corresponding alpha carbons of 1.45 and 1.55 A, respectively. These helices in L7/L12 are within a patch of highly conserved residues on the surface of L7/L12 whose role is as yet uncertain. We raise the possibility that they may constitute a binding site for nucleic acids, most probably RNA. Consistent with this hypothesis are calculations of the electrostatic charge potential surrounding the protein, which show a region of positive potential centered on the first of these helices.     

Feedback regulation of the rplJL-rpoBC ribosomal protein operon of Escherichia coli requires a region of mRNA secondary structure

The rplJ-rpoBC (L10) operon of Escherichia coli is regulated in part through translational repression (feedback regulation) by ribosomal protein L10 or a complex of ribosomal proteins L10 and L7/L12 (L10-L7/L12). We have constructed mutants in the untranslated leader region of a rplJ-lacZ fusion by oligonucleotide-directed mutagenesis. The mutations include several deletions and a number of single base changes, all of which fail to exhibit normal feedback regulation. Chemical probing of part of the rplJ mRNA leader in the mutagenized region confirms that all of the mutations lie in a stem structure located 140 nucleotides upstream from the translation start-site. The structure includes a 12 base-pair stem, a four base stem-loop, and a six base bulge-loop. Point mutations that abolish feedback regulation are presumed to disrupt this stem structure. Pseudorevertants of selected point mutations were constructed by combining pairs of single base mutations. In these cases, both the secondary structure of the RNA and feedback regulation were restored. The results allow us to define a region of secondary structure in the rplJ mRNA leader that is necessary for feedback regulation.