Localization of proteins L4, L5, L20 and L25 on the ribosomal surface by immuno-electron microscopy

Summary Ribosomal proteins L4, L5, L20 and L25 have been localized on the surface of the 50S ribosomal subunit of Escherichia coli by immuno-electron microscopy. The two 5S RNA binding proteins L5 and L25 were both located at the central protuberance extending towards its base, at the interface side of the 50S particle. L5 was localized on the side of the central protuberance that faces the L1 protuberance, whereas L25 was localized on the side that faces the L7/L12 stalk. Proteins L4 and L20 were both located at the back of the 50S subunit; L4 was located in the vicinity of proteins L23 and L29, and protein L20 was localized between proteins L17 and L10 and is thus located below the origin of the L7/L12 stalk.     

Ribosomal proteins L7/L12 localized at a single region of the large subunit by immune electron microscopy.

Ribosomal proteins $\text{L}\text{7}\text{L}\text{12}$ have been mapped by immune electron microscopy. These multiple copy proteins are located at a single region extending from the large subunit, known as the $\text{L}\text{7}\text{L}\text{12}$ stalk. The $\text{L}\text{7}\text{L}\text{12}$ stalk is approximately 100 Å long, about 40 Å wide and extends at an angle of approximately 50 ° from one side of the central protuberance of the large subunit. In the monomeric 70 S ribosome, the portion of the $\text{L}\text{7}\text{L}\text{12}$ stalk proximal to the 50 S subunit is located in the vicinity of the 30 S-50 S interface.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibody binding to the stalk was shown to be solely dependent upon the presence of $\text{L}\text{7}\text{L}\text{12}$ by the following experiments. Sucrose gradient analysis was used to demonstrate that large subunits depleted of $\text{L}\text{7}\text{L}\text{12}$ were unable to bind anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ restored the ability of $\text{L}\text{7}\text{L}\text{12}$-depleted cores to react with anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies. Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies pre-absorbed with $\text{L}\text{7}\text{L}\text{12}$ did not react with 50 S subunits.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies used in these experiments reacted only with the $\text{L}\text{7}\text{L}\text{12}$ stalk and with no other region of the subunit. This was shown by electron microscopy and by immune electron microscopy in the following ways. Electron microscopy of 50 S subunits, $\text{L}\text{7}\text{L}\text{12}$-depleted 50 S cores, and reconstituted 50 S subunits was used to demonstrate that stripping removes the $\text{L}\text{7}\text{L}\text{12}$ stalk from more than 95% of the subunits, and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ into depleted cores restores the $\text{L}\text{7}\text{L}\text{12}$ stalk. Double-labelling experiments, using monomeric subunits with two or more attached anti-$\text{L}\text{7}\text{L}\text{12}$ immunoglobulins, were used to demonstrate, independently of 50 S subunit morphology, that $\text{L}\text{7}\text{L}\text{12}$ are located only on the $\text{L}\text{7}\text{L}\text{12}$ stalk.     

Gel electrophoretic studies on ribosomal proteins L7L12 and the Escherichia coli 50 s subunit

Three forms of the 50 S ribosomal subunit of Escherichia coli have been separated by agarose/acrylamide gel electrophoresis. The slowest migrating form, S-50 S, corresponded to native 50 S subunits and contained four copies of proteins $\text{L}\text{7}\text{L}\text{12}$. Removal of the four copies of this protein produced a more rapidly migrating form, M-50 S. The M-50 S form was then converted to the fastest migrating form, F-50 S, by removal of additional proteins, including L10 and L11. A one-step removal of a pentameric complex of four copies of $\text{L}\text{7}\text{L}\text{12}$ plus L10 converted the S-50 S subunit directly to the F-50 S subunit. These proteins recombined specifically with the appropriate protein-deficient 50 S subunit at 3 °C to reform the S-50 S subunit, i.e. the M-50 S subunit was converted back to the S-50 S form by the addition of purified proteins $\text{L}\text{7}\text{L}\text{12}$; and the F-50 S subunit bound the pentameric complex of $\text{L}\text{7}\text{L}\text{12}$ and L10 to form S-50 S. The binding of the pentameric complex, isolated by glycerol gradient centrifugation, supports the model that all four copies of proteins $\text{L}\text{7}\text{L}\text{12}$ are together in one part of the ribosome called the “$\text{L}\text{7}\text{L}\text{12}$ stalk”. Only the four copies of $\text{L}\text{7}\text{L}\text{12}$ were removed from the 50 S subunit in low salt (0.125 m-NH₄Cl) plus 50% ethanol at 0 °C. These ribosomes (in the M-50 S form) had less than 5% of the peptide-synthesizing activity of untreated control ribosomes as measured by a poly(U) translation system in vitro. Peptide-synthesizing activity was restored, upon addition of $\text{L}\text{7}\text{L}\text{12}$, back to the treated ribosomes to give 50 S subunits (S-50 S) with a full complement of four copies of $\text{L}\text{7}\text{L}\text{12}$. Antibody to proteins $\text{L}\text{7}\text{L}\text{12}$ bound only to the S-50 S subunits, producing four new bands separated by gel electrophoresis. The bands represented complexes of one, two, three and four antibodies bound to a 50 S subunit. This result was obtained using either 50 S subunits or 70 S tight couples and indicated that all four copies of $\text{L}\text{7}\text{L}\text{12}$ are either located at a single site in the $\text{L}\text{7}\text{L}\text{12}$ stalk or, much less likely, are divided between two symmetrical sites. Proteins $\text{L}\text{7}\text{L}\text{12}$ were not only accessible to their specific antibody but could also be removed from 70 S ribosomes and polyribosomes without causing their dissociation into subunits. The ribosomes and polyribosomes had an increased gel electrophoretic mobility which was reversed by addition of proteins $\text{L}\text{7}\text{L}\text{12}$.     

Requirement for the Escherichia coli ribosomal protein L11 in peptide chain termination.

Subparticles of the Escherichia coli 50 S ribosome subunit containing varying amounts of the protein L11 have been prepared. These core particles have been used to form 70 S couples containing f[³H]Met-tRNA as a substrate for the peptidyl hydrolysis reaction of in vitro termination. Studies with antibodies against L11 suggested previously that the protein was involved in this event. The peptidyl transferase of the 50 S subunit core particles containing no more than 6% of the normal complement of L11 was fully active. The 70 S couples formed from 50 S cores lacking L11 showed some decrease in their ability to bind fMet-tRNA. Ribosomes lacking the proteins $\text{L}\text{7}\text{L}\text{12}$ retained about 50% of their activity for the peptidyl-tRNA hydrolysis event of in vitro termination. Cores lacking both $\text{L}\text{7}\text{L}\text{12}$ and L11 were almost as active as those lacking only $\text{L}\text{7}\text{L}\text{12}$. L11 is, therefore, not absolutely required for peptidyl-tRNA hydrolysis at termination in vitro. The ribosome subparticles lacking L11 have been reconstituted with $\text{L}\text{7}\text{L}\text{12}$. Despite the absence of L11, they regained significant activity for the codon-directed in vitro termination reaction.     

Monoclonal antibodies to Escherichia coli ribosomal proteins L9 and L10. Effects on ribosome function and localization of L9 on the surface of the 50 S ribosomal subunit

Monoclonal antibodies against Escherichia coli ribosomal proteins L9 and L10 were obtained and their specificity confirmed by Western blot analysis of total ribosomal protein. This was particularly important for the L9 antibody, since the immunizing antigen mixture contained predominantly L11. Each antibody recognized both 70 S ribosomes and 50 S subunits. Affinity-purified antibodies were tested for their effect on in vitro assays of ribosome function. Anti-L10 and anti-L9 inhibited poly(U)-directed polyphenylalanine synthesis almost completely. The antibodies had no effect on subunit association or dissociation and neither antibody inhibited peptidyltransferase activity. Both antibodies inhibited the binding of the ternary complex that consisted of aminoacyl-tRNA, guanylyl beta, gamma-methylenediphosphonate, and elongation factor Tu, and the binding of elongation factor G to the ribosome. The intact antibodies were more potent inhibitors than the Fab fragments. In contrast to the previously established location of L10 at the base of the L7/L12 stalk near the factor-binding site, the site of anti-L9 binding to 50 S subunits was shown by immune electron microscopy to be on the L1 lateral protuberance opposite the L7/L12 stalk as viewed in the quasisymmetric projection. The inhibition of factor binding by both antibodies, although consistent with established properties of L10 in the ribosome, suggests a long range effect on subunit structure that is triggered by the binding of anti-L9.     

Structural alteration of rRNA in the L7/L12 region of 50s ribosome on removal of L7/L12 proteins

Core particles of 50S ribosomes depleted of $\text{L7}\text{L12}$ proteins are degraded by RNase I at a considerably slower rate than intact 50S ribosomes. The normal rate is restored on incorporating $\text{L7}\text{L12}$ proteins into the core particles. The capacity of the core particles to inhibit the RNase I-catalyzed hydrolysis of poly A and to bind ethidium bromide is also greater with core particles than with intact 50S ribosomes. It appears from these results that the region(s) of rRNA in the vicinity of $\text{L7}\text{L12}$ proteins has less ordered structure which, on removal of $\text{L7}\text{L12}$ proteins, becomes more organized. Apparently, binding of $\text{L7}\text{L12}$ proteins to the 50S core leads to the destabilization of double-stranded regions of rRNA.     

Analysis of kethoxal bound to ribosomal proteins from Escherichia coli 70S reacted ribosomes

To investigate ribosome topography and possible function, 70S ribosomes of $\text{Escherichia coli}$ were reacted with the dicarbonyl compound kethoxal. Ribosomal protein was extracted after reaction, and through two dimensional gel electrophoresis, the reactive proteins of the two subunits were identified. From the 30S subunit, the most reacted proteins were S2, S3, S4, S5 and S7 and from the 50S subunit, L1, L5, L16, L17, L18 and L27. The results with kethoxal are compared with other modifiers of ribosomal proteins.     

RNA-protein interactions in the ribosome: VIII. Co-operative interactions in the 50 S subunit of Escherichia coli

Six 50 S ribosomal subunit proteins, each unable to interact independently with the 23 S RNA, were shown to associate specifically with ribonucleoprotein complexes consisting of intact 23 S RNA, or fragments derived from it, and one or more RNA-binding proteins. In particular, L21 and L22 depend for attachment upon L20 and L24, respectively; L5, L10 and L11 interact individually with complexes containing L2 and L16; and one or both proteins of the $\text{L}\text{17}\text{L}\text{27}$ mixture are stimulated to bind in the presence of L1, L3, L6, L13 and L23. Moreover, L14 alone was found to interact with a fragment from the 3′ end of the 23 S RNA, even though it cannot bind to 23 S RNA. By correlating the data reported here with the findings of others, it has been possible to formulate a partial in vitro assembly map of the Escherichia coli 50 S subunit encompassing both the 5 S and 23 S RNAs as well as 21 of the 34 subunit proteins.     

Regulation of the in vitro synthesis of E. coli ribosomal protein L12.

The $\text{in}\text{vitro}$ DNA dependent synthesis of ribosomal protein L12 and the β subunit of RNA polymerase has been investigated using DNA from a plasmid which contains the genetic information for ribosomal protein L12 and the β subunit of RNA polymerase. This DNA, however, lacks the promoter region and the genetic information for the first 26 amino acids of ribosomal protein L10. It was found that L12 and the β subunit of RNA polymerase are efficiently synthesized $\text{in}\text{vitro}$ from this DNA. These results suggest that L12 and the β subunit of RNA polymerase can be synthesized from a promoter situated within the L10 gene.     

Involvement of 50S ribosomal proteins L6 and L10 in the ribosome dependent GTPase activity of elongation factor G

CsCI-prepared 50S cores in the presence of groups of individual split proteins were tested for their capacity to support EF-G dependent GTP hydrolysis. The activity of cores prepared at 40 mM Mg²⁺ could be restored by adding $\text{L7}\text{L12}$ and L10 together, each in an amount of two copies per 50S particle, which abolishes the difference in activity between L7 and L12. In the range of 20-2 mM Mg²⁺, 50S cores lose the protein L6, which is also required for GTP hydrolysis. L10 cannot replace L6, or $\text{vice versa}$.     

Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit

Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes from Trypanosoma brucei with reduced RNA and increased protein mass to provide insights into the biogenesis of the mitoribosomal large subunit. Structural characterization of a stable assembly intermediate revealed 22 assembly factors, some of which have orthologues/counterparts/homologues in mammalian genomes. These assembly factors form a protein network that spans a distance of 180 Å, shielding the ribosomal RNA surface. The central protuberance and L7/L12 stalk are not assembled entirely and require removal of assembly factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt‐EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl‐carrier protein plays a role in docking the L1 stalk, which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly while the exit tunnel is blocked. Together, this extensive network of accessory factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.     

A complex of acidic ribosomal proteins. Evidence of a four-to-one complex of proteins in the Bacillus stearothermophilus ribosome

Two acidic proteins from the 50 S subunit of Bacillus stearothermophilus ribosomes, namely B-L13 (homologous to Escherichia coli protein $\text{L}\text{7}\text{L}\text{12}$) and B-L8, form a complex. Radioactive B-L13, added to ribosomes before dissociation, does not appear in the complex after electrophoresis, so the (B-L13 · B-L8) complex must exist in the ribosome before dissociation. Digestion of B. stearothermophilus ribosomes with polyacrylamide-bound trypsin causes the appearance of new B-L8 and B-L13 spots on two-dimensional polyacrylamide gel electrophoresis, in a pattern which suggests that single molecules of B-L13 are being sequentially cleaved from a four-to-one complex of B-L13 and B-L8.     

RNA-protein interactions in the ribosome. II. Binding of ribosomal proteins to isolated fragments of the 16 S RNA

Specific fragments of the 16 S ribosomal RNA of Escherichia coli have been isolated and tested for their ability to interact with proteins of the 30 S ribosomal subunit. The 12 S RNA, a 900-nucleotide fragment derived from the 5′-terminal portion of the 16 S RNA, was shown to form specific complexes with proteins S4, S8, S15, and S20. The stoichiometry of binding at saturation was determined in each case. Interaction between the 12 S RNA and protein fraction $\text{S}\text{16}\text{S}\text{17}$ was detected in the presence of S4, S8, S15 and S20; only these proteins were able to bind to this fragment, even when all 21 proteins of the 30 S subunit were added to the reaction mixture. Protein S4 also interacted specifically with the 9 S RNA, a fragment of 500 nucleotides that corresponds to the 5′-terminal third of the 16 S RNA, and protein S15 bound independently to the 4 S RNA, a fragment containing 140 nucleotides situated toward the middle of the RNA molecule. None of the proteins interacted with the 600-nucleotide 8 S fragment that arose from the 3′-end of the 16 S RNA.When the 16 S RNA was incubated with an unfractionated mixture of 30 S subunit proteins at 0 °C, 10 to 12 of the proteins interacted with the ribosomal RNA to form the reconstitution intermediate (RI) particle. Limited hydrolysis of this particle with T₁ ribonuclease yielded 14 S and 8 S subparticles whose RNA components were indistinguishable from the 12 S and 8 S RNAs isolated from digests of free 16 S RNA. The 14 S subparticle contained proteins S6 and S18 in addition to the RNA-binding proteins S4, S8, S15, S20 and $\text{S}\text{16}\text{S}\text{17}$. The 8 S subparticle contained proteins S7, S9, S13 and S19. These findings serve to localize the sites at which proteins incapable of independent interaction with 16 S RNA are fixed during the early stages of 30 S subunit assembly.     

Investigation of structure of the ribosomal L12/P stalk

This review contains recent data on the structure of the functionally important ribosomal domain, L12/P stalk, of the large ribosomal subunit. It is the most mobile site of the ribosome; it has been found in ribosomes of all living cells, and it is involved in the interaction between ribosomes and translation factors. The difference between the structures of the ribosomal proteins forming this protuberance (despite their general resemblance) determines the specificity of interaction between eukaryotic and prokaryotic ribosomes and the respective protein factors of translation. In this review, works on the structures of ribosomal proteins forming the L12/P-stalk in bacteria, archaea, and eukaryotes and data on structural aspects of interactions between these proteins and rRNA are described in detail.     

DNA-dependent in vitro synthesis of Escherichia coli ribosomal protein L10 and the formation of an L10L12 complex

The $\text{in vitro}$ synthesis of ribosomal protein L10 has been demonstrated using λrif^d18 DNA as template. The L10 synthesized $\text{in vitro}$ forms a complex with ribosomal protein L12 and the L10 in this complex can be immunoprecipitated with L12 antiserum.     

Conformational variability in Escherichia coli 70S ribosome as revealed by 3D cryo-electron microscopy

During protein biosynthesis, ribosomes are believed to go through a cycle of conformational transitions. We have identified some of the most variable regions of the E. coli 70S ribosome and its subunits, by means of cryo-electron microscopy and three-dimensional (3D) reconstruction. Conformational changes in the smaller 30S subunit are mainly associated with the functionally important domains of the subunit, such as the neck and the platform, as seen by comparison of heat-activated, non-activated and 50S-bound states. In the larger 50S subunit the most variable regions are the L7/L12 stalk, central protuberance and the L1-protein, as observed in various tRNA-70S ribosome complexes. Difference maps calculated between 3D maps of ribosomes help pinpoint the location of ribosomal regions that are most strongly affected by conformational transitions. These results throw direct light on the dynamic behavior of the ribosome and help in understanding the role of these flexible domains in the translation process.     

Characterization of two acidic proteins of Saccharomyces cerevisiae ribosome

In $\text{Saccharomyces}\text{cerevisiae}$ ribosomes two proteins, L44 and L45, of strong acidic character are detected. These proteins, presumably equivalent to bacterial L7 and L12, have been purified and have given a total cross reaction when tested by double immunodiffusion. Reaction with fluorescamine has shown that the amino terminal group of the polypeptide is blocked in protein L44 and free in protein L45. Tryptic analysis of the two proteins shows that three out of nine peptides are in identical position in both patterns, three more are easily related and the last three are clearly different. The data indicate that proteins L44 and L45 are closely related but not totally identical.     

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.     

Interaction of membrane surface charges with the reconstituted ADP/ATP-carrier from mitochondria

Various modulating influences of negative and positive membrane charges on binding and transport properties of the reconstituted ADP/ATP carrier from mitochondria were investigated. The results are interpreted in terms of functional and structural asymmetries of the adenine nucleotide carrier embedded in the liposomal membrane. The surface potential of liposomes was measured directly either by potential-dependent adsorption of the fluorescent dye $\text{2-p-}\text{toluidinylnaphthalene}$ 6-sulfonate (TNS) or by the $\text{p}\text{K}$ shift of the lipophilic pH indicator pentadecylumbelliferone. These results were correlated with the following observations. (1) Negative surface potentials increase the apparent dissociation constant, $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$, for binding of the negatively charged inhbitor carboxyatractylate to the reconstituted carrier protein. (2) Surface potentials modulate the apparent transport affinity, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, of the reconstituted adenine nucleotide carrier for ADP and ATP. The interaction of surface charges with the transport function was investigated with carrier proteins oriented both right-side-out and inside-out. Thus the influence of the surface potential on the function of the ADP/ATP carrier could be determined for the internal and external active sites of the translocator on the outer side of the membrane. Large discrepancies were observed not only between the potentials measured directly (fluorescent dyes) and those measured indirectly (binding and transport affinities), but also between the different surface potentials determined from the influence on the alternatively oriented carrier proteins. The effect of surface charges was rather weak on the cytosolic side of the translocator, whereas there was a strong influence of surface charges on the active site at the matrix side. The most obvious explanation, i.e., screening of negative membrane charges by positively charged amino acid residues at the protein surface, could be ruled out. Besides the modulation of binding affinities for substrates and inhibitors, an additional side-specific effect of surface charges on the transport velocity was observed. Again, the influence on the internal active site of the ADP/ATP carrier was found to be much higher than that on the cytosolic site. The observed effects can be explained by a definite structural asymmetry of the carrier embedded in the liposomal membrane. That site which is physiologically exposed to the cytosol is located at a considerable distance from the plane of the membrane, whereas the opposite site seems to be in close proximity to the membrane surface. Moreover, a spatial equivalence of carboxyatractylate binding site and nucleotide binding site at the external side of the carrier protein was concluded.     

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.