New insights into the interaction of ribosomal protein L1 with RNA

The RNA-binding ability of ribosomal protein L1 is of profound interest, since L1 has a dual function as a ribosomal structural protein that binds rRNA and as a translational repressor that binds its own mRNA. Here, we report the crystal structure at 2.6 A resolution of ribosomal protein L1 from the bacterium Thermus thermophilus in complex with a 38 nt fragment of L1 mRNA from Methanoccocus vannielii. The conformation of RNA-bound T.thermophilus L1 differs dramatically from that of the isolated protein. Analysis of four copies of the L1-mRNA complex in the crystal has shown that domain II of the protein does not contribute to mRNA-specific binding. A detailed comparison of the protein-RNA interactions in the L1-mRNA and L1-rRNA complexes identified amino acid residues of L1 crucial for recognition of its specific targets on the both RNAs. Incorporation of the structure of bacterial L1 into a model of the Escherichia coli ribosome revealed two additional contact regions for L1 on the 23S rRNA that were not identified in previous ribosome models.     

Structure of 5S rRNA within the Escherichia coli ribosome: iodine-induced cleavage patterns of phosphorothioate derivatives.

The protection patterns of 5S rRNA in solution, within the ribosomal 50S subunit, 70S ribosomes, and functional complexes, were assessed with the phosphorothioate method. About 20% of the analyzed positions (G9-G107) showed strong assembly defects: A phosphorothioate at one of these positions significantly impaired the incorporation of 5S rRNA into 50S particles. The reverse has also been observed: A phosphorothioate is preferred over a phosphate residue in the assembly process at a few positions. The results further demonstrate that 5S rRNA undergoes conformational changes during the assembly in the central protuberance of the 50S subunit and upon association with the small ribosomal subunit forming a 70S ribosome. In striking contrast, when the 70S ribosomes are once formed, the contact pattern of the 5S rRNA is the same in various functional states such as initiation-like complexes and pre- and posttranslocational states.     

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.     

Atomic structures at last: the ribosome in 2000

Last year, atomic structures of the 50S ribosomal subunit from Haloarcula marismortui and of the 30S ribosomal subunit from Thermus thermophilus were published. A year before that, a 7.8 A resolution electron density map of the 70S ribosome from T. thermophilus appeared. This information is revolutionizing our understanding of protein synthesis.     

Structure of the mammalian ribosome-channel complex at 17A resolution

The co-translational translocation of proteins into the endoplasmic reticulum (ER) lumen and the biogenesis of membrane proteins require ribosome binding to a membrane channel formed by the Sec61p complex. We now report the 17A structure of a mammalian ribosome-channel complex derived from ER membranes. Atomic models of the ribosomal subunits were aligned to the programmed ribosome from Thermus thermophilus, to provide a common reference frame. The T.thermophilus ribosome, and by extension all known high resolution subunit models, were then docked within our map of the ribosome-channel complex. The structure shows that the ribosome contains a putative tRNA in the exit site, and a comparison with a non-programmed, yeast ribosome suggests that the L1 stalk may function as a gate in the tRNA exit path. We have localized six major expansion segments in the large subunit of the vertebrate ribosome including ES27, and suggest a function for ES30.The large ribosomal subunit is linked to the channel by four connections. We identified regions in the large subunit rRNA and four proteins that may help form the connections. These regions of the ribosome probably serve as a template to guide the assembly of the asymmetric translocation channel. Three of the connections form a picket fence that separates the putative translocation pore from the attachment site of an additional membrane component. The ribosome-channel connections also create an open junction that would allow egress of a nascent chain into the cytosol. At a threshold that is appropriate for the entire complex, the channel is rather solid and the lumenal half of the putative translocation pore is closed. These data suggest that the flow of small molecules across the membrane may be impeded by the channel itself, rather than the ribosome-channel junction.     

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.     

A short fragment of 23S rRNA containing the binding sites for two ribosomal proteins, L24 and L4, is a key element for rRNA folding during early assembly

Previously we described an in vitro selection variant abbreviated SERF (in vitro selection from random rRNA fragments) that identifies protein binding sites within large RNAs. With this method, a small rRNA fragment derived from the 23S rRNA was isolated that binds simultaneously and independently the ribosomal proteins L4 and L24 from Escherichia coli. Until now the rRNA structure within the ternary complex L24-rRNA-L4 could not be studied due to the lack of an appropriate experimental strategy. Here we tackle the issue by separating the various complexes via native gel-electrophoresis and analyzing the rRNA structure by in-gel iodine cleavage of phosphorothioated RNA. The results demonstrate that during the transition from either the L4 or L24 binary complex to the ternary complex the structure of the rRNA fragment changes significantly. The identified protein binding sites are in excellent agreement with the recently reported crystal structure of the 50S subunit. Because both proteins play a prominent role in early assembly of the large subunit, the results suggest that the identified rRNA fragment is a key element for the folding of the 23S RNA during early assembly. The introduced in-gel cleavage method should be useful when an RNA structure within mixed populations of different but related complexes should be studied.     

L22 ribosomal protein and effect of its mutation on ribosome resistance to erythromycin

The ribosomal protein L22 is a core protein of the large ribosomal subunit interacting with all domains of the 23S rRNA. The triplet Met82-Lys83-Arg84 deletion in L22 from Escherichia coli renders cells resistant to erythromycin which is known as an inhibitor of the nascent peptide chain elongation. The crystal structure of the Thermus thermophilus L22 mutant with equivalent triplet Leu82-Lys83-Arg84 deletion has been determined at 1.8A resolution. The superpositions of the mutant and the wild-type L22 structures within the 50S subunits from Haloarcula marismortui and Deinococcus radiodurans show that the mutant beta-hairpin is bent inward the ribosome tunnel modifying the shape of its narrowest part and affecting the interaction between L22 and 23S rRNA. 23S rRNA nucleotides of domain V participating in erythromycin binding are located on the opposite sides of the tunnel and are brought to those positions by the interaction of the 23S rRNA with the L22 beta-hairpin. The mutation in the L22 beta-hairpin affects the orientation and distances between those nucleotides. This destabilizes the erythromycin-binding "pocket" formed by 23S rRNA nucleotides exposed at the tunnel surface. It seems that erythromycin, while still being able to interact with one side of the tunnel but not reaching the other, is therefore unable to block the polypeptide growth in the drug-resistant ribosome.     

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.     

How does the mRNA pass through the ribosome?

A working model of the mRNA path through the ribosome is proposed. According to the model, the template goes around the small ribosomal subunit along the region where its 'head' is separated from other parts of the subunit. The 5'-end of the mRNA fragment covered by the ribosome is located near the 3'-terminus of 16S rRNA, whereas the 3'-terminal residues of the fragment are situated on the outer surface of the subunit, opposite its 'side ledge'. When associated with the 50S subunit, the 30S subunit is oriented in such a manner that the decoding center faces the L7/L12 stalk. Implications of the proposed working model of the mRNA topography for the function of the ribosome are discussed.     

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.     

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.     

Following the dynamics of changes in solvent accessibility of 16 S and 23 S rRNA during ribosomal subunit association using synchrotron-generated hydroxyl radicals

We have probed the structure and dynamics of ribosomal RNA in the Escherichia coli ribosome using equilibrium and time-resolved hydroxyl radical (OH) RNA footprinting to explore changes in the solvent-accessible surface of the rRNA with single-nucleotide resolution. The goal of these studies is to better understand the structural transitions that accompany association of the 30 S and 50 S subunits and to build a foundation for the quantitative analysis of ribosome structural dynamics during translation. Clear portraits of the subunit interface surfaces for 16 S and 23 S rRNA were obtained by constructing difference maps between the OH protection maps of the free subunits and that of the associated ribosome. In addition to inter-subunit contacts consistent with the crystal structure, additional OH protections are evident in regions at or near the subunit interface that reflect association-induced conformational changes. Comparison of these data with the comparable difference maps of the solvent-accessible surface of the rRNA calculated for the Thermus thermophilus X-ray crystal structures shows extensive agreement but also distinct differences. As a prelude to time-resolved OH footprinting studies, the reactivity profiles obtained using Fe(II)EDTA and X-ray generated OH were comprehensively compared. The reactivity patterns are similar except for a small number of nucleotides that have decreased reactivity to OH generated from Fe(II)EDTA compared to X-rays. These nucleotides are generally close to ribosomal proteins, which can quench diffusing radicals by virtue of side-chain oxidation. Synchrotron X-ray OH footprinting was used to monitor the kinetics of association of the 30 S and 50 S subunits. The rates individually measured for the inter-subunit contacts are comparable within experimental error. The application of this approach to the study of ribosome dynamics during the translation cycle is discussed.     

Localization of the protein L2 in the 50 S subunit and the 70 S E. coli ribosome

The protein L2 is found in all ribosomes and is one of the best conserved proteins of this mega-dalton complex. The protein was localized within both the isolated 50 S subunit and the 70 S ribosome of the Escherichia coli bacteria with the neutron-scattering technique of spin-contrast variation. L2 is elongated, exposing one end of the protein to the surface of the intersubunit interface of the 50 S subunit. The protein changes its conformation slightly when the 50 S subunit reassociates with the 30 S subunit to form a 70 S ribosome, becoming more elongated and moving approximately 30 A into the 50 S matrix. The results support a recent observation that L2 is essential for the association of the ribosomal subunits and might participate in the binding and translocation of the tRNAs.     

Cross-linking of streptomycin to the 50S subunit of Escherichia coli with phenyldiglyoxal

[3H]Dihydrostreptomycin was covalently linked to the 50S subunit of Escherichia coli K12A19 with the bifunctional cross-linking reagent phenyldiglyoxal. The cross-linking was abolished under conditions that prevent the specific interaction of streptomycin with the ribosome. The binding primarily involved the ribosomal RNA and also a limited number of proteins, namely, L2, L6, and L17. This suggests that the binding domain for streptomycin is close to the peptidyl transferase center, in the valley between the central protuberance and the wider lateral protuberance of the 50S subunit. This domain faces the binding domain for streptomycin which we have previously characterized on the 30S subunit [Melan?on, P., Boileau, G., & Brakier-Gingras, L. (1984) Biochemistry 23, 6697-6703]. Our results indicate that the 50S subunit is involved in the binding of streptomycin to the bacterial ribosome, in addition to the 30S subunit which is generally considered as the specific target of the antibiotic. They are consistent with the occurrence of a single binding site for streptomycin on the ribosome, comprised of regions of both subunits.     

Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5.

Ribosomal protein L5 binds specifically to 5S rRNA to form a complex that is a precursor to 60S subunit assembly in vivo. Analyses in yeast cells, mammalian cells, and Xenopus embryos have shown that the accumulation of L5 is not coordinated with the expression of other ribosomal proteins. In this study, the primary structure and developmental expression of Xenopus ribosomal protein L5 were examined to determine the basis for its distinct regulation. These analyses showed that L5 expression could either coincide with 5S rRNA synthesis and ribosome assembly or be controlled independently of these events at different stages of Xenopus development. L5 synthesis during oogenesis was uncoupled from the accumulation of 5S rRNa but coincided with subunit assembly. In early embryos, the inefficient translation of L5 mRNA resulted in the accumulation of a stable L5-5S rRNA complex before ribosome assembly at later stages of development. Additional results demonstrated that L5 protein synthesized in vitro bound specifically to 5S rRNA.     

Structure of the base of the L7/L12 stalk of the Haloarcula marismortui large ribosomal subunit: analysis of L11 movements

Initiation factors, elongation factors, and release factors all interact with the L7/L12 stalk of the large ribosomal subunit during their respective GTP-dependent cycles on the ribosome. Electron density corresponding to the stalk is not present in previous crystal structures of either 50 S subunits or 70 S ribosomes. We have now discovered conditions that result in a more ordered factor-binding center in the Haloarcula marismortui (H.ma) large ribosomal subunit crystals and consequently allows the visualization of the full-length L11, the N-terminal domain (NTD) of L10 and helices 43 and 44 of 23 S rRNA. The resulting model is currently the most complete reported structure of a L7/L12 stalk in the context of a ribosome. This region contains a series of intermolecular interfaces that are smaller than those typically seen in other ribonucleoprotein interactions within the 50 S subunit. Comparisons of the L11 NTD position between the current structure, which is has an NTD splayed out with respect to previous structures, and other structures of ribosomes in different functional states demonstrates a dynamic range of L11 NTD movements. We propose that the L11 NTD moves through three different relative positions during the translational cycle: apo-ribosome, factor-bound pre-GTP hydrolysis and post-GTP hydrolysis. These positions outline a pathway for L11 NTD movements that are dependent on the specific nucleotide state of the bound ligand. These three states are represented by the orientations of the L11 NTD relative to the ribosome and suggest that L11 may play a more specialized role in the factor binding cycle than previously appreciated.     

Identification of 50S components neighboring 23S rRNA nucleotides A2448 and U2604 within the peptidyl transferase center of Escherichia coli ribosomes

The 23S rRNA nucleotides 2604-12 and 2448-58 fall within the central loop of domain V, which forms a major part of the peptidyl transferase center of the ribosome. We report the synthesis of radioactive, photolabile 2'-O-methyloligoRNAs, PHONTs 1 and 2, complementary to these nucleotides and their exploitation in identifying 50S ribosomal subunit components neighboring their target sites. Photolysis of the 50S complex with PHONT 1, complementary to nts 2604-12, leads to target site-specific photoincorporation into protein L2 and 23S rRNA nucleotides A886, Alpha1918, A1919, G1922-C1924, U2563, U2586, and C2601. Photolysis of the 50S complex with PHONT 2, complementary to nts 2448-58, leads to target site-specific probe photoincorporation into proteins L2, L3, one or more of proteins L17, L18, L21, and of proteins L9, L15, L16, and 23S rRNA nucleotides C2456 and psi2457. Chemical footprinting studies show that 2'-O-methyloligoRNA binding causes little distortion of the peptidyl transferase center but do provide suggestive evidence for the location of flexible regions within 23S rRNA. The significance of these results for the structure of the peptidyl transferase center is considered.