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The ribosome is a large macromolecular complex that must be assembled efficiently and accurately for the viability of all organisms. In bacteria, this process must be robust and tunable to support life in diverse conditions from the ice of arctic glaciers to thermal hot springs. Assembly of the Small ribosomal SUbunit (SSU) of Escherichia coli has been extensively studied and is highly temperature-dependent. However, a lack of data on SSU assembly for other bacteria is problematic given the importance of the ribosome in bacterial physiology. To broaden the understanding of how optimal growth temperature may affect SSU assembly, in vitro SSU assembly of two thermophilic bacteria, Geobacillus kaustophilus and Thermus thermophilus, was compared with that of E. coli. Using these phylogenetically, morphologically, and environmentally diverse bacteria, we show that SSU assembly is highly temperature-dependent and efficient SSU assembly occurs at different temperatures for each organism. Surprisingly, the assembly landscape is characterized by at least two distinct intermediate populations in the organisms tested. This novel, second intermediate, is formed in the presence of the full complement of r-proteins, unlike the previously observed RI* particle formed in the absence of late-binding r-proteins in E. coli. This work reveals multiple distinct intermediate populations are present during SSU assembly in vitro for several bacteria, yielding insights into RNP formation and possible antimicrobial development toward this common SSU target.

Methylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of the Sorangium cellulosum So ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum in Escherichia coli . Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core β-strands 6 and 7, which forms part of the S -adenosyl- l -methionine (SAM) binding pocket and contributes to base flipping by the m 1 A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition by S -adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate.

Highlights • –1PRF occurs when ribosomes move over a slippery sequence. • A frameshifting pseudoknot/stem-loop element stalls ribosomes in a metastable state. • –1PRF may contribute to the quality-control machinery in eukaryotes. • Trans-acting factors (proteins, miRNAs, or antibiotics) can modulate –1PRF., Programmed −1 ribosomal frameshifting (−1PRF) is an mRNA recoding event commonly utilized by viruses and bacteria to increase the information content of their genomes. Recent results have implicated −1PRF in quality control of mRNA and DNA stability in eukaryotes. Biophysical experiments demonstrated that the ribosome changes the reading frame while attempting to move over a slippery sequence of the mRNA – when a roadblock formed by a folded downstream segment in the mRNA stalls the ribosome in a metastable conformational state. The efficiency of −1PRF is modulated not only by cis-regulatory elements in the mRNA but also by trans-acting factors such as proteins, miRNAs, and antibiotics. These recent results suggest a molecular mechanism and new important cellular roles for −1PRF.

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. This structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.

Ribosomal proteins S4 and S5 participate in the decoding and assembly processes on the ribosome and the interaction with specific antibiotic inhibitors of translation. Many of the characterized mutations affecting these proteins decrease the accuracy of translation, leading to a ribosomal-ambiguity phenotype. Structural analyses of ribosomal complexes indicate that the tRNA selection pathway involves a transition between the closed and open conformations of the 30S ribosomal subunit and requires disruption of the interface between the S4 and S5 proteins. In agreement with this observation, several of the mutations that promote miscoding alter residues located at the S4-S5 interface. Here, the Escherichia coli rpsD and rpsE genes encoding the S4 and S5 proteins were targeted for mutagenesis and screened for accuracy-altering mutations. While a majority of the 38 mutant proteins recovered decrease the accuracy of translation, error-restrictive mutations were also recovered; only a minority of the mutant proteins affected rRNA processing, ribosome assembly, or interactions with antibiotics. Several of the mutations affect residues at the S4-S5 interface. These include five nonsense mutations that generate C-terminal truncations of S4. These truncations are predicted to destabilize the S4-S5 interface and, consistent with the domain closure model, all have ribosomal-ambiguity phenotypes. A substantial number of the mutations alter distant locations and conceivably affect tRNA selection through indirect effects on the S4-S5 interface or by altering interactions with adjacent ribosomal proteins and 16S rRNA.

The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. The decoding process of the ribosome has been extensively studied structurally at high resolution. Ribosome crystals are particularly sensitive to X-ray radiation, a difficulty that led to the development of data collection at cryogenic temperature, which is now the standard in macromolecular crystallography. Structural analysis of ribosomes using electron microscopy equally requires cryogenic cooling of the specimen. The advent of X-ray free electron lasers (XFELs) enabled to probe delicate crystals of ribosome complexes at ambient temperature, before they are destroyed by the fsec X-ray pulse. To explore the structural basis of ribosome decoding at near-physiological temperature, we determined the ambient-temperature X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 Angstroem resolution using data obtained at the Linac Coherent Light Source (LCLS). We found that the ambient-temperature structure was largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493, which directly participate in decoding. However, small differences in our structure include an alternate conformation of cytosine 1397 near the mRNA channel and the A-site.

The ribosome is the factory for protein biosynthesis, consisting of 3 different ribosomal RNA (rRNA) molecules (16S, 23S, and 5S rRNAs in prokaryotes) and more than 50 different ribosomal proteins. Because almost all organisms have multiple operons for rRNA genes (rrn operons), mutational analysis of ribosomes has inevitable technical difficulties, particularly for analyzing the functions of the 16S and 23S rRNAs, which form part of the core structure for the small (30S) and large (50S) subunits, respectively. In this chapter, we introduce six major strategies that allow researchers to perform mutational studies of the prokaryotic ribosome, particularly by focusing on the analysis of the 16S and 23S rRNA molecules. Although conventional mutational studies allow only for a small number of nucleotide changes simultaneously, recent approach developed by our group circumvents this problem in the Escherichia coli 16S rRNA gene, allowing for changes of up to 20% of the total nucleotides by interspecies exchange of the gene with that from foreign (non-E. coli) bacteria. The outcome of this novel technique has led to the discovery of an unexpected, nontranslational function (ribonuclease inhibitor) in the 16S rRNA molecule. The introduction of such a large sequence perturbation into the central core of the ribosome will open up a new era of ribosomal engineering to create highly functional ribosomes or phenotypic improvements of the host cell, which would be advantageous for biotechnological applications.

Chloroplast ribosomes, which originated from cyanobacteria, comprise a large subunit (50S) and a small subunit (30S) containing ribosomal RNAs (rRNAs) and various ribosomal proteins. Genes for many chloroplast ribosomal proteins, as well as proteins with auxiliary roles in ribosome biogenesis or functioning, reside in the nucleus. Here, we identified Arabidopsis (Arabidopsis thaliana) CHLOROPLAST RIBOSOME ASSOCIATED (CRASS), a member of the latter class of proteins, based on the tight coexpression of its mRNA with transcripts for nucleus-encoded chloroplast ribosomal proteins. CRASS was acquired during the evolution of embryophytes and is localized to the chloroplast stroma. Loss of CRASS results in minor defects in development, photosynthetic efficiency, and chloroplast translation activity under controlled growth conditions, but these phenotypes are greatly exacerbated under stress conditions induced by the translational inhibitors lincomycin and chloramphenicol or by cold treatment. The CRASS protein comigrates with chloroplast ribosomal particles and coimmunoprecipitates with the 16S rRNA and several chloroplast ribosomal proteins, particularly the plastid ribosomal proteins of the 30S subunit (PRPS1 and PRPS5). The association of CRASS with PRPS1 and PRPS5 is independent of rRNA and is not detectable in yeast two-hybrid experiments, implying that either CRASS interacts indirectly with PRPS1 and PRPS5 via another component of the small ribosomal subunit or that it recognizes structural features of the multiprotein/rRNA particle. CRASS plays a role in the biogenesis and/or stability of the chloroplast ribosome that becomes critical under certain stressful conditions when ribosomal activity is compromised.

Ribosomal RNA (rRNA) is a major component of ribosomes and is fundamental to the process of translation. In bacteria, 16S rRNA is a component of the small ribosomal subunit and plays a critical role in mRNA decoding. rRNA maturation entails the removal of intervening spacer sequences contained within the pre-rRNA transcript by nucleolytic enzymes. Enzymatic activities involved in maturation of the 5'-end of 16S rRNA have been identified, but those involved in 3'-end maturation of 16S rRNA are more enigmatic. Here, we investigate molecular details of 16S rRNA maturation using purified in vivo-formed small subunit (SSU) assembly intermediates (pre-SSUs) from wild-type Escherichia coli that contain precursor 16S rRNA (17S rRNA). Upon incubation of pre-SSUs with E. coli S100 cell extracts or purified enzymes implicated in 16S rRNA processing, the 17S rRNA is processed into additional intermediates and mature 16S rRNA. These results illustrate that exonucleases RNase R, RNase II, PNPase, and RNase PH can process the 3'-end of pre-SSUs in vitro. However, the endonuclease YbeY did not exhibit nucleolytic activity with pre-SSUs under these conditions. Furthermore, these data demonstrate that multiple pathways facilitate 16S rRNA maturation with pre-SSUs in vitro, with the dominant pathways entailing complete processing of the 5'-end of 17S rRNA prior to 3'-end maturation or partial processing of the 5'-end with concomitant processing of the 3'-end. These results reveal the multifaceted nature of SSU biogenesis and suggest that E. coli may be able to escape inactivation of any one enzyme by using an existing complementary pathway.

In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit. Ribosomal protein S1 forms a wall of the tunnel between RNAP and the 30S subunit, consistent with its role in directing mRNAs onto the ribosome. The nucleic-acid-binding cleft of RNAP samples distinct conformations, suggesting different functional states during transcription-translation coupling. The architecture of the 30S•RNAP complex provides a structural basis for co-localization of the transcriptional and translational machineries, and inform future mechanistic studies of coupled transcription and translation.

In this report, originally proposed apratoxin E (30S-7), revised apratoxin E (30R-7), and (30S)/(30R)-oxoapratoxin E (30S)-38/(30R)-38 were efficiently prepared by two synthetic methods. The chiral lactone 10, recycled from the degradation of saponin glycosides, was utilized to prepare the key nonpeptide fragment 9. Our alternative convergent assembly strategy was applied to the divergent synthesis of revised apratoxin E and its three analogues. Moreover, ring-closing metathesis (RCM) was for the first time found to be an efficient strategy for the macrocyclization of apratoxins.

Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy. Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNAPhe that are in perfect agreement with previous indirect estimates, and once fMet-tRNAfMet has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.

How HIV-1 genomic RNA (gRNA) is packaged into assembling virus remains unclear. Here, we use biochemical andin situapproaches to identify the complex in which the capsid protein Gag first associates with gRNA, termed the packaging initiation complex. First, we show that in the absence of assembling Gag, non-nuclear non-translating gRNA is nearly absent from the soluble fraction of provirus-expressing cells, and is found instead primarily in complexes >30S. When we express a Gag mutant known to be arrested at packaging initiation, we find only one complex containing Gag and gRNA; thus, this complex corresponds to the packaging initiation complex. This ∼80S complex also contains two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6, and therefore corresponds to a co-opted host RNA granule and a previously described capsid assembly intermediate. Additionally, we find this granule-derived packaging initiation complex in HIV-1-infected H9 T cells, and demonstrate that wild-type Gag forms both the packaging initiation complex and a larger granule-derived complex corresponding to a late packaging/assembly intermediate. We also demonstrate that packaging initiation complexes are far more numerous than P bodiesin situ. Finally, we show that Gag enters the ∼80S granule to form the packaging initiation complex via a two-step mechanism. In a step that is independent of a gRNA-binding domain, Gag enters a broad class of RNA granules, most of which lack gRNA. In a second step that is dependent on the gRNA-binding nucleocapsid domain of Gag or a heterologous gRNA-binding domain, Gag enters a gRNA-containing subset of these granules. Thus, we conclude that packaging in cells does not result from random encounters between Gag and gRNA; instead our data support a fundamentally different model in which Gag is directed to gRNA within a unique host RNA granule to initiate this critical event in HIV-1 replication.Nontechnical SummaryTo form infectious virus, the HIV-1 capsid protein Gag must associate with and package the viral genomic RNA (gRNA) during the virus assembly process. HIV-1 Gag first associates with gRNA in the cytoplasm, forming a complex termed the packaging initiation complex; this complex subsequently targets to the plasma membrane where Gag completes the assembly and packaging process before releasing the virus from the cell. Although the packaging initiation complex is critical for infectious virus formation, its identity and composition, and the mechanism by which it is formed, remain unknown. Here we identify the packaging initiation complex, and demonstrate that it corresponds to a host RNA granule that is co-opted by the virus. RNA granules are diverse complexes utilized by host cells for all aspects of RNA storage and metabolism besides translation. Our study also defines the mechanism by which HIV-1 Gag enters this host RNA granule to form the packaging initiation complex, and reveal that it involves two steps that depend on different regions of Gag. Our finding that Gag co-opts a poorly studied host complex to first associate with gRNA during packaging provides a new paradigm for understanding this critical event in the viral life cycle.

After having translated short upstream open reading frames, ribosomes can re-initiate translation on the same mRNA. This process, referred to as re-initiation, controls the translation of a large fraction of mammalian cellular mRNAs, many of which are important in cancer. Key ribosomal binding proteins involved in re-initiation are the eukaryotic translation initiation factor 2D (eIF2D) or the homologous complex of MCT-1/DENR. We determined the structures of these factors bound to the human 40S ribosomal subunit in complex with initiator tRNA positioned on an mRNA start codon in the P-site using a combination of cryoelectron microscopy and X-ray crystallography. The structures, supported by biochemical experiments, reveal how eIF2D emulates the function of several canonical translation initiation factors by using three independent, flexibly connected RNA binding domains to simultaneously monitor codon-anticodon interactions in the ribosomal P-site and position the initiator tRNA.

As a typical endoribonuclease, YoeB mediates cellular adaptation in diverse bacteria by degrading mRNAs on its activation. Although the catalytic core of YoeB is thought to be identical to well-studied nucleases, this enzyme specifically targets mRNA substrates that are associated with ribosomes in vivo. However, the molecular mechanism of mRNA recognition and cleavage by YoeB, and the requirement of ribosome for its optimal activity, largely remain elusive. Here, we report the structure of YoeB bound to 70S ribosome in pre-cleavage state, revealing that both the 30S and 50S subunits participate in YoeB binding. The mRNA is recognized by the catalytic core of YoeB, of which the general base/acid (Glu46/His83) are within hydrogen-bonding distance to their reaction atoms, demonstrating an active conformation of YoeB on ribosome. Also, the mRNA orientation involves the universally conserved A1493 and G530 of 16S rRNA. In addition, mass spectrometry data indicated that YoeB cleaves mRNA following the second position at the A-site codon, resulting in a final product with a 3′–phosphate at the newly formed 3′ end. Our results demonstrate a classical acid-base catalysis for YoeB-mediated RNA hydrolysis and provide insight into how the ribosome is essential for its specific activity. Published version

International audience; In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress-induced proteins to ribosomes. This process typically involves the formation of 100S ribosome dimers. Here, we present the structures of hibernating ribosomes from human pathogen Staphylococcus aureus containing a long variant of the hibernation-promoting factor (SaHPF) that we solved using cryo-electron microscopy. Our reconstructions reveal that the N-terminal domain (NTD) of SaHPF binds to the 30S subunit as observed for shorter variants of HPF in other species. The C-terminal domain (CTD) of SaHPF protrudes out of each ribosome in order to mediate dimerization. Using NMR, we characterized the interactions at the CTD-dimer interface. Secondary interactions are provided by helix 26 of the 16S ribosomal RNA We also show that ribosomes in the 100S particle adopt both rotated and unrotated conformations. Overall, our work illustrates a specific mode of ribosome dimerization by long HPF, a finding that may help improve the selectivity of antimicrobials.

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA‐mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli . Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA. Hfq assists ribosome assembly and associates with pre‐30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq‐mediated regulation of ribosomes is independent of its function as sRNA‐regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm‐like protein Hfq beyond its function in small RNA‐mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation.

Synthetic ribosomesin vitrocan facilitate engineering translation of novel polymers, identifying ribosome biogenesis central components, and paving the road to constructing replicating systems from defined biochemical components. Here, we report functional syntheticEscherichia coli30S ribosomal subunits constructed using a defined, purified cell free system under physiological conditions. We test hypotheses about key components of natural ribosome biogenesis pathway as required for efficient function – including integration of 16S rRNA modification, cofactors facilitated ribosome assembly and protein synthesis in the same compartmentin vitro. We observe ~17% efficiency for fully synthetic 30S and ~70% efficiency fromin vitrotranscribed 16S rRNA assembled with natural proteins. We observe up to 5 fold improvement over previous crude extracts. We suggest extending the minimal list of components required for central-dogma replication from the 151 gene products previously reported to at least 180 to allow the speed and accuracy of macromolecular synthesis to approach nativeE. colivalues.

Ribosomal protein uS4 is an essential ribosomal component involved in multiple functions, including mRNA decoding. Structural analyses indicate that during decoding, the interface between the C-terminus of uS4 and protein uS5 is disrupted and in agreement with this, C-terminal uS4 truncation mutants are readily isolated on the basis of their increased miscoding phenotypes. The same mutants can also display defects in small subunit assembly and 16S rRNA processing and some are temperature sensitive for growth. Starting with one such temperature sensitive Escherichia coli uS4 mutant, we have isolated temperature insensitive derivatives carrying additional, intragenic mutations that restore the C-terminus and ameliorate the ribosomal defects. At least one of these suppressors has no detectable ribosome biogenesis phenotype, yet still miscodes, suggesting that the C-terminal requirements for ribosome assembly are less rigid than for mRNA decoding. In contrast to the uS4 C-terminal mutants that increase miscoding, two Salmonella enterica uS4 mutants with altered C-termini have been reported as being error-restrictive. Here, reconstruction experiments demonstrate that contrary to the previous reports, these mutants have a distinct error-prone, increased misreading phenotype, consistent with the behavior of the equivalent E. coli mutants and their likely structural effects on uS4-uS5 interactions.

Research into post‐transcriptional control of mRNAs by small noncoding RNAs (sRNAs) in the model bacteria Escherichia coli and Salmonella enterica has mainly focused on sRNAs that associate with the RNA chaperone Hfq. However, the recent discovery of the protein ProQ as a common binding partner that stabilizes a distinct large class of structured sRNAs suggests that additional RNA regulons exist in these organisms. The cellular functions and molecular mechanisms of these new ProQ‐dependent sRNAs are largely unknown. Here, we report in Salmonella Typhimurium the mode‐of‐action of RaiZ, a ProQ‐dependent sRNA that is made from the 3′ end of the mRNA encoding ribosome‐inactivating protein RaiA. We show that RaiZ is a base‐pairing sRNA that represses in trans the mRNA of histone‐like protein HU‐α. RaiZ forms an RNA duplex with the ribosome‐binding site of hupA mRNA, facilitated by ProQ, to prevent 30S ribosome loading and protein synthesis of HU‐α. Similarities and differences between ProQ‐ and Hfq‐mediated regulation will be discussed.

Recent work suggests that bacterial YjeQ (RsgA) participates in the late stages of assembly of the 30S subunit and aids the assembly of the decoding center but also binds the mature 30S subunit with high affinity. To determine the function and mechanisms of YjeQ in the context of the mature subunit, we determined the cryo-EM structure of the fully assembled 30S subunit in complex with YjeQ at 5.8-A resolution. We found that binding of YjeQ stabilizes helix 44 into a conformation similar to that adopted by the subunit during proofreading. This finding indicates that, along with acting as an assembly factor, YjeQ has a role as a checkpoint protein, consisting of testing the proofreading ability of the 30S subunit. The structure also informs the mechanism by which YjeQ implements the release from the 30S subunit of a second assembly factor, called RbfA. Finally, it reveals how the 30S subunit stimulates YjeQ GTPase activity and leads to release of the protein. Checkpoint functions have been described for eukaryotic ribosome assembly factors; however, this work describes an example of a bacterial assembly factor that tests a specific translation mechanism of the 30S subunit.

To build replicating systems with new functions, the engineering of existing biological machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE might be the basis for a radically alterable, lifelike system after optimization. Here, we regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. We show that using stable isotope labeling with amino acids, mass spectrometry based quantitative proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when coexpressed in batch format PURE system. By optimizing DNA template concentrations and adapting a miniaturized Fluid Array Device with optimized feeding solution, we were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in coexpression pool varied from ∼1.5 to 5-fold compared to the batch mode, with up to ∼2.4 μM yield for a single r-protein. Reconstituted ribosomes under physiological condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ∼13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. This work also points to what is still needed to obtain self-replicating synthetic ribosomes in situ in the PURE system.

Although trans-translation mediated by tmRNA-SmpB has long been known as the sole system to relieve bacterial stalled ribosomes, ArfA has recently been identified as an alternative factor for ribosome rescue in Escherichia coli. This process requires hydrolysis of nascent peptidyl-tRNA by RF2, which usually acts as a stop codon-specific peptide release factor. It poses a fascinating question of how ArfA and RF2 recognize and rescue the stalled ribosome. Here, we mapped the location of ArfA in the stalled ribosome by directed hydroxyl radical probing. It revealed an ArfA-binding site around the neck region of the 30S subunit in which the N- and C-terminal regions of ArfA are close to the decoding center and the mRNA entry channel, respectively. ArfA and RF2 sequentially enter the ribosome stalled in either the middle or 3′ end of mRNA, whereas RF2 induces a productive conformational change of ArfA only when ribosome is stalled at the 3′ end of mRNA. On the basis of these results, we propose that ArfA functions as the sensor to recognize the target ribosome after RF2 binding.

During ribosomal translocation, a process central to the elongation phase of protein synthesis, movement of mRNA and tRNAs requires large-scale rotation of the head domain of the small (30S) subunit of the ribosome. It has generally been accepted that the head rotates by pivoting around the neck helix (h28) of 16S rRNA, its sole covalent connection to the body domain. Surprisingly, we observe that the calculated axis of rotation does not coincide with the neck. Instead, comparative structure analysis across 55 ribosome structures shows that 30S head movement results from flexing at two hinge points lying within conserved elements of 16S rRNA. Hinge 1, although located within the neck, moves by straightening of the kinked helix h28 at the point of contact with the mRNA. Hinge 2 lies within a three-way helix junction that extends to the body through a second, noncovalent connection; its movement results from flexing between helices h34 and h35 in a plane orthogonal to the movement of hinge 1. Concerted movement at these two hinges accounts for the observed magnitudes of head rotation. Our findings also explain the mode of action of spectinomycin, an antibiotic that blocks translocation by binding to hinge 2.

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.

The origins and evolution of the ribosome, 3–4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be “observed” by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call “insertion fingerprints.” Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel.

Association of the two ribosomal subunits during the process of translation initiation is a crucial step of protein synthesis. The two subunits (30S and 50S) of the bacterial 70S ribosome are held together by 12 dynamic bridges involving RNA-RNA, RNA-protein, and protein-protein interactions. The process of bridge formation, such as whether all these bridges are formed simultaneously or in a sequential order, is poorly understood. To understand such processes, we have developed and implemented a class of microfluidic devices that mix two components to completion within 0.4 ms and spray the mixture in the form of microdroplets onto an electron microscopy grid, yielding a minimum reaction time of 9.4 ms before cryofixation. Using these devices, we have obtained cryo-EM data corresponding to reaction times of 9.4 and 43 ms and have determined 3D structures of ribosomal subunit association intermediates. Molecular analyses of the cryo-EM maps reveal that eight intersubunit bridges (bridges B1a, B1b, B2a, B2b, B3, B7a, B7b, and B8) form within 9.4 ms, whereas the remaining four bridges (bridges B2c, B4, B5, and B6) take longer than 43 ms to form, suggesting that bridges are formed in a stepwise fashion. Our approach can be used to characterize sequences of various dynamic functional events on complex macromolecular assemblies such as ribosomes.

Summary The multisubunit eukaryotic translation initiation factor 3, among which the subunit b (eIF3b) is a major scaffold protein, plays essential roles in protein synthesis. Here, we report the crystal structure of the WD40 domain of Chaetomium thermophilum eIF3b, revealing a nine-bladed β-propeller fold. Sequence analysis indicates that this propeller architecture is common to all eIF3b orthologs. Revisiting the cryoelectron microscopy (cryo-EM) map of the 43S preinitiation complex suggests an interaction of the eIF3b with the 40S ribosomal subunit involving the ribosomal protein S9e and the 18S rRNA. This model is strongly supported by the direct binding of eIF3b to 40S ribosomes and to the isolated ribosomal protein rpS9e in vitro.

Ribosome is a bionanomachine that facilitates an orderly translation process during protein synthesis in living cells. Real-time monitoring of conformational changes in ribosomal subunits in aqueous solution is important to understand the regulatory mechanism of protein synthesis, because conformational changes in ribosome in E. coli have been predicted to operate the switch from translation initiation to an elongation process during translation. We performed an energy dissipation measurement by using a quartz-crystal microbalance-admittance (QCM-A) technique for in situ monitoring of conformational changes in pre-30S translation initiation complex in response to the binding of fMet-tRNA(fMet) in aqueous solution. The addition of fMet-tRNA(fMet) caused changes in the physical property (increased dehydration and elasticity) in 30S ribosomal subunit in the presence of mRNA and IF2/guanosine 5'-triphosphate (GTP) on the QCM plate. Furthermore, two sequential changes triggered by the addition of fMet-tRNA(fMet) were observed in 30S ribosomal subunit bound to mRNA in the presence of IF2/GTP and IF3. These observations suggest that the structural changes in 30S ribosomal subunit caused by the binding of fMet-tRNA(fMet) with IF2/GTP in the presence of IF3 could act as a switch to regulate the orderly processing in the construction of translation initiation complex, because the structural distinction can be a guidepost in the process for the relevant biomolecules.

Aminoglycosides are potent, broad spectrum, ribosome-targeting antibacterials whose clinical efficacy is seriously threatened by multiple resistance mechanisms. Here, we report the structural basis for 30S recognition by the novel plasmid-mediated aminoglycoside-resistance rRNA methyltransferase A (NpmA). These studies are supported by biochemical and functional assays that define the molecular features necessary for NpmA to catalyze m(1)A1408 modification and confer resistance. The requirement for the mature 30S as a substrate for NpmA is clearly explained by its recognition of four disparate 16S rRNA helices brought into proximity by 30S assembly. Our structure captures a "precatalytic state" in which multiple structural reorganizations orient functionally critical residues to flip A1408 from helix 44 and position it precisely in a remodeled active site for methylation. Our findings provide a new molecular framework for the activity of aminoglycoside-resistance rRNA methyltransferases that may serve as a functional paradigm for other modification enzymes acting late in 30S biogenesis.

The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3′ domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5′ end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation. Electronic supplementary material The online version of this article (doi:10.1007/s13238-014-0044-1) contains supplementary material, which is available to authorized users.

Ribosomes are large and highly charged macromolecular complexes consisting of RNA and proteins. Here, we address the electrostatic and nonpolar properties of ribosomal proteins that are important for ribosome assembly and interaction with other cellular components and may influence protein folding on the ribosome. We examined 50 S ribosomal subunits from 10 species and found a clear distinction between the net charge of ribosomal proteins from halophilic and non-halophilic organisms. We found that ∼67% ribosomal proteins from halophiles are negatively charged, whereas only up to ∼15% of ribosomal proteins from non-halophiles share this property. Conversely, hydrophobicity tends to be lower for ribosomal proteins from halophiles than for the corresponding proteins from non-halophiles. Importantly, the surface electrostatic potential of ribosomal proteins from all organisms, especially halophiles, has distinct positive and negative regions across all the examined species. Positively and negatively charged residues of ribosomal proteins tend to be clustered in buried and solvent-exposed regions, respectively. Hence, the majority of ribosomal proteins is characterized by a significant degree of intramolecular charge segregation, regardless of the organism of origin. This key property enables the ribosome to accommodate proteins within its complex scaffold regardless of their overall net charge.

Ribosomes, as the center of protein translation in the cell, require careful regulation via multiple pathways. While regulation of ribosomal synthesis and function has been widely studied on the transcriptional and translational “levels,” the biological roles of ribosomal post-translational modifications (PTMs) are largely not understood. Here, we explore this matter by using quantitative mass spectrometry to compare the prevalence of ribosomal methylation and acetylation for yeast in the log phase and the stationary phase of growth. We find that of the 27 modified peptides identified, two peptides experience statistically significant changes in abundance: a 1.9-fold decrease in methylation for k(Me)VSGFKDEVLETV of ribosomal protein S1B (RPS1B), and a 10-fold increase in dimethylation for r(DiMe)GGFGGR of ribosomal protein S2 (RPS2). While the biological role of RPS1B methylation has largely been unexplored, RPS2 methylation is a modification known to have a role in processing and export of ribosomal RNA. This suggests that yeast in the stationary phase increase methylation of RPS2 in order to regulate ribosomal synthesis. These results demonstrate the utility of mass spectrometry for quantifying dynamic changes in ribosomal PTMs.

Ribosomal proteins S4 and S5 participate in the decoding and assembly processes on the ribosome and the interaction with specific antibiotic inhibitors of translation. Many of the characterized mutations affecting these proteins decrease the accuracy of translation, leading to a ribosomal-ambiguity phenotype. Structural analyses of ribosomal complexes indicate that the tRNA selection pathway involves a transition between the closed and open conformations of the 30S ribosomal subunit and requires disruption of the interface between the S4 and S5 proteins. In agreement with this observation, several of the mutations that promote miscoding alter residues located at the S4-S5 interface. Here, the Escherichia coli rpsD and rpsE genes encoding the S4 and S5 proteins were targeted for mutagenesis and screened for accuracy-altering mutations. While a majority of the 38 mutant proteins recovered decrease the accuracy of translation, error-restrictive mutations were also recovered; only a minority of the mutant proteins affected rRNA processing, ribosome assembly, or interactions with antibiotics. Several of the mutations affect residues at the S4-S5 interface. These include five nonsense mutations that generate C-terminal truncations of S4. These truncations are predicted to destabilize the S4-S5 interface and, consistent with the domain closure model, all have ribosomal-ambiguity phenotypes. A substantial number of the mutations alter distant locations and conceivably affect tRNA selection through indirect effects on the S4-S5 interface or by altering interactions with adjacent ribosomal proteins and 16S rRNA.

The bacterial ribosome is a complex of three strands of RNA and approximately 55 proteins. During protein synthesis, the ribosome interacts with other proteins, numbered in the hundreds, forming some stable and some transient complexes. The stoichiometries of these complexes and of partially assembled ribosomes are often unknown. We describe the development of a flexible standard for the determination of stoichiometries of ribosomal particles and complexes. A core QconCAT, an artificial protein consisting of concatenated signature peptides derived from the ribosomal proteins L2, L4, L13, S4, S7, and S8, was developed. The core QconCAT DNA construct incorporates restriction sites for the insertion of cassettes encoding signature peptides from additional proteins under study. Two cassettes encoding signature peptides from the remaining 30S and 50S ribosomal proteins were prepared, and the resulting QconCATs were expressed, digested, and analyzed by mass spectrometry. The majority of Escherichia coli ribosomal proteins are small and basic; therefore, tryptic digestion alone yields insufficient signature peptides for quantification of all of the proteins. The ribosomal QconCATs therefore rely on a dual-enzyme strategy: endoproteinase Lys-C digestion and analysis followed by trypsin digestion and further analysis. The utility of technology was demonstrated by a determination of the effect of gentamicin on the protein composition of the E. coli ribosome.

The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4–RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein–RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA–protein complexes. Three-colour fluorescence resonance energy transfer and molecular dynamics simulations are used to study the events occurring early in assembly of the 30S ribosome; within a non-native intermediate S4 ribosomal protein–16S RNA structure, S4 is capable of altering the RNA helix dynamics to facilitate conformation changes that enable subsequent protein binding. The assembly of protein–RNA complexes can involve the sculpting of RNA structure as proteins are sequentially bound, but direct demonstration of the details of these changes has been difficult. Sarah Woodson and colleagues have used sophisticated three-colour fluorescence resonance energy transfer (FRET) and modelling to look at the events occurring early in the assembly of the 30S ribosome. They find that when the S4 ribosomal protein interacts with the 16S rRNA, there is a stable, on-path, non-native intermediate, and that the S4 protein can alter the RNA helix dynamics to facilitate conformational changes that enable subsequent protein binding.

Ribosomal protein L4 is a regulator of protein synthesis in the Escherichia coli S10 operon, which contains genes of 11 ribosomal proteins. In this work, we have investigated regulatory functions of ribosomal protein L4 of the thermophilic archaea Methanococcus jannaschii. The S10-like operon from M. jannaschii encodes not 11, but only five ribosomal proteins (L3, L4, L23, L2, S19), and the first protein is L3 instead of S10. We have shown that MjaL4 and its mutant form lacking an elongated loop specifically inhibit expression of the first gene of the S10-like operon from the same organism in a coupled transcription-translation system in vitro. By deletion analysis, an L4-binding regulatory site has been found on MjaL3 mRNA, and a fragment of mRNA with length of 40 nucleotides has been prepared that is necessary and sufficient for the specific interaction with the MjaL4 protein.

During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is facilitated by large-scale conformational changes within the ribosomal complex and catalyzed by elongtion factor G (EF-G). Previous structural analysis of the interaction of EF-G with the ribosome used either model complexes containing no tRNA or only a single tRNA, or complexes where EF-G was directly bound to ribosomes in the posttranslocational state. Here, we present a multiparticle cryo-EM reconstruction of a translocation intermediate containing two tRNAs trapped in transit, bound in chimeric intrasubunit ap/P and pe/E hybrid states. The downstream ap/P-tRNA is contacted by domain IV of EF-G and P-site elements within the 30S subunit body, whereas the upstream pe/E-tRNA maintains tight interactions with P-site elements of the swiveled 30S head. Remarkably, a tight compaction of the tRNA pair can be seen in this state. The translocational intermediate presented here represents a previously missing link in understanding the mechanism of translocation, revealing that the ribosome uses two distinct molecular ratchets, involving both intra- and intersubunit rotational movements, to drive the synchronous movement of tRNAs and mRNA.

Plastids (chloroplasts) possess 70S ribosomes that are very similar in structure and function to the ribosomes of their bacterial ancestors. While most components of the bacterial ribosome (ribosomal RNAs [rRNAs] and ribosomal proteins) are well conserved in the plastid ribosome, little is known about the factors mediating the biogenesis of plastid ribosomes. Here, we have investigated a putative homolog of the bacterial RbfA (for ribosome-binding factor A) protein that was identified as a coldshock protein and an auxiliary factor acting in the 59 maturation of the 16S rRNA. The unicellular green alga Chlamydomonas reinhardtii and the vascular plant Arabidopsis (Arabidopsis thaliana) both encode a single RbfA-like protein in their nuclear genomes. By generating specific antibodies against this protein, we show that the plant RbfA-like protein functions exclusively in the plastid, where it is associated with thylakoid membranes. Analysis of mutants for the corresponding gene (termed RBF1) reveals that the gene function is essential for photoautotrophic growth. Weak mutant alleles display reduced levels of plastid ribosomes, a specific depletion in 30S ribosomal subunits, and reduced activity of plastid protein biosynthesis. Our data suggest that, while the function in ribosome maturation and 16S rRNA 59 end processing is conserved, the RBF1 protein has assumed an additional role in 39 end processing. Together with the apparent absence of a homologous protein from plant mitochondria, our findings illustrate that the assembly process of the 70S ribosome is not strictly conserved and has undergone some modifications during organelle evolution. © 2014 American Society of Plant Biologists. All Rights Reserved.

Eukaryotic ribosome assembly involves a plethora of factors, which ensure that a correctly folded ribosome contains all ribosomal protein components. Among these assembly factors, Yar1 has recently emerged as a molecular chaperone for ribosomal protein rpS3 of the small ribosomal subunit (40S) in yeast. In complex with its chaperone, rpS3 is imported into the nucleus and protected from aggregation. How rpS3 and other ribosomal proteins are initially sequestered and subsequently integrated into pre-ribosomal particles is currently poorly understood. Here, we present the crystal structure of yeast rpS3 in complex with its chaperone Yar1 at 2.8Å resolution. The crystal structure rationalizes how Yar1 can protect rpS3 from aggregation while facilitating nuclear import and suggests a mechanism for a stepwise exchange of molecular partners that ribosomal proteins interact with during ribosome assembly.

Graphical abstract, Accurate unbound solution 3D-structures of ligands provide unique opportunities for medicinal chemistry and, in particular, a context to understand binding thermodynamics and kinetics. Previous methods of deriving these 3D-structures have had neither the accuracy nor resolution needed for drug design and have not yet realized their potential. Here, we describe and apply a NMR methodology to the aminoglycoside streptomycin that can accurately quantify accessible 3D-space and rank the occupancy of observed conformers to a resolution that enables medicinal chemistry understanding and design. Importantly, it is based upon conventional small molecule NMR techniques and can be performed in physiologically-relevant solvents. The methodology uses multiple datasets, an order of magnitude more experimental data than previous NMR approaches and a dynamic model during refinement, is independent of computational chemistry and avoids the problem of virtual conformations. The refined set of solution 3D-shapes for streptomycin can be grouped into two major families, of which the most populated is almost identical to the 30S ribosomal subunit bioactive shape. We therefore propose that accurate unbound ligand solution conformations may, in some cases, provide a subsidiary route to bioactive shape without crystallography. This experimental technique opens up new opportunities for drug design and more so when complemented with protein co-crystal structures, SAR data and pharmacophore modeling.

Upon high throughput screening of 6700 microbial fermentation extracts, we discovered a compound, designated orthoformimycin, capable of inhibiting protein synthesis in vitro with high efficiency. The molecule, whose structure was elucidated by chemical, spectrometric, and spectroscopic methods, contains an unusual orthoformate moiety (hence the name) and belongs to a novel class of translation inhibitors. This antibiotic does not affect any function of the 30S ribosomal subunit but binds to the 50S subunit causing inhibition of translation elongation and yielding polypeptide products of reduced length. Analysis by fluorescence stopped flow kinetics revealed that EF-G-dependent mRNA translocation is inhibited by orthoformimycin, whereas, surprisingly, translocation of the aminoacyl-tRNA seems to be unaffected. © 2013 American Chemical Society.

During translation, initiation factor (IF) 3 binds to the small, 30S, ribosomal subunit and regulates the fidelity with which the initiator tRNA and mRNA start codon substrates are selected into the 30S initiation complex (30S IC). The molecular mechanism through which IF3 promotes recognition and signaling of correct substrate selection, however, remains poorly defined. Using single-molecule fluorescence resonance energy transfer, here we show that 30S IC-bound Escherichia coli IF3 exists in a dynamic equilibrium between at least three conformations. We have found that recognition of a proper anticodon-codon interaction between initiator tRNA and the start codon within a completely assembled 30S IC selectively shifts this equilibrium towards a single IF3 conformation. Our results strongly support a conformational selection model in which the conformation of IF3 that is selectively stabilized within a completely and correctly assembled 30S IC facilitates further progress along the initiation pathway.

Translation initiation is a dynamic and complicated process requiring the building a 70S initiation complex (70S-IC) composed of a ribosome, mRNA, and an initiator tRNA. During the formation of the 70S-IC, initiation factors (IFs: IF1, IF2, and IF3) interact with a ribosome to form a 30S initiation complex (30S-IC) and a 70S-IC. Although some spectroscopic analyses have been performed, the mechanism of binding and dissociation of IFs remains unclear. Here, we employed a 27 MHz quartz crystal microbalance (QCM) to evaluate the process of bacterial IC formation in translation initiation by following frequency changes (mass changes). IFs (IF1, IF2, and IF3), N-terminally fused to biotin carboxyl carrier protein (bio-BCCP), were immobilized on a Neutravidin-covered QCM plate. By using bio-BCCP-IF2 immobilized to the QCM, three steps of the formation of ribosomal initiation complex could be sequentially observed as simple mass changes in real time: binding of a 30S complex to the immobilized IF2, a recruitment of 50S to the 30S-IC, and formation of the 70S-IC. The kinetic parameters implied that the release of IF2 from the 70S-IC could be the rate-limiting step in translation initiation. The IF3-immobilized QCM revealed that the affinity of IF3 for the 30S complex decreased upon the addition of mRNA and fMet-tRNA(fMet) but did not lead to complete dissociation from the 30S-IC. These results suggest that IF3 binds and stays bound to ICs, and its interaction mode is altered during the formation of 30S-IC and 70S-IC and is finally induced to dissociate from ICs by 50S binding. This methodology demonstrated here is applicable to investigate the role of IFs in translation initiation driven by other pathways.

The increase in antibiotic-resistant microorganisms has driven a search for new antibiotic targets and novel antimicrobial agents. A large number of different antibiotics target bacterial ribosomal subunit formation. Several specific ribonucleases are important in the processing of rRNA during subunit biogenesis. This work demonstrates that the ribonuclease inhibitor, vanadyl ribonucleoside complex (VRC), can inhibit RNases involved in ribosomal subunit formation. The ribosomal subunit synthesis rate was significantly decreased and ribosomal RNA from the subunit precursors was degraded. VRC had no inhibitory effect on translation. VRC also potentiated the inhibitory effects of an aminoglycoside and a macrolide antibiotic.

Cinergie – Il Cinema e le altre Arti, No 3 (2013)

The L1 stalk of the large ribosomal subunit undergoes large-scale movements coupled to the translocation of deacylated tRNA during protein synthesis. We use quantitative comparative structural analysis to localize the origins of L1 stalk movement and to understand its dynamic interactions with tRNA and other structural elements of the ribosome. Besides its stacking interactions with the tRNA elbow, stalk movement is directly linked to intersubunit rotation, rotation of the 30S head domain and contact of the acceptor arm of deacylated tRNA with helix 68 of 23S rRNA. Movement originates from pivoting at stacked non-canonical base pairs in a Family A three-way junction and bending in an internal G-U-rich zone. Use of these same motifs as hinge points to enable such dynamic events as rotation of the 30S subunit head domain and in flexing of the anticodon arm of tRNA suggests that they represent general strategies for movement of functional RNAs., Translocation of the tRNA on the ribosome is associated with large-scale molecular movements of the ribosomal L1 stalk. Here the authors identify the key determinants that allow these dramatic movements, and suggest they represent general strategies used to enable large-scale motions in functional RNAs.

The pathogen-associated 16S rRNA methyltransferase NpmA catalyzes m 1 A1408 modification to block the action of structurally diverse aminoglycoside antibiotics. Here, we describe the development of a fluorescence polarization binding assay and its use, together with complementary functional assays, to dissect the mechanism of NpmA substrate recognition. These studies reveal that electrostatic interactions made by the NpmA β2/3 linker collectively are critical for docking of NpmA on a conserved 16S rRNA tertiary surface. In contrast, other NpmA regions (β5/β6 and β6/β7 linkers) contain several residues critical for optimal positioning of A1408 but are largely dispensable for 30S binding. Our data support a model for NpmA action in which 30S binding and adoption of a catalytically competent state are distinct: docking on 16S rRNA via the β2/3 linker necessarily precedes functionally critical 30S substrate-driven conformational changes elsewhere in NpmA. This model is also consistent with catalysis being completely positional in nature, as the most significant effects on activity arise from changes that impact binding or stabilization of the flipped A1408 conformation. Our results provide a molecular framework for aminoglycoside resistance methyltransferase action that may serve as a functional paradigm for related enzymes and a starting point for development of inhibitors of these resistance determinants.