Using Cross-links to Study Ribosomal Dynamics

Abstract After publications of 3-D models of a static ribosome and its large and small subunits, one of the next tasks is to recognize movable ribosomal elements responsible for mechanical shifts during protein synthesis. Statistic analysis of available cross-linking data allowed us to reveal three well separated groups of motions in the ribosome: I, mean magnitude of 10?; II, most abundant, centered at 20? and of wide dispersion, and III, sparsely populated, with large distances up to 95?. The last group, III, comprises elements, like the L7/12-stalk and the L1- protuberance, that adopt different positions in crystallographic or electron micrographic structures, and neighboring hairpins 88 and 89, indicating mobility. We demonstrate that the cross- linking method can be applied to study ribosomal dynamics, including large-scale functional movements and, in particular, to estimate which structures participate in molecular switches.     

Structure of the chloroplast ribosome: novel domains for translation regulation

Gene expression in chloroplasts is controlled primarily through the regulation of translation. This regulation allows coordinate expression between the plastid and nuclear genomes, and is responsive to environmental conditions. Despite common ancestry with bacterial translation, chloroplast translation is more complex and involves positive regulatory mRNA elements and a host of requisite protein translation factors that do not have counterparts in bacteria. Previous proteomic analyses of the chloroplast ribosome identified a significant number of chloroplast-unique ribosomal proteins that expand upon a basic bacterial 70S-like composition. In this study, cryo-electron microscopy and single-particle reconstruction were used to calculate the structure of the chloroplast ribosome to a resolution of 15.5 Å. Chloroplast-unique proteins are visualized as novel structural additions to a basic bacterial ribosome core. These structures are located at optimal positions on the chloroplast ribosome for interaction with mRNAs during translation initiation. Visualization of these chloroplast-unique structures on the ribosome, combined with mRNA cross-linking, allows us to propose a model for translation initiation in chloroplasts in which chloroplast-unique ribosomal proteins interact with plastid-specific translation factors and RNA elements to facilitate regulated translation of chloroplast mRNAs.     

Protein-protein cross-linking of the 50 S ribosomal subunit of Escherichia coli using 2-iminothiolane. Identification of cross-links by immunoblotting techniques

We have investigated the protein-protein cross-links formed within the 50 S subunit of the Escherichia coli ribosome using 2-iminothiolane as the cross-linking reagent. The members of the cross-links have been identified by immunoblotting from one-dimensional and two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gels using antisera specific for the individual ribosomal proteins. This method also allowed a quantitation of the yield of cross-linking for each cross-link. A total of 14 cross-links have been identified: L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L30, L17-L32, L19-L25, L20-L21, L22-L32, and L23-L34. Our results are compared with those of Traut and coworkers (Traut, R. R., Tewari, D. S., Sommer, A., Gavino, G. R., Olson, H. M., and Glitz, D. G. (1986) in Structure, Function and Genetics of Ribosomes (Hardesty, B. and Kramer, G., eds) pp. 286-308, Springer-Verlag, New York). Our cross-linking data allow us to propose the approximate locations of eight proteins of the 50 S ribosomal subunit that so far have not been localized by immunoelectron microscopy and they thus contribute considerably to our knowledge of ribosome structure.     

Structures of deacylated tRNA mimics bound to the E site of the large ribosomal subunit

During translation, tRNAs cycle through three binding sites on the ribosome: the A, the P, and the E sites. We have determined the structures of complexes between the Haloarcula marismortui large ribosomal subunit and two different E site substrates: a deacylated tRNA acceptor stem minihelix and a CCA-acceptor end. Both of these tRNA mimics contain analogs of adenosine 76, the component responsible for a large proportion of E site binding affinity. They bind in the center of the loop-extension of protein L44e, and make specific contacts with both L44e and 23S rRNA including bases that are conserved in all three kingdoms of life. These contacts are consistent with the footprinting, protection, and cross-linking data that have identified the E site biochemically. These structures explain the specificity of the E site for deacylated tRNAs, as it is too small to accommodate any relevant aminoacyl-tRNA. The orientation of the minihelix suggests that it may mimic the P/E hybrid state. It appears that the E site on the 50S subunit was formed by only RNA in the last common ancestor of the three kingdoms, since the proteins at the E sites of H. marismortui and Deinucoccus radiodurans large subunits are not homologous.     

Ribosomal proteins cross-linked to the initiator AUG codon of a mRNA in the translation initiation complex by UV-irradiation

Eukaryotic ribosomal proteins constituting the binding site for the initiator codon AUG on the ribosome at the translation initiation step were investigated by UV-induced cross-linking between protein and mRNA. The 80S-initiation complex was formed in a rabbit reticulocyte cell-free system in the presence of sparsomycin with radiolabeled Omega-fragment as a template, which was a 73-base 5'-leader sequence of tobacco mosaic virus RNA having AUG at the extreme 3'-terminal end and extended with 32pCp. Two radioactive peaks were sedimented by sucrose gradient centrifugation, one being the 80S initiation complex formed at the 3'-terminal AUG codon, and the other presumably a "disome" with an additional 80S ribosome bound at an upstream AUU codon, formed when Omega-fragment was incubated with sparsomycin [Filipowicz and Henni (1979) Proc. Natl. Acad. Sci. USA 76, 3111-3115]. Cross-links between ribosomal proteins and the radiolabeled Omega-fragment were induced in situ by UV-irradiation at 254 nm. After extensive nuclease digestion of the complexes, ribosomal proteins were separated by two-dimensional gel electrophoresis. Autoradiography identified the proteins S7, S10, S25, S29, and L5 of the 80S initiation complex and S7, S25, S29 and L5 of that in the disome as 32P-labeled proteins. Together with the results of cross-linking experiments of other investigators and recently solved crystal structures of prokaryotic ribosomes, the spatial arrangement of eukaryotic ribosomal proteins at the AUG-binding domain is discussed.     

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.     

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.     

Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: the IRES functions as an RNA-based translation factor

Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs. An IRES element in cricket paralysis virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA. Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome. The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites. Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome. CrPV-like IRES elements seem to act as RNA-based translation factors.     

Localization and dynamic behavior of ribosomal protein L30e

The ribosomal protein L30e is an indispensable component of the eukaryotic 80S ribosome, where it is part of the large (60S) ribosomal subunit. Here, we determined the localization of L30e in the cryo-EM map of the 80S wheat germ (wg) ribosome at a resolution of 9.5 A. L30e is part of the interface between large and small subunits, where it dynamically participates in the formation of the two intersubunit bridges eB9 and B4.     

Role of ribosomal protein L27 in peptidyl transfer

The current view of ribosomal peptidyl transfer is that the ribosome is a ribozyme and that ribosomal proteins are not involved in catalysis of the chemical reaction. This view is largely based on the first crystal structures of bacterial large ribosomal subunits that did not show any protein components near the peptidyl transferase center (PTC). Recent crystallographic data on the full 70S ribosome from Thermus thermophilus, however, show that ribosomal protein L27 extends with its N-terminus into the PTC in accordance with independent biochemical data, thus raising the question of whether the ribozyme picture is strictly valid. We have carried out extensive computer simulations of the peptidyl transfer reaction in the T. thermophilus ribosome to address the role of L27. The results show a reaction rate similar to that obtained in earlier simulations of the Haloarcula marismortui reaction. Furthermore, deletion of L27 is predicted to only give a minor rate reduction, in agreement with biochemical data, suggesting that the ribozyme view is indeed valid. The N-terminus of L27 is predicted to interact with the A76 phosphate group of the A-site tRNA, thereby explaining the observed impairment of A-site substrate binding for ribosomes lacking L27. Simulations are also reported for the reaction with puromycin, an A-site tRNA analogue which lacks the A76 phosphate group. The calculated energetics shows that this substrate can cause a downward p K a shift of L27 and that the reaction proceeds faster with the L27 N-terminus deprotonated, in contrast to the situation with aminoacyl-tRNA substrates. These results could explain the observed differences in pH dependence between the puromycin and C-puromycin reactions, where the former reaction has been seen to depend on an additional ionizing group besides the attacking amine, and our model predicts this ionizing group to be the N-terminal amine of L27.     

Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

Yeast ribosomal protein L12 is a substrate of protein-arginine methyltransferase 2

Type III protein-arginine methyltransferase from the yeast Saccharomyces cerevisiae (RMT2) was expressed in Escherichia coli and purified to apparent homogeneity. The cytosolic, ribosomal, and ribosome salt wash fractions from yeast cells lacking RMT2 were used as substrates for the recombinant RMT2. Using S-adenosyl-l-methionine as co-substrate, RMT2 methylated a protein in the ribosome salt wash fraction. The same protein in the ribosomal fraction was also methylated by RMT2 after pretreating the sample with endonuclease. Amino acid analysis affirmed that the labeling products were delta-N-monomethylarginines. The methylated protein from the ribosomal or the ribosome salt wash fraction was isolated by two-dimensional gel electrophoresis and identified as ribosomal protein L12 by mass spectrometry. Using synthetic peptides, recombinant L12, and its mutant as substrates, we pinpointed Arg(67) on ribosomal protein L12 as the methyl acceptor. L12 was isolated from wild type yeast cells that have been grown in the presence of S-adenosyl-l-[methyl-(3)H]methionine and subjected to amino acid analysis. The results indicate that L12 contains delta-N-monomethylarginines.     

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.     

The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control

The Escherichia coli autoregulatory ribosomal protein L20 consists of two structurally distinct domains. The C-terminal domain is globular and sits on the surface of the large ribosomal subunit whereas the N-terminal domain has an extended shape and penetrates deep into the RNA-rich core of the subunit. Many other ribosomal proteins have analogous internal or terminal extensions. However, the biological functions of these extended domains remain obscure. Here we show that the N-terminal tail of L20 is important for ribosome assembly in vivo. Indeed, a truncated version of L20 without its N-terminal tail is unable to complement the deletion of rplT, the gene encoding L20. In addition, this L20 truncation confers a lethal-dominant phenotype, suggesting that the N-terminal domain is essential for cell growth because it could be required for ribosome assembly. Supporting this hypothesis, partial deletions of the N-terminal tail of the protein are shown to cause a slow-growth phenotype due to altered ribosome assembly in vivo as large amounts of intermediate 40S ribosomal particles accumulate. In addition to being a ribosomal protein, L20 also acts as an autogenous repressor. Using L20 truncations, we also show that the N-terminal tail of L20 is dispensable for autogenous control.     

Functional Interaction between Ribosomal Protein L6 and RbgA during Ribosome Assembly

RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit assembly. How RbgA functions in this process is still poorly understood. To gain insight into the function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein to function more effectively in ribosome maturation. Our results suggest that RbgA functions to properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins.     

A small protein unique to bacteria organizes rRNA tertiary structure over an extensive region of the 50 S ribosomal subunit

A number of small, basic proteins penetrate into the structure of the large subunit of the ribosome. While these proteins presumably aid in the folding of the rRNA, the extent of their contribution to the stability or function of the ribosome is unknown. One of these small, basic proteins is L36, which is highly conserved in Bacteria, but is not present in Archaea or Eucarya. Comparison of ribosome crystal structures shows that the space occupied by L36 in a bacterial ribosome is empty in an archaeal ribosome. To ask what L36 contributes to ribosome stability and function, we have constructed an Escherichia coli strain lacking ribosomal protein L36; cell growth is slowed by 40-50% between 30 degrees C and 42 degrees C. Ribosomes from this deletion strain sediment normally and have a full complement of proteins, other than L36. Chemical protection experiments comparing rRNA from wild-type and L36-deficient ribosomes show the expected increase in reagent accessibility in the immediate vicinity of the L36 binding site, but suggest that a cooperative network of rRNA tertiary interactions has been disrupted along a path extending 60 A deep into the ribosome. These data argue that L36 plays a significant role in organizing 23 S rRNA structure. Perhaps the Archaea and Eucarya have compensated for their lack of L36 by maintaining more stable rRNA tertiary contacts or by adopting alternative protein-RNA interactions elsewhere in the ribosome.     

Janus chaperones: Assistance of both RNA- and protein-folding by ribosomal proteins

Ribosomal proteins assist the assembly and increase the stability of ribosomal RNA, without requiring ATP for their action. Some ribosomal proteins are also known to have essential functions outside the ribosome, i.e. promiscuity of functions that appears to correlate with their structural disorder. Here we addressed if certain ribosomal proteins with RNA chaperone activity and with a significant level of disorder also have protein-chaperone activity in vitro. Four proteins of the large subunit of Escherichia coli ribosome, L15, L16, L18 and L19 have been tested in three chaperone assays, in which all of them exhibited potent chaperone activity, commensurable with that of heat shock protein 90 kDa. These observations highlight possible novel aspects of the promiscuous functions of ribosomal proteins outside of the ribosome.     

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.     

Ribosomal protein L3: gatekeeper to the A site

Ribosomal protein L3 (L3) is an essential and indispensable component for formation of the peptidyltransferase center. Atomic resolution ribosome structures reveal two extensions of L3 protruding deep into the core of the large subunit. The central extension of L3 in Saccharomyces cerevisiae was investigated using a combination of molecular genetic, biochemical, chemical probing, and molecular modeling methods. A reciprocal relationship between ribosomal affinity for eEF-1A stimulated binding of aa-tRNA and for eEF2 suggests that the central extension of L3 may function as an allosteric switch in coordinating binding of the elongation factors. Opening of the aa-tRNA accommodation corridor promoted resistance to the A site-specific translational inhibitor anisomycin, suggesting a competitive model for anisomycin resistance. These changes were also found to inhibit peptidyltransferase activity, stimulating programmed -1 ribosomal frameshifting and promoting virus propagation defects. These studies provide a basis for deeper insight into rational design of small molecule antiviral therapeutics.     

In vivo studies on the incorporation of microinjected acidic proteins of the large ribosomal subunit from Escherichia coli and artermia salina into oocyte ribosomes from Xenopus laevis.

Tritium-labeled acidic proteins from the large ribosomal subunit of Artermia salina or Escherichia coli were microinjected into the cytoplasm of stage IV/V oocytes from Xenopus laevis. eL12 from the large ribosomal subunit of A. salina but not L7/L12 or L7/L12--L10 from E. coli is specifically incorporated into 60S ribosomal subunits of oocytes. This incorporation is not significantly inhibited by actinomycin D. Incorporation of eL12 into the 60S subunits occurs in enucleated oocytes, suggesting that active ribosomal ribonucleic acid synthesis and ribosome assembly as well are not prerequired for this reaction. Apparently the incorporation proceeds via an exchange reaction between a free cytoplasmic pool of eL12 and ribosomal eL12.