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1.  Functional Interaction between Ribosomal Protein L6 and RbgA during Ribosome Assembly 
PLoS Genetics  2014;10(10):e1004694.
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.
Author Summary
Ribosomes are complex macromolecular machines that carry out the essential function of protein synthesis in the cell. The assembly of ribosomal subunits is a multistep process that involves the accurate and timely assembly of 3 rRNA molecules and>50 ribosomal-proteins. In recent years many ribosome assembly factors have been identified in bacterial and eukaryotic cells; however, their precise functions in ribosome biogenesis are poorly understood. We have previously shown that the GTPase RbgA, a protein conserved from bacteria to humans, is essential for ribosome assembly in Bacillus subtilis. Here, we show that growth defect caused by a mutation in RbgA is partially suppressed by mutations in ribosomal protein L6. The suppressor strains accumulate novel ribosomal intermediates that appear to suppress the RbgA defect by weakening the interaction of L6 for the ribosome and facilitating RbgA dependent assembly. Our work provides evidence for a functional interaction between ribosome assembly factor RbgA and ribosomal protein L6 during assembly, a function that is likely important for mitochondrial, chloroplast, and eukaryotic ribosome assembly as well.
doi:10.1371/journal.pgen.1004694
PMCID: PMC4199504  PMID: 25330043
2.  Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1 
eLife  2013;2:e00411.
Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.
DOI: http://dx.doi.org/10.7554/eLife.00411.001
eLife digest
Ribosomes are complex molecular machines that join amino acids together to form proteins. The order of amino acids in the protein is specified by a strand of messenger RNA (mRNA), and the process of decoding the mRNA into a string of amino acids is called translation. A ribosome consists of two subunits—one large, one small—that come together at a particular site on the mRNA strand called the translation initiation site. The ribosome then moves along the mRNA—joining together amino acids brought to it by transfer RNA (tRNA)—until it reaches a termination site and releases the protein.
The ribosome has three sites; the first amino acid to be delivered by a tRNA molecule to the ribosome occupies the site in the middle—also called the P site—and the second amino acid is delivered to the A site. Once the first two amino acids have been joined together, the ribosome moves along the mRNA so that the first amino acid now occupies the third site, called the E or exit site, and the second amino acid occupies the P site, leaving the A site vacant. The third amino acid is then delivered to the A site, and the whole process repeats itself until the ribosome reaches the termination site. Proteins called release factors are responsible for terminating the translation process and releasing the translated string of amino acids, which folds to form a protein. In bacteria this task can by performed by two releases factors, known as RF1 and RF2. However, the release factor must itself be released to leave the ribosome free to translate another strand of mRNA.
Pallesen et al. have used cryo-electron microscopy (cryo-EM) to study how a third release factor, RF3, helps to release RF1 from the ribosome in bacteria. In cells, RF3 usually forms a complex with a molecule called GDP, and the cryo-EM studies show that this molecule is released shortly after the RF3•GDP complex enters the ribosome. Once inside the ribosome, RF3 comes into contact with RF1 and with a protein called L12 that is part of the ribosome. A molecule called GTP—which is well known as a source of energy within cells—then binds to RF3, and this causes the shape of the ribosome to change. This change of shape results in the release of RF1 and the formation of a new RF3•GDP complex, which then leaves the ribosome.
Further work is needed to fully understand the role of L12 in these events, but a detailed understanding of the mechanism for terminating the translation of mRNA by the ribosome is coming into view.
DOI: http://dx.doi.org/10.7554/eLife.00411.002
doi:10.7554/eLife.00411
PMCID: PMC3677378  PMID: 23755360
Ribosome; Cryo-EM; Structure; RF1; RF3; L7/L12; E. coli
3.  A Computational Investigation on the Connection between Dynamics Properties of Ribosomal Proteins and Ribosome Assembly 
PLoS Computational Biology  2012;8(5):e1002530.
Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and experimental evidence suggests that prokaryotic ribosomal proteins undergo conformational changes during assembly. However, to date, no studies have attempted to elucidate these conformational changes. The present work utilizes computational methods to analyze protein dynamics and to investigate the linkage between dynamics and binding of these proteins during the assembly of the ribosome. Ribosomal proteins are known to be positively charged and we find the percentage of positive residues in r-proteins to be about twice that of the average protein: Lys+Arg is 18.7% for E. coli and 21.2% for T. thermophilus. Also, positive residues constitute a large proportion of RNA contacting residues: 39% for E. coli and 46% for T. thermophilus. This affirms the known importance of charge-charge interactions in the assembly of the ribosome. We studied the dynamics of three primary proteins from E. coli and T. thermophilus 30S subunits that bind early in the assembly (S15, S17, and S20) with atomic molecular dynamic simulations, followed by a study of all r-proteins using elastic network models. Molecular dynamics simulations show that solvent-exposed proteins (S15 and S17) tend to adopt more stable solution conformations than an RNA-embedded protein (S20). We also find protein residues that contact the 16S rRNA are generally more mobile in comparison with the other residues. This is because there is a larger proportion of contacting residues located in flexible loop regions. By the use of elastic network models, which are computationally more efficient, we show that this trend holds for most of the 30S r-proteins.
Author Summary
Ribosomes are complex cellular machines that synthesize new proteins in the cell. The accurate and efficient assembly of ribosomal proteins (r-proteins) and ribosomal RNA (rRNA) to form a functional ribosome is important for cell growth, metabolic reactions, and other cellular processes. Additionally, some antibacterial drugs are believed to target the bacterial ribosome during its construction. Hence, ribosomal assembly has been an active research topic for many years because understanding the assembly mechanisms can provide insight into protein/RNA recognitions important in many other cellular processes, as well as optimize the development of antibacterial therapeutics. Experimental studies thus far have provided still limited understanding about the assembly process. To further understand the assembly process, we have computationally studied the dynamic properties that r-proteins exhibit during assembly and the relationship between dynamics, physical properties, and binding propensity. We observe significant charged interactions between r-proteins and rRNA. We also detect a strong correlation between contact residues and their dynamic mobilities. Protein residues contacting with rRNA are observed to be more mobile in comparison with other residues. We also relate the location of the r-protein in the fully assembled ribosome to its susceptibility for large conformational changes prior to binding.
doi:10.1371/journal.pcbi.1002530
PMCID: PMC3359968  PMID: 22654657
4.  An RNA-Binding Complex Involved in Ribosome Biogenesis Contains a Protein with Homology to tRNA CCA-Adding Enzyme 
PLoS Biology  2013;11(10):e1001669.
The structure of a complex of two ribosome synthesis factors and identification of their ribosomal binding sites provides insights into early stages of ribosome biogenesis.
A multitude of proteins and small nucleolar RNAs transiently associate with eukaryotic ribosomal RNAs to direct their modification and processing and the assembly of ribosomal proteins. Utp22 and Rrp7, two interacting proteins with no recognizable domain, are components of the 90S preribosome or the small subunit processome that conducts early processing of 18S rRNA. Here, we determine the cocrystal structure of Utp22 and Rrp7 complex at 1.97 Å resolution and the NMR structure of a C-terminal fragment of Rrp7, which is not visible in the crystal structure. The structure reveals that Utp22 surprisingly resembles a dimeric class I tRNA CCA-adding enzyme yet with degenerate active sites, raising an interesting evolutionary connection between tRNA and rRNA processing machineries. Rrp7 binds extensively to Utp22 using a deviant RNA recognition motif and an extended linker. Functional sites on the two proteins were identified by structure-based mutagenesis in yeast. We show that Rrp7 contains a flexible RNA-binding C-terminal tail that is essential for association with preribosomes. RNA–protein crosslinking shows that Rrp7 binds at the central domain of 18S rRNA and shares a neighborhood with two processing H/ACA snoRNAs snR30 and snR10. Depletion of snR30 prevents the stable assembly of Rrp7 into preribosomes. Our results provide insight into the evolutionary origin and functional context of Utp22 and Rrp7.
Author Summary
Ribosomes are large RNA–protein complexes that manufacture proteins in all living organisms. Synthesis of large and small ribosomal subunits is a fundamental and enormous task that requires activities of approximately 200 assembly factors in eukaryotic cells. These factors transiently associate with the ribosome, forming a series of pre-ribosomal particles. We currently have a poor understanding of the structure and assembly of ribosome precursors. Utp22 and Rrp7 are two interacting proteins present in early precursors of the small ribosomal subunit. In this study, we determined the structure of the Utp22 and Rrp7 complex by X-ray crystallography and NMR and dissected their functional domains by mutagenesis. The structure of Utp22 reveals an unexpected structural similarity to the tRNA CCA-adding enzyme, providing insight into the evolutionary origin of Utp22. Utp22 apparently lacks any enzymatic activity and functions instead as a structural building block. Rrp7 associates extensively with Utp22 and appears to be anchored to pre-ribosomes via a flexible RNA-binding tail. We used RNA–protein crosslinking to identify the binding site and neighboring factor of Rrp7 on pre-ribosomes. Our study provides a detailed insight into the structure of small ribosomal subunit precursors.
doi:10.1371/journal.pbio.1001669
PMCID: PMC3794860  PMID: 24130456
5.  Final Pre-40S Maturation Depends on the Functional Integrity of the 60S Subunit Ribosomal Protein L3 
PLoS Genetics  2014;10(3):e1004205.
Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3′ end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles.
Author Summary
Recent progress has provided us with detailed knowledge of the structure and function of eukaryotic ribosomes. However, our understanding of the intricate processes of pre-ribosome assembly and the transition to translation-competent ribosomal subunits remains incomplete. The early and intermediate steps of ribosome assembly occur successively in the nucleolus and nucleoplasm. The pre-ribosomal subunits are then exported to the cytoplasm where final maturation steps, notably including D site cleavage of the 20S pre-rRNA to mature 18S rRNA, confer subunit joining and translation competence. Recent evidence indicates that pre-40S subunits are subject to a quality control step involving the GTP-dependent translation initiation factor eIF5B/Fun12, in the context of 80S-like ribosomes. Here, we demonstrate the involvement of 60S subunits in promoting 20S pre-rRNA cleavage. In particular, we show that a specific point mutation in the 60S subunit ribosomal protein L3 (rpl3[W255C]) leads to the accumulation of pre-40S particles that contain the 20S pre-rRNA but are translation-competent. Notably, this mutation prevents the stimulation of the GTPase activity of eIF5B/Fun12, which is also required for site D cleavage. We conclude that L3 plays an important role in regulating the function of eIF5B/Fun12 during 3′ end processing of 18S rRNA at site D, in the context of 80S ribosomes that have not yet engaged in translation.
doi:10.1371/journal.pgen.1004205
PMCID: PMC3945201  PMID: 24603549
6.  Structural Analysis of Base Substitutions in Thermus thermophilus 16S rRNA Conferring Streptomycin Resistance 
Streptomycin is a bactericidal antibiotic that induces translational errors. It binds to the 30S ribosomal subunit, interacting with ribosomal protein S12 and with 16S rRNA through contacts with the phosphodiester backbone. To explore the structural basis for streptomycin resistance, we determined the X-ray crystal structures of 30S ribosomal subunits from six streptomycin-resistant mutants of Thermus thermophilus both in the apo form and in complex with streptomycin. Base substitutions at highly conserved residues in the central pseudoknot of 16S rRNA produce novel hydrogen-bonding and base-stacking interactions. These rearrangements in secondary structure produce only minor adjustments in the three-dimensional fold of the pseudoknot. These results illustrate how antibiotic resistance can occur as a result of small changes in binding site conformation.
doi:10.1128/AAC.02857-14
PMCID: PMC4136021  PMID: 24820088
7.  STRUCTURAL INSIGHTS INTO PRE-TRANSLOCATION RIBOSOME MOTIONS 
Subsequent to the peptidyl transfer step of the translation elongation cycle, the initially formed pre-translocation ribosome, which we refer to here as R1, undergoes a ratchet-like intersubunit rotation in order to sample a rotated conformation, referred to here as RF, that is an obligatory intermediate in the translocation of tRNAs and mRNA through the ribosome during the translocation step of the translation elongation cycle. RF and the R1 to RF transition are currently the subject of intense research, driven in part by the potential for developing novel antibiotics which trap RF or confound the R1 to RF transition. Currently lacking a 3D atomic structure of the RF endpoint of the transition, as well as a preliminary conformational trajectory connecting R1 and RF, the dynamics of the mechanistically crucial R1 to RF transition remain elusive. The current literature reports fitting of only a few ribosomal RNA (rRNA) and ribosomal protein (r-protein) components into cryogenic electron microscopy (cryo-EM) reconstructions of the Escherichia coli ribosome in RF. In this work we now fit the entire Thermus thermophilus 16S and 23S rRNAs and most of the remaining T. thermophilus r-proteins into a cryo-EM reconstruction of the E. coli ribosome in RF in order to build an almost complete model of the T. thermophilus ribosome in RF thus allowing a more detailed view of this crucial conformation. The resulting model validates key predictions from the published literature; in particular it recovers intersubunit bridges known to be maintained throughout the R1 to RF transition and results in new intersubunit bridges that are predicted to exist only in RF. In addition, we use a recently reported E. coli ribosome structure, apparently trapped in an intermediate state along the R1 to RF transition pathway, referred to here as R2, as a guide to generate a T. thermophilus ribosome in the R2 state. This demonstrates a multiresolution method for morphing large complexes and provides us with a structural model of R2 in the species of interest. The generated structural models form the basis for probing the motion of the deacylated tRNA bound at the peptidyl-tRNA binding site (P site) of the pre-translocation ribosome as it moves from its so-called classical P/P configuration to its so-called hybrid P/E configuration as part of the R1 to RF transition. We create a dynamic model of this process which provides structural insights into the functional significance of R2 as well as detailed atomic information to guide the design of further experiments. The results suggest extensibility to other steps of protein synthesis as well as to spatially larger systems.
PMCID: PMC3371265  PMID: 21121048
8.  Structural Insights Into Ribosome Recycling Factor Interactions with the 70S Ribosome 
Journal of molecular biology  2008;376(5):1334-1347.
SUMMARY
At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with Elongation Factor G (EF-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 (PTC). Upon binding of either E. coli or T. thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix H69 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 H69 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.
doi:10.1016/j.jmb.2007.12.048
PMCID: PMC2712656  PMID: 18234219
9.  The Escherichia coli GTPase CgtAE Is Involved in Late Steps of Large Ribosome Assembly†  
Journal of Bacteriology  2006;188(19):6757-6770.
The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtAE, in late steps of large ribosomal subunit biogenesis. CgtAE belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtAE cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtAE mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtAE is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtAE. We also show that purified CgtAE associates with purified ribosomal particles in the GTP-bound form. Finally, CgtAE cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.
doi:10.1128/JB.00444-06
PMCID: PMC1595513  PMID: 16980477
10.  Elongation factor G bound to the ribosome in an intermediate state of translocation 
Science (New York, N.Y.)  2013;340(6140):10.1126/science.1235490.
A key step of translation by the ribosome is translocation, which involves the movement of mRNA and tRNA with respect to the ribosome. This allows a new round of protein chain elongation by placing the next mRNA codon in the A site of the 30S subunit. Translocation proceeds via an intermediate state in which the acceptor ends of the tRNAs have moved with respect to the 50S subunit but not the 30S subunit, to form hybrid states. The GTPase elongation factor G (EF-G) catalyzes the subsequent movement of mRNA and tRNA with respect to the 30S subunit. Here we present a crystal structure at 3 Å resolution of the Thermus thermophilus ribosome with a tRNA in the hybrid P/E state bound to EF-G with a GTP analog. The structure provides insights into structural changes that facilitate translocation and suggests a common GTPase mechanism for EF-G and elongation factor Tu.
doi:10.1126/science.1235490
PMCID: PMC3836249  PMID: 23812720
11.  Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase 
PLoS Biology  2014;12(5):e1001866.
Kinetics and cryo-electronmicroscopy data provide insights into GTPase ObgE’s role as a ribosome anti-association factor that is modulated by nutrient availability, coupling growth control to ribosome biosynthesis and protein translation.
Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S·ObgE·GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p)ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p)ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues.
Author Summary
GTPases commonly act as molecular switches in biological systems. By oscillating between two conformational states, depending on the type of guanine nucleotide bound (GTP or GDP), GTPases are essential regulators of many aspects of cell biology. Additional levels of regulation can be acquired through the synthesis of other guanine nucleotide derivatives that target GTPases; for instance, when nutrients are limited, bacterial cells produce guanine tetraphosphate/pentaphosphate—(p)ppGpp—as part of the “stringent response” to adjust the balance between growth and survival. ObgE is a GTPase with many reported cellular functions that include ribosome biogenesis, but none of its functions is understood at the molecular level. Here we characterize, both biochemically and structurally, the binding of ObgE to its cellular partner, the 50S ribosomal subunit. Our results show that ObgE is an anti-association factor, which binds to the 50S subunit to block the formation of the 70S ribosome, thereby inhibiting the initiation of translation. Furthermore, the binding and anti-association activities of ObgE are regulated by guanine nucleotides, as well as by (p)ppGpp. We thus propose that ObgE is a checkpoint protein in the assembly of the 50S subunit, which senses the cellular energy stress via levels of (p)ppGpp and links ribosome assembly to other global growth control pathways.
doi:10.1371/journal.pbio.1001866
PMCID: PMC4028186  PMID: 24844575
12.  Identification of Novel Escherichia coli Ribosome-Associated Proteins Using Isobaric Tags and Multidimensional Protein Identification Techniques▿ †  
Journal of Bacteriology  2007;189(9):3434-3444.
Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16°C and 37°C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles.
doi:10.1128/JB.00090-07
PMCID: PMC1855874  PMID: 17337586
13.  Domain I of ribosomal protein L1 is sufficient for specific RNA binding 
Nucleic Acids Research  2007;35(21):7389-7395.
Ribosomal protein L1 has a dual function as a ribosomal protein binding 23S rRNA and as a translational repressor binding its mRNA. L1 is a two-domain protein with N- and C-termini located in domain I. Earlier it was shown that L1 interacts with the same targets on both rRNA and mRNA mainly through domain I. We have suggested that domain I is necessary and sufficient for specific RNA-binding by L1. To test this hypothesis, a truncation mutant of L1 from Thermus thermophilus, representing domain I, was constructed by deletion of the central part of the L1 sequence, which corresponds to domain II. It was shown that the isolated domain I forms stable complexes with specific fragments of both rRNA and mRNA. The crystal structure of the isolated domain I was determined and compared with the structure of this domain within the intact protein L1. This comparison revealed a close similarity of both structures. Our results confirm our suggestion that in protein L1 its domain I alone is sufficient for specific RNA binding, whereas domain II stabilizes the L1-rRNA complex.
doi:10.1093/nar/gkm898
PMCID: PMC2175363  PMID: 17962298
14.  Structural Basis for the Rescue of Stalled Ribosomes: Structure of YaeJ Bound to the Ribosome 
Science (New York, N.Y.)  2012;335(6074):1370-1372.
In bacteria, the hybrid transfer-messenger RNA (tmRNA) rescues ribosomes stalled on defective messenger RNAs (mRNAs). However, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmRNA-independent manner. Here, we report a 3.2 angstrom–resolution crystal structure of the rescue factor YaeJ bound to the Thermus thermophilus 70S ribosome in complex with the initiator tRNAifMet and a short mRNA. The structure reveals that the C-terminal tail of YaeJ functions as a sensor to discriminate between stalled and actively translating ribosomes by binding in the mRNA entry channel downstream of the A site between the head and shoulder of the 30S subunit. This allows the N-terminal globular domain to sample different conformations, so that its conserved GGQ motif is optimally positioned to catalyze the hydrolysis of peptidyl-tRNA. This structure gives insights into the mechanism of YaeJ function and provides a basis for understanding how it rescues stalled ribosomes.
doi:10.1126/science.1217443
PMCID: PMC3377438  PMID: 22422986
15.  Structural motifs of the bacterial ribosomal proteins S20, S18 and S16 that contact rRNA present in the eukaryotic ribosomal proteins S25, S26 and S27A, respectively 
Nucleic Acids Research  2009;38(6):2089-2098.
The majority of constitutive proteins in the bacterial 30S ribosomal subunit have orthologues in Eukarya and Archaea. The eukaryotic counterparts for the remainder (S6, S16, S18 and S20) have not been identified. We assumed that amino acid residues in the ribosomal proteins that contact rRNA are to be constrained in evolution and that the most highly conserved of them are those residues that are involved in forming the secondary protein structure. We aligned the sequences of the bacterial ribosomal proteins from the S20p, S18p and S16p families, which make multiple contacts with rRNA in the Thermus thermophilus 30S ribosomal subunit (in contrast to the S6p family), with the sequences of the unassigned eukaryotic small ribosomal subunit protein families. This made it possible to reveal that the conserved structural motifs of S20p, S18p and S16p that contact rRNA in the bacterial ribosome are present in the ribosomal proteins S25e, S26e and S27Ae, respectively. We suggest that ribosomal protein families S20p, S18p and S16p are homologous to the families S25e, S26e and S27Ae, respectively.
doi:10.1093/nar/gkp1170
PMCID: PMC2847233  PMID: 20034956
16.  RNA Mimicry by the Fap7 Adenylate Kinase in Ribosome Biogenesis 
PLoS Biology  2014;12(5):e1001860.
The structure of a ribosome assembly factor in complex bound to a ribosomal protein uncovers a chaperoning function that uses RNA mimicry and is regulated by ATP hydrolysis.
During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53–MDM2 pathway. This work presents the functional and structural characterization of the Fap7–Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation.
Author Summary
Ribosomes are the cellular machines responsible for all protein synthesis. In eukaryotes, the assembly of ribosomes from their protein and RNA components is extremely complicated and involves more than 200 nonribosomal factors—three times the number of proteins in the mature complex. Among these factors, the Fap7 protein is particularly intriguing because it interacts with the small subunit ribosomal protein Rps14 and it exhibits adenylate kinase activity—a molecular function more commonly associated with regulating ATP/ADP levels than assembling protein–RNA complexes. Combining structural and biochemical analysis of the Rps14–Fap7 complex, we show that Fap7 uses protein side chains to mimic RNA contacts, rendering the interaction of Rps14 with ribosomal RNA or with Fap7 competitive and mutually exclusive. Once bound, Rps14 blocks the substrate-binding cavity of Fap7, and ATP hydrolysis will then break the Fap7–Rps14 complex apart. At the same time, the ribosome structure at the location where Rps14 binds is disrupted when the Fap7/Rps14 complex is formed, and this process is regulated by ATP binding and hydrolysis. Our model is thus that Fap7 temporarily removes Rps14 from the ribosome to enable a conformational change of the ribosomal RNA that is needed for the final maturation step of the small ribosomal subunit.
doi:10.1371/journal.pbio.1001860
PMCID: PMC4019466  PMID: 24823650
17.  Structural aspects of RbfA action during small ribosomal subunit assembly 
Molecular cell  2007;28(3):434-445.
Summary
Ribosome binding factor A (RbfA) is a bacterial cold-shock response protein, required for an efficient processing of the 5′end of the 16S ribosomal RNA (rRNA) during assembly of the small (30S) ribosomal subunit. Here we present a crystal structure of Thermus thermophilus RbfA and a three-dimensional cryo-electron microscopic (EM) map of the T. thermophilus 30S·RbfA complex. RbfA binds to the 30S subunit in a position overlapping the binding sites of the A- and P-site tRNAs, and RbfA’s functionally important C-terminus extends toward the 5′ end of the 16S rRNA. In the presence of RbfA, a portion of the 16S rRNA encompassing helix 44, which is known to be directly involved in mRNA decoding and tRNA binding, is displaced. These results shed light on the role played by RbfA during maturation of the 30S subunit, and also indicate how RbfA provides cells with a translational advantage under conditions of cold shock.
doi:10.1016/j.molcel.2007.08.026
PMCID: PMC2118056  PMID: 17996707
18.  Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms 
The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region.
Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.
doi:10.1107/S0907444913006422
PMCID: PMC3663118  PMID: 23695237
translation initiation factor 2; Thermus thermophilus; GTP; GDP
19.  Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium, Thermus thermophilus 
Journal of Bacteriology  2006;188(20):7111-7122.
Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel+; wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNASer aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).
doi:10.1128/JB.00574-06
PMCID: PMC1636220  PMID: 17015650
20.  GTPases IF2 and EF-G bind GDP and the SRL RNA in a mutually exclusive manner 
Scientific Reports  2012;2:843.
Translational GTPases (trGTPases) are involved in all four stages of protein biosynthesis: initiation, elongation, termination and ribosome recycling. The trGTPases Initiation Factor 2 (IF2) and Elongation Factor G (EF-G) respectively orchestrate initiation complex formation and translocation of the peptidyl-tRNA:mRNA complex through the bacterial ribosome. The ribosome regulates the GTPase cycle and efficiently discriminates between the GDP- and GTP-bound forms of these proteins. Using Isothermal Titration Calorimetry, we have investigated interactions of IF2 and EF-G with the sarcin-ricin loop of the 23S rRNA, a crucial element of the GTPase-associated center of the ribosome. We show that binding of IF2 and EF-G to a 27 nucleotide RNA fragment mimicking the sarcin-ricin loop is mutually exclusive with that of GDP, but not of GTP, providing a mechanism for destabilization of the ribosome-bound GDP forms of translational GTPases.
doi:10.1038/srep00843
PMCID: PMC3496166  PMID: 23150791
21.  Mutational Analysis of 16S and 23S rRNA Genes of Thermus thermophilus 
Journal of Bacteriology  2005;187(14):4804-4812.
Structural studies of the ribosome have benefited greatly from the use of organisms adapted to extreme environments. However, little is known about the mechanisms by which ribosomes or other ribonucleoprotein complexes have adapted to functioning under extreme conditions, and it is unclear to what degree mutant phenotypes of extremophiles will resemble those of their counterparts adapted to more moderate environments. It is conceivable that phenotypes of mutations affecting thermophilic ribosomes, for instance, will be influenced by structural adaptations specific to a thermophilic existence. This consideration is particularly important when using crystal structures of thermophilic ribosomes to interpret genetic results from nonextremophilic species. To address this issue, we have conducted a survey of spontaneously arising antibiotic-resistant mutants of the extremely thermophilic bacterium Thermus thermophilus, a species which has featured prominently in ribosome structural studies. We have accumulated over 20 single-base substitutions in T. thermophilus 16S and 23S rRNA, in the decoding site and in the peptidyltransferase active site of the ribosome. These mutations produce phenotypes that are largely identical to those of corresponding mutants of mesophilic organisms encompassing a broad phylogenetic range, suggesting that T. thermophilus may be an ideal model system for the study of ribosome structure and function.
doi:10.1128/JB.187.14.4804-4812.2005
PMCID: PMC1169515  PMID: 15995195
22.  Control of ribosomal subunit rotation by elongation factor G 
Science (New York, N.Y.)  2013;340(6140):1235970.
Protein synthesis by the ribosome requires the translocation of transfer RNAs and messenger RNA by one codon after each peptide bond is formed, a reaction that requires ribosomal subunit rotation and is catalyzed by the guanosine triphosphatase (GTPase) elongation factor G (EF-G). We determined 3 Å resolution x-ray crystal structures of EF-G complexed with a non-hydrolyzable GTP analogue and bound to the Escherichia coli ribosome in different states of ribosomal subunit rotation. The structures reveal that EF-G binding to the ribosome stabilizes switch regions in the GTPase active site, resulting in a compact EF-G conformation that favors an intermediate state of ribosomal subunit rotation. These structures suggest that EF-G controls the translocation reaction by cycles of conformational rigidity and relaxation preceding and following GTP hydrolysis.
doi:10.1126/science.1235970
PMCID: PMC4274944  PMID: 23812721
23.  Stability of the ‘L12 stalk’ in ribosomes from mesophilic and (hyper)thermophilic Archaea and Bacteria 
Nucleic Acids Research  2006;34(20):5800-5814.
The ribosomal stalk complex, consisting of one molecule of L10 and four or six molecules of L12, is attached to 23S rRNA via protein L10. This complex forms the so-called ‘L12 stalk’ on the 50S ribosomal subunit. Ribosomal protein L11 binds to the same region of 23S rRNA and is located at the base of the ‘L12 stalk’. The ‘L12 stalk’ plays a key role in the interaction of the ribosome with translation factors. In this study stalk complexes from mesophilic and (hyper)thermophilic species of the archaeal genus Methanococcus and from the Archaeon Sulfolobus solfataricus, as well as from the Bacteria Escherichia coli, Geobacillus stearothermophilus and Thermus thermophilus, were overproduced in E.coli and purified under non-denaturing conditions. Using filter-binding assays the affinities of the archaeal and bacterial complexes to their specific 23S rRNA target site were analyzed at different pH, ionic strength and temperature. Affinities of both archaeal and bacterial complexes for 23S rRNA vary by more than two orders of magnitude, correlating very well with the growth temperatures of the organisms. A cooperative effect of binding to 23S rRNA of protein L11 and the L10/L124 complex from mesophilic and thermophilic Archaea was shown to be temperature-dependent.
doi:10.1093/nar/gkl751
PMCID: PMC1635324  PMID: 17053098
24.  Optimization of Ribosome Structure and Function by rRNA Base Modification 
PLoS ONE  2007;2(1):e174.
Background
Translating mRNA sequences into functional proteins is a fundamental process necessary for the viability of organisms throughout all kingdoms of life. The ribosome carries out this process with a delicate balance between speed and accuracy. This work investigates how ribosome structure and function are affected by rRNA base modification. The prevailing view is that rRNA base modifications serve to fine tune ribosome structure and function.
Methodology/Principal Findings
To test this hypothesis, yeast strains deficient in rRNA modifications in the ribosomal peptidyltransferase center were monitored for changes in and translational fidelity. These studies revealed allele-specific sensitivity to translational inhibitors, changes in reading frame maintenance, nonsense suppression and aa-tRNA selection. Ribosomes isolated from two mutants with the most pronounced phenotypic changes had increased affinities for aa-tRNA, and surprisingly, increased rates of peptidyltransfer as monitored by the puromycin assay. rRNA chemical analyses of one of these mutants identified structural changes in five specific bases associated with the ribosomal A-site.
Conclusions/Significance
Together, the data suggest that modification of these bases fine tune the structure of the A-site region of the large subunit so as to assure correct positioning of critical rRNA bases involved in aa-tRNA accommodation into the PTC, of the eEF-1A•aa-tRNA•GTP ternary complex with the GTPase associated center, and of the aa-tRNA in the A-site. These findings represent a direct demonstration in support of the prevailing hypothesis that rRNA modifications serve to optimize rRNA structure for production of accurate and efficient ribosomes.
doi:10.1371/journal.pone.0000174
PMCID: PMC1766470  PMID: 17245450
25.  Crystal Structures of EF-G-Ribosome Complexes Trapped in Intermediate States of Translocation 
Science (New York, N.Y.)  2013;340(6140):1236086.
Translocation of mRNA and tRNA through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. The crystal structures of three Thermus ribosome·tRNA·mRNA·EF-G complexes trapped with GDPNP or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30S subunit head and body. In all complexes, the tRNA acceptor ends occupy the 50S subunit E site, while their anticodon stem-loops move with the head of the 30S subunit to positions between the P and E sites, forming chimeric intermediate states. Two universally conserved bases of 16S rRNA that intercalate between bases of the mRNA may act as “pawls” of a translocational ratchet. These findings provide new insights into the molecular mechanism of ribosomal translocation.
doi:10.1126/science.1236086
PMCID: PMC3979973  PMID: 23812722

Results 1-25 (950861)