Domain III of Saccharomyces cerevisiae 25 S ribosomal RNA: its role in binding of ribosomal protein L25 and 60 S subunit formation

Domain III of Saccharomyces cerevisiae 25 S rRNA contains the recognition site for the primary rRNA-binding ribosomal protein L25, which belongs to the functionally conserved EL23/L25 family of ribosomal proteins. The EL23/L25 binding region is very complex, consisting of several irregular helices held together by long-distance secondary and tertiary interactions. Moreover, it contains the eukaryote-specific V9 (D7a) expansion segment. Functional characterisation of the structural elements of this site by a detailed in vitro and in vivo mutational analysis indicates the presence of two separate regions that are directly involved in L25 binding. In particular, mutation of either of two conserved nucleotides in the loop of helix 49 significantly reduces in vitro L25 binding, thus strongly supporting their role as attachment sites for the r-protein. Two other helices appear to be primarily required for the correct folding of the binding site. Mutations that abolish in vitro binding of L25 block accumulation of 25 S rRNA in vivo because they stall pre-rRNA processing at the level of its immediate precursor, the 27 S(B) pre-rRNA. Surprisingly, several mutations that do not significantly affect L25 binding in vitro cause the same lethal defect in 27 S(B) pre-rRNA processing. Deletion of the V9 expansion segment also leads to under-accumulation of mature 25 S rRNA and a twofold reduction in growth rate. We conclude that an intact domain III, including the V9 expansion segment, is essential for normal processing and assembly of 25 S rRNA.     

Role of protein L25 and its contact with protein L16 in maintaining the active state of <Emphasis Type="Italic">Escherichia coli</Emphasis> ribosomes <Emphasis Type="Italic">in vivo</Emphasis>

A ribosomal protein of the L25 family specifically binding to 5S rRNA is an evolutionary feature of bacteria. Structural studies showed that within the ribosome this protein contacts not only 5S rRNA, but also the C-terminal region of protein L16. Earlier we demonstrated that ribosomes from the ΔL25 strain of Escherichia coli have reduced functional activity. In the present work, it is established that the reason for this is a fraction of functionally inactive 50S ribosomal subunits. These subunits have a deficit of protein L16 and associate very weakly with 30S subunits. To study the role of the contact of these two proteins in the formation of the active ribosome, we created a number of E. coli strains containing protein L16 with changes in its C-terminal region. We found that some mutations (K133L or K127L/K133L) in this protein lead to a noticeable slowing of cell growth and decrease in the activity of their translational apparatus. As in the case of the ribosomes from the ΔL25 strain, the fraction of 50S subunits, which are deficient in protein L16, is present in the ribosomes of the mutant strains. All these data indicate that the contact with protein L25 is important for the retention of protein L16 within the E. coli ribosome in vivo. In the light of these findings, the role of the protein of the L25 family in maintaining the active state of the bacterial ribosome is discussed.     

NMR structure of bacterial ribosomal protein l20: implications for ribosome assembly and translational control

L20 is a specific protein of the bacterial ribosome, which is involved in the early assembly steps of the 50S subunit and in the feedback control of the expression of its own gene. This dual function involves specific interactions with either the 23S rRNA or its messenger RNA. The solution structure of the free Aquifex aeolicus L20 has been solved. It is composed of an unstructured N-terminal domain comprising residues 1-58 and a C-terminal alpha-helical domain. This is in contrast with what is observed in the bacterial 50S subunit, where the N-terminal region folds as an elongated alpha-helical region. The solution structure of the C-terminal domain shows that several solvent-accessible, conserved residues are clustered on the surface of the molecule and are probably involved in RNA recognition. In vivo studies show that this domain is sufficient to repress the expression of the cistrons encoding L35 and L20 in the IF3 operon. The ability of L20 C-terminal domain to specifically recognise RNA suggests an assembly mechanism for L20 into the ribosome. The pre-folded C-terminal domain would make a primary interaction with a specific site on the 23S rRNA. The N-terminal domain would then fold within the ribosome, participating in its correct 3D assembly.     

GTPase activation of elongation factors Tu and G on the ribosome

The GTPase activity of elongation factors Tu and G is stimulated by the ribosome. The factor binding site is located on the 50S ribosomal subunit and comprises proteins L7/12, L10, L11, the L11-binding region of 23S rRNA, and the sarcin-ricin loop of 23S rRNA. The role of these ribosomal elements in factor binding, GTPase activation, or functions in tRNA binding and translocation, and their relative contributions, is not known. By comparing ribosomes depleted of L7/12 and reconstituted ribosomes, we show that, for both factors, interactions with L7/12 and with other ribosomal residues contribute about equally and additively to GTPase activation, resulting in an overall 10(7)-fold stimulation. Removal of L7/12 has little effect on factor binding to the ribosome. Effects on other factor-dependent functions, i.e., A-site binding of aminoacyl-tRNA and translocation, are fully explained by the inhibition of GTP hydrolysis. Based on these results, we propose that L7/12 stimulates the GTPase activity of both factors by inducing the catalytically active conformation of the G domain. This effect appears to be augmented by interactions of other structural elements of the large ribosomal subunit with the switch regions of the factors.     

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.     

Functional conservation between structurally diverse ribosomal proteins from Drosophila melanogaster and Saccharomyces cerevisiae: fly L23a can substitute for yeast L25 in ribosome assembly and function

The proposed Drosophila melanogaster L23a ribosomal protein features a conserved C-terminal amino acid signature characteristic of other L23a family members and a unique N-terminal extension [Koyama et al. (Poly(ADP-ribose) polymerase interacts with novel Drosophila ribosomal proteins, L22 and l23a, with unique histone-like amino-terminal extensions. Gene 1999; 226: 339–345)], absent from Saccharomyces cerevisiae L25 that nearly doubles the size of fly L23a. The ability of fly L23a to replace the role of yeast L25 in ribosome biogenesis was determined by creating a yeast strain carrying an L25 chromosomal gene disruption and a plasmid-encoded FLAG-tagged L23a gene. Though affected by a reduced growth rate, the strain is dependent on fly L23a-FLAG function for survival and growth, demonstrating functional compatibility between the fly and yeast proteins. Pulse-chase experiments reveal a delay in rRNA processing kinetics, most notably at a late cleavage step that converts precursor 27S rRNA into mature 25S rRNA, likely contributing to the strain's slower growth pattern. Yet, given the essential requirement for L23(a)/L25 in ribosome biogenesis, there is a remarkable tolerance for accommodating the fly L23a N-terminal extension within the structure of the yeast ribosome. A search of available databases shows that the unique N-terminal extension is shared by multiple insect lineages. An evolutionary perspective on L23a structure and function within insect lineages is discussed.     

Structural diversity in bacterial ribosomes: mycobacterial 70S ribosome structure reveals novel features

Here we present analysis of a 3D cryo-EM map of the 70S ribosome from Mycobacterium smegmatis, a saprophytic cousin of the etiological agent of tuberculosis in humans, Mycobacterium tuberculosis. In comparison with the 3D structures of other prokaryotic ribosomes, the density map of the M. smegmatis 70S ribosome reveals unique structural features and their relative orientations in the ribosome. Dramatic changes in the periphery due to additional rRNA segments and extra domains of some of the peripheral ribosomal proteins like S3, S5, S16, L17, L25, are evident. One of the most notable features appears in the large subunit near L1 stalk as a long helical structure next to helix 54 of the 23S rRNA. The sharp upper end of this structure is located in the vicinity of the mRNA exit channel. Although the M. smegmatis 70S ribosome possesses conserved core structure of bacterial ribosome, the new structural features, unveiled in this study, demonstrates diversity in the 3D architecture of bacterial ribosomes. We postulate that the prominent helical structure related to the 23S rRNA actively participates in the mechanisms of translation in mycobacteria.     

A conserved motif is prerequisite for the interaction of NAC with ribosomal protein L23 and nascent chains

In eukaryotes, newly synthesized proteins interact co-translationally with a multitude of different ribosome-bound factors and chaperones including the conserved heterodimeric nascent polypeptide-associated complex (NAC) and a Hsp40/70-based chaperone system. These factors are thought to play an important role in protein folding and targeting, yet their specific ribosomal localizations, which are prerequisite for their functions, remain elusive. This study describes the ribosomal localization of NAC and the molecular details by which NAC is able to contact the ribosome and gain access to nascent polypeptides. We identified a conserved RRK(X)nKK ribosome binding motif within the beta-subunit of NAC that is essential for the entire NAC complex to attach to ribosomes and allow for its interaction with nascent polypeptide chains. The motif localizes within a potential loop region between two predicted alpha-helices in the N terminus of betaNAC. This N-terminal betaNAC ribosome-binding domain was completely portable and sufficient to target an otherwise cytosolic protein to the ribosome. NAC modified with a UV-activatable cross-linker within its ribosome binding motif specifically cross-linked to L23 ribosomal protein family members at the exit site of the ribosome, providing the first evidence of NAC-L23 interaction in the context of the ribosome. Mutations of L23 reduced NAC ribosome binding in vivo and in vitro, whereas other eukaryotic ribosome-associated factors such as the Hsp70/40 chaperones Ssb or Zuotin were unaffected. We conclude that NAC employs a conserved ribosome binding domain to position itself on the L23 ribosomal protein adjacent to the nascent polypeptide exit site.     

Structure of the L1 protuberance in the ribosome

The L1 protuberance of the 50S ribosomal subunit is implicated in the release/disposal of deacylated tRNA from the E site. The apparent mobility of this ribosomal region has thus far prevented an accurate determination of its three-dimensional structure within either the 50S subunit or the 70S ribosome. Here we report the crystal structure at 2.65 A resolution of ribosomal protein L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus. This structure fills a major gap in current models of the 50S ribosomal subunit. The conformations of L1 and of the rRNA fragment differ dramatically from those within the crystallographic model of the T. thermophilus 70S ribosome. Incorporation of the L1-rRNA complex into the structural models of the T. thermophilus 70S ribosome and the Deinococcus radiodurans 50S subunit gives a reliable representation of most of the L1 protuberance within the ribosome.     

Thiostrepton-resistant mutants of Thermus thermophilus

Ribosomal protein L11 and its associated binding site on 23S rRNA together comprise one of the principle components that mediate interactions of translation factors with the ribosome. This site is also the target of the antibiotic thiostrepton, which has been proposed to act by preventing important structural transitions that occur in this region of the ribosome during protein synthesis. Here, we describe the isolation and characterization of spontaneous thiostrepton-resistant mutants of the extreme thermophile, Thermus thermophilus. All mutations were found at conserved positions in the flexible N-terminal domain of L11 or at conserved positions in the L11-binding site of 23S rRNA. A number of the mutant ribosomes were affected in in vitro EF-G-dependent GTP hydrolysis but all showed resistance to thiostrepton at levels ranging from high to moderate. Structure probing revealed that some of the mutations in L11 result in enhanced reactivity of adjacent rRNA bases to chemical probes, suggesting a more open conformation of this region. These data suggest that increased flexibility of the factor binding site results in resistance to thiostrepton by counteracting the conformation-stabilizing effect of the antibiotic.     

The central core region of yeast ribosomal protein L11 is important for subunit joining and translational fidelity

Yeast ribosomal protein L11 is positioned at the intersubunit cleft of the large subunit central protuberance, forming an intersubunit bridge with the small subunit protein S18. Mutants were engineered in the central core region of L11 which interacts with Helix 84 of the 25S rRNA. Numerous mutants in this region conferred 60S subunit biogenesis defects. Specifically, many mutations of F96 and the A66D mutant promoted formation of halfmers as assayed by sucrose density ultracentrifugation. Halfmer formation was not due to deficiency in 60S subunit production, suggesting that the mutants affected subunit-joining. Chemical modification analyses indicated that the A66D mutant, but not the F96 mutants, promoted changes in 25S rRNA structure, suggesting at least two modalities for subunit joining defects. 25S rRNA structural changes were located both adjacent to A66D (in H84), and more distant (in H96-7). While none of the mutants significantly affected ribosome/tRNA binding constants, they did have strong effects on cellular growth at both high and low temperatures, in the presence of translational inhibitors, and promoted changes in translational fidelity. Two distinct mechanisms are proposed by which L11 mutants may affect subunit joining, and identification of the amino acids associated with each of these processes are presented. These findings may have implications for our understanding of multifaceted diseases such as Diamond–Blackfan anemia which have been linked in part with mutations in L11.     

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.     

Yeast ribosomal protein L25 binds to an evolutionary conserved site on yeast 26S and E. coli 23S rRNA.

The binding site of the yeast 60S ribosomal subunit protein L25 on 26S rRNA was determined by RNase protection experiments. The fragments protected by L25 originate from a distinct substructure within domain IV of the rRNA, encompassing nucleotides 1465-1632 and 1811-1861. The protected fragments are able to rebind to L25 showing that they constitute the complete protein binding site. This binding site is remarkably conserved in all 23/26/28S rRNAs sequenced to date including Escherichia coli 23S rRNA. In fact heterologous complexes between L25 and E. coli 23S rRNA could be formed and RNase protection studies on these complexes demonstrated that L25 indeed recognizes the conserved structure. Strikingly the L25 binding site on 23S rRNA is virtually identical to the previously identified binding site of E. coli ribosomal protein EL23. Therefore EL23 is likely to be the prokaryotic counterpart of L25 in spite of the limited homology displayed by the amino acid sequences of the two proteins.     

Identification of a rRNA/chloramphenicol interaction site within the peptidyltransferase center of the 50 S subunit of the Escherichia coli ribosome.

We have used oligodeoxyribonucleotide probes to investigate possible interactions between chloramphenicol and portions of the rRNA contained within the peptidyltransferase center of the Escherichia coli ribosome. Oligodeoxyribonucleotide probes complementary to bases 2448-2454, 2468-2482, and 2497-2505 of 23 S rRNA were hybridized to 50 S subunits in situ. Probe binding was qualitatively assessed by sucrose gradient centrifugation. Each probe was shown to bind specifically with its intended binding site through digestion of the rRNA within the RNA/DNA hetero-duplexes with RNase H and analysis of the digestion fragments using gel electrophoresis. Competitive binding experiments were conducted between each probe and the antibiotics chloramphenicol and erythromycin. The binding of a probe complementary to bases 2497-2505 was attenuated by 70% upon the binding of chloramphenicol. A probe complementary to bases 2468-2482 showed an increase in binding of 14% while binding of a probe complementary to bases 2448-2454 was not affected by chloramphenicol binding. Erythromycin did not affect the binding of any of these probes to 50 S subunits. These results suggest that bases within the 2497-2505 region of 23 S rRNA in E. coli may be involved in a chloramphenicol/rRNA interaction.     

Translational regulation via L11: molecular switches on the ribosome turned on and off by thiostrepton and micrococcin

The thiopeptide class of antibiotics targets the GTPase-associated center (GAC) of the ribosome to inhibit translation factor function. Using X-ray crystallography, we have determined the binding sites of thiostrepton (Thio), nosiheptide (Nosi), and micrococcin (Micro), on the Deinococcus radiodurans large ribosomal subunit. The thiopeptides, by binding within a cleft located between the ribosomal protein L11 and helices 43 and 44 of the 23S rRNA, overlap with the position of domain V of EF-G, thus explaining how this class of drugs perturbs translation factor binding to the ribosome. The presence of Micro leads to additional density for the C-terminal domain (CTD) of L7, adjacent to and interacting with L11. The results suggest that L11 acts as a molecular switch to control L7 binding and plays a pivotal role in positioning one L7-CTD monomer on the G' subdomain of EF-G to regulate EF-G turnover during protein synthesis.     

Establishment of Arabidopsis thaliana ribosomal protein RPL23A-1 as a functional homologue of Saccharomyces cerevisiae ribosomal protein L25

Arabidopsis thaliana ribosomal protein (r-protein) RPL23A-1 shows 54% amino acid sequence identity to the Saccharomyces cerevisiae equivalent r-protein, L25. AtRPL23A-1 also shows high amino acid sequence identity to members of the L23/L25 r-protein family in other species. R-protein L25 in S. cerevisiae has been identified as a primary rRNA-binding protein that directly binds to a specific site on yeast 26S rRNA. It is translocated to the nucleolus where it binds to 26S rRNA during early large ribosome subunit assembly; this binding is thought to play an important role in ribosome assembly. The S. cerevisiae mutant strain YCR61 expresses L25 when grown on galactose, but not glucose, medium. Transformation of YCR61 with a shuttle vector containing the AtRPL23A-1 cDNA allowed transformed colonies to grow in and on glucose selection medium. R-protein AtRPL23A-1 can complement the L25 mutation, demonstrating the functional equivalence of the two r-proteins and introducing AtRPL23A-1 as the first plant member of the L23/L25 r-protein family.     

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.     

Solution structure of ribosomal protein L16 from Thermus thermophilus HB8

Ribosomal protein L16 is an essential component of the bacterial ribosome. It organizes the architecture of aminoacyl tRNA binding site in the ribosome 50S subunit. The three-dimensional structure of L16 from Thermus thermophilus HB8 was determined by NMR. In solution, L16 forms an alpha+beta sandwich structure combined with two additional beta sheets located at the loop regions connecting the two layers. The terminal regions and a central loop region did not show any specific secondary structure. The structured part of L16 could be superimposed well on the C(alpha) model of L16 determined in the crystal structure of the ribosome 50S subunit. By overlaying the L16 solution structure onto the coordinates of the ribosome crystal structure, we constructed the combined model that represents the ribosome-bound state of L16 in the detailed structure. The model showed that L16 possesses residues in contact with helices 38, 39, 42, 43 and 89 of 23S rRNA and helix 4 of 5S rRNA. This suggests its broad effect on the ribosome architecture. Comparison of L16 with the L10e protein, which is the archaeal counterpart, showed that they share a common fold, but differ in some regions of functional importance, especially in the N-terminal region. All known mutation sites in L16 that confer resistance to avilamycin and evernimicin were positioned so that their side-chains were exposed to solvent in the internal cavity of the ribosome. This suggests the direct participation of L16 as a part of the binding site for antibiotics.