FAST, a method based on split-GFP for the detection in solution of proteins synthesized in cell-free expression systems.

Cell-free protein synthesis (CFPS) systems offer a versatile platform for a wide range of applications. However, the traditional methods for detecting proteins synthesized in CFPS, such as radioactive labeling, fluorescent tagging, or electrophoretic separation, may be impractical, due to environmental hazards, high costs, technical complexity, and time consuming procedures. These limitations underscore the need for new approaches that streamline the detection process, facilitating broader application of CFPS. By harnessing the reassembly capabilities of two GFP fragments-specifically, the GFP1-10 and GFP11 fragments-we have crafted a method that simplifies the detection of in vitro synthesized proteins called FAST (Fluorescent Assembly of Split-GFP for Translation Tests). FAST relies on the fusion of the small tag GFP11 to virtually any gene to be expressed in CFPS. The in vitro synthesized protein:GFP11 can be rapidly detected in solution upon interaction with an enhanced GFP1-10 fused to the Maltose Binding Protein (MBP:GFP1-10). This interaction produces a fluorescent signal detectable with standard fluorescence readers, thereby indicating successful protein synthesis. Furthermore, if required, detection can be coupled with the purification of the fluorescent complex using standardized MBP affinity chromatography. The method's versatility was demonstrated by fusing GFP11 to four distinct E. coli genes and analyzing the resulting protein synthesis in both a homemade and a commercial E. coli CFPS system. Our experiments confirmed that the FAST method offers a direct correlation between the fluorescent signal and the amount of synthesized protein:GFP11 fusion, achieving a sensitivity threshold of 8 ± 2 pmol of polypeptide, with fluorescence plateauing after 4 h. Additionally, FAST enables the investigation of translation inhibition by antibiotics in a dose-dependent manner. In conclusion, FAST is a new method that permits the rapid, efficient, and non-hazardous detection of protein synthesized within CFPS systems and, at the same time, the purification of the target protein.

Novel CRISPR-based detection of Leishmania species.

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10-2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

Identifying RO9021 as a Potential Inhibitor of PknG from Mycobacterium tuberculosis: Combinative Computational and In Vitro Studies.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). Despite being considered curable and preventable, the increase of antibiotic resistance is becoming a serious public health problem. Mtb is a pathogen capable of surviving in macrophages, causing long-term latent infection where the mycobacterial serine/threonine protein kinase G (PknG) plays a protective role. Therefore, PknG is an important inhibitory target to prevent Mtb from entering the latency stage. In this study, we use a pharmacophore-based virtual screening and biochemical assays to identify the compound RO9021 (CHEMBL3237561) as a PknG inhibitor. In detail, 1.5 million molecules were screened using a scalable cloud-based setup, identifying 689 candidates, which were further subjected to additional screening employing molecular docking. Molecular docking spotted 62 compounds with estimated binding affinities of -7.54 kcal/mol (s.d. = 0.77 kcal/mol). Finally, 14 compounds were selected for in vitro experiments considering previously reported biological activities and commercial availability. In vitro assays of PknG activity showed that RO9021 inhibits the kinase activity similarly to AX20017, a known inhibitor. The inhibitory effect was found to be dose dependent with a relative IC50 value of 4.4 ± 1.1 µM. Molecular dynamics simulations predicted that the PknG-RO9021 complex is stable along the tested timescale. Altogether, our study indicates that RO9021 is a noteworthy drug candidate for further developing new anti-TB drugs that hold excellent reported pharmacokinetic parameters.

The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions.

During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states. Entry into the OFF state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly and growth. Here, we determine how (p)ppGpp impacts RA-GTPase-ribosome interactions. We show that RA-GTPases preferentially bind to 5'-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell to shut down ribosome biogenesis during stress. Stopped-flow fluorescence and association assays reveal that when bound to (p)ppGpp, the association of RA-GTPases to ribosomal subunits is destabilized, both in vitro and within bacterial cells. Consistently, structural analysis of the ppGpp-bound RA-GTPase RsgA reveals an OFF-state conformation similar to the GDP-bound state, with the G2/switch I loop adopting a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated ribosome assembly and growth control. IMPORTANCE The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown. Our previous work revealed that (p)ppGpp can bind to and inhibit the enzymatic activity of four ribosome-associated GTPases (RA-GTPases), enzymes that facilitate maturation of the 50S and 30S ribosomal subunits. Here, we examine how this occurs mechanistically and demonstrate that this interaction prevents the accommodation of RA-GTPases on ribosomal subunits both in vitro and within bacterial cells, with the ppGpp-bound state structurally mimicking the inactive GDP-bound conformation of the enzyme. We additionally reveal that these GTPase enzymes have a greater affinity for OFF-state-inducing nucleotides, which is a mechanism likely to control ribosome assembly during growth. With this, we further our understanding of how ribosome function is controlled by (p)ppGpp, enabling bacterial survival during stress.

A low-cost and open-source protocol to produce key enzymes for molecular detection assays.

Here, we describe a detailed step-by-step protocol for the expression, purification, quantification, and activity determination of key enzymes for molecular detection of pathogens. Based on previous reports, we optimized the protocol for LbCas12a, Taq DNA polymerase, M-MLV reverse transcriptase, and TEV protease to make it compatible with minimal laboratory equipment, broadly available in low- and middle-income countries. The enzymes produced with this protocol have been successfully used for molecular detection applications. For complete details on the use and execution of this protocol, please refer to Alcántara et al. (2021a, 2021b).

Unlocking SARS-CoV-2 detection in low- and middle-income countries.

Low- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local production and implementation of molecular testing. Here, we provide detailed methods and validation of a molecular toolkit that can be readily produced and deployed using laboratory equipment available in LMICs. Our results show that lab-scale production of enzymes and nucleic acids can supply over 50,000 tests per production batch. The optimized one-step RT-PCR coupled to CRISPR-Cas12a-mediated detection showed a limit of detection of 102 ge/µL in a turnaround time of 2 h. The clinical validation indicated an overall sensitivity of 80%-88%, while for middle and high viral load samples (Cq ≤ 31) the sensitivity was 92%-100%. The specificity was 96%-100% regardless of viral load. Furthermore, we show that the toolkit can be used with the mobile laboratory Bento Lab, potentially enabling LMICs to implement detection services in unattended remote regions.

UnCovid: A versatile, low-cost, and open-source protocol for SARS-CoV-2 RNA detection.

Here, we describe a detailed step-by-step protocol to detect SARS-CoV-2 RNA using RT-PCR-mediated amplification and CRISPR/Cas-based visualization. The optimized assay uses basic molecular biology equipment such as conventional thermocyclers and transilluminators for qualitative detection. Alternatively, a fluorescence plate reader can be used for quantitative measurements. The protocol detects two regions of the SARS-CoV-2 genome in addition to the human RNaseP sample control. Aiming to reach remote regions, this work was developed to use the portable molecular workstation from BentoLab. For complete details on the use and execution of this protocol, please refer to Alcántara et al., 2021.

A Complementary Mechanism of Bacterial mRNA Translation Inhibition by Tetracyclines.

Tetracycline has positively impacted human health as well as the farming and animal industries. Its extensive usage and versatility led to the spread of resistance mechanisms followed by the development of new variants of the antibiotic. Tetracyclines inhibit bacterial growth by impeding the binding of elongator tRNAs to the ribosome. However, a small number of reports indicated that Tetracyclines could also inhibit translation initiation, yet the molecular mechanism remained unknown. Here, we use biochemical and computational methods to study how Oxytetracycline (Otc), Demeclocycline (Dem), and Tigecycline (Tig) affect the translation initiation phase of protein synthesis. Our results show that all three Tetracyclines induce Initiation Factor IF3 to adopt a compact conformation on the 30S ribosomal subunit, similar to that induced by Initiation Factor IF1. This compaction was faster for Tig than Dem or Otc. Furthermore, all three tested tetracyclines affected IF1-bound 30S complexes. The dissociation rate constant of IF1 in early 30S complexes was 14-fold slower for Tig than Dem or Otc. Late 30S initiation complexes (30S pre-IC or IC) exhibited greater IF1 stabilization by Tig than for Dem and Otc. Tig and Otc delayed 50S joining to 30S initiation complexes (30S ICs). Remarkably, the presence of Tig considerably slowed the progression to translation elongation and retained IF1 in the resulting 70S initiation complex (70S IC). Molecular modeling of Tetracyclines bound to the 30S pre-IC and 30S IC indicated that the antibiotics binding site topography fluctuates along the initiation pathway. Mainly, 30S complexes show potential contacts between Dem or Tig with IF1, providing a structural rationale for the enhanced affinity of the antibiotics in the presence of the factor. Altogether, our data indicate that Tetracyclines inhibit translation initiation by allosterically perturbing the IF3 layout on the 30S, retaining IF1 during 70S IC formation, and slowing the transition toward translation elongation. Thus, this study describes a new complementary mechanism by which Tetracyclines may inhibit bacterial protein synthesis.

The dynamic cycle of bacterial translation initiation factor IF3.

Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation.

Long-range allostery mediates cooperative adenine nucleotide binding by the Ski2-like RNA helicase Brr2.

Brr2 is an essential Ski2-like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N-terminal helicase cassette and a structurally similar but enzymatically inactive C-terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide-binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide-binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C-terminal cassette is more than two orders of magnitude higher than that of the active N-terminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C-terminal cassette reduces nucleotide binding at the N-terminal cassette 70 Å away. Molecular dynamics simulations identified structural communication lines that likely mediate these long-range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine-tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicing.

Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation.

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.

How the initiating ribosome copes with ppGpp to translate mRNAs.

During host colonization, bacteria use the alarmones (p)ppGpp to reshape their proteome by acting pleiotropically on DNA, RNA, and protein synthesis. Here, we elucidate how the initiating ribosome senses the cellular pool of guanosine nucleotides and regulates the progression towards protein synthesis. Our results show that the affinity of guanosine triphosphate (GTP) and the inhibitory concentration of ppGpp for the 30S-bound initiation factor IF2 vary depending on the programmed mRNA. The TufA mRNA enhanced GTP affinity for 30S complexes, resulting in improved ppGpp tolerance and allowing efficient protein synthesis. Conversely, the InfA mRNA allowed ppGpp to compete with GTP for IF2, thus stalling 30S complexes. Structural modeling and biochemical analysis of the TufA mRNA unveiled a structured enhancer of translation initiation (SETI) composed of two consecutive hairpins proximal to the translation initiation region (TIR) that largely account for ppGpp tolerance under physiological concentrations of guanosine nucleotides. Furthermore, our results show that the mechanism enhancing ppGpp tolerance is not restricted to the TufA mRNA, as similar ppGpp tolerance was found for the SETI-containing Rnr mRNA. Finally, we show that IF2 can use pppGpp to promote the formation of 30S initiation complexes (ICs), albeit requiring higher factor concentration and resulting in slower transitions to translation elongation. Altogether, our data unveil a novel regulatory mechanism at the onset of protein synthesis that tolerates physiological concentrations of ppGpp and that bacteria can exploit to modulate their proteome as a function of the nutritional shift happening during stringent response and infection.

DNA aptamers for the recognition of HMGB1 from Plasmodium falciparum.

Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable, and present along all blood-stages of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in a few seconds. The specificity of aptamer binding to the HMG-box of P. falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a candidate biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P. falciparum detection.

Antibiotics Targeting the 30S Ribosomal Subunit: A Lesson from Nature to Find and Develop New Drugs.

The use of antibiotics has revolutionized medicine, greatly improving our capacity to save millions of lives from otherwise deadly bacterial infections. Unfortunately, the health-associated benefits provided by antibiotics have been counteracted by bacteria developing or acquiring resistance mechanisms. The negative impact to public health is now considered of high risk due to the rapid spreading of multi-resistant strains. More than 60 % of clinically relevant antibiotics of natural origin target the ribosome, the supramolecular enzyme which translates the genetic information into proteins. Although many of these antibiotics bind the small ribosomal subunit, only a few are reported to inhibit the initiation of protein synthesis, with none reaching commercial availability. Counterintuitively, translation initiation is the most divergent phase of protein synthesis between prokaryotes and eukaryotes, a fact which is a solid premise for the successful identification of drugs with reduced probability of undesired effects to the host. Such a paradox is one of its kind and deserves special attention. In this review, we explore the inhibitors that bind the 30S ribosomal subunit focusing on both the compounds with proved effects on the translation initiation step and the underreported translation initiation inhibitors. In addition, we explore recent screening tests and approaches to discover new drugs targeting translation.

Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin.

Aminoglycoside antibiotics are widely used to treat infectious diseases. Among them, streptomycin and kanamycin (and derivatives) are of importance to battle multidrug-resistant (MDR) Mycobacterium tuberculosis. Both drugs bind the small ribosomal subunit (30S) and inhibit protein synthesis. Genetic, structural, and biochemical studies indicate that local and long-range conformational rearrangements of the 30S subunit account for this inhibition. Here, we use intramolecular FRET between the C- and N-terminus domains of the flexible IF3 to monitor real-time perturbations of their binding sites on the 30S platform. Steady and pre-steady state binding experiments show that both aminoglycosides bring IF3 domains apart, promoting an elongated state of the factor. Binding of Initiation Factor IF1 triggers closure of IF3 bound to the 30S complex, while both aminoglycosides revert the IF1-dependent conformation. Our results uncover dynamic perturbations across the 30S subunit, from the A-site to the platform, and suggest that both aminoglycosides could interfere with prokaryotic translation initiation by modulating the interaction between IF3 domains with the 30S platform.

Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A.

Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC.

Directional transition from initiation to elongation in bacterial translation.

The transition of the 30S initiation complex (IC) to the translating 70S ribosome after 50S subunit joining provides an important checkpoint for mRNA selection during translation in bacteria. Here, we study the timing and control of reactions that occur during 70S IC formation by rapid kinetic techniques, using a toolbox of fluorescence-labeled translation components. We present a kinetic model based on global fitting of time courses obtained with eight different reporters at increasing concentrations of 50S subunits. IF1 and IF3 together affect the kinetics of subunit joining, but do not alter the elemental rates of subsequent steps of 70S IC maturation. After 50S subunit joining, IF2-dependent reactions take place independent of the presence of IF1 or IF3. GTP hydrolysis triggers the efficient dissociation of fMet-tRNA(fMet) from IF2 and promotes the dissociation of IF2 and IF1 from the 70S IC, but does not affect IF3. The presence of non-hydrolyzable GTP analogs shifts the equilibrium towards a stable 70S-mRNA-IF1-IF2-fMet-tRNA(fMet) complex. Our kinetic analysis reveals the molecular choreography of the late stages in translation initiation.

Novel insights into the architecture and protein interaction network of yeast eIF3.

Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs). The largest of these factors, eIF3, forms the scaffold for other initiation factors, promoting their binding to the 40S ribosomal subunit. Biochemical and structural studies on eIF3 need highly pure eIF3. However, natively purified eIF3 comprise complexes containing other proteins such as eIF5. Therefore we have established in vitro reconstitution protocols for Saccharomyces cerevisiae eIF3 using its five recombinantly expressed and purified subunits. This reconstituted eIF3 complex (eIF3(rec)) exhibits the same size and activity as the natively purified eIF3 (eIF3(nat)). The homogeneity and stoichiometry of eIF3(rec) and eIF3(nat) were confirmed by analytical size exclusion chromatography, mass spectrometry, and multi-angle light scattering, demonstrating the presence of one copy of each subunit in the eIF3 complex. The reconstituted and native eIF3 complexes were compared by single-particle electron microscopy showing a high degree of structural conservation. The interaction network between eIF3 proteins was studied by means of limited proteolysis, analytical size exclusion chromatography, in vitro binding assays, and isothermal titration calorimetry, unveiling distinct protein domains and subcomplexes that are critical for the integrity of the protein network in yeast eIF3. Taken together, the data presented here provide a novel procedure to obtain highly pure yeast eIF3, suitable for biochemical and structural analysis, in addition to a detailed picture of the network of protein interactions within this complex.

Structural and functional characterization of the bacterial translocation inhibitor GE82832.

The structure of GE82832, a translocation inhibitor produced by a soil microorganism, is shown to be highly related to that of dityromycin, a bicyclodecadepsipeptide antibiotic discovered long ago whose characterization had never been pursued beyond its structural elucidation. GE82832 and dityromycin were shown to interfere with both aminoacyl-tRNA and mRNA movement and with the Pi release occurring after ribosome- and EF-G-dependent GTP hydrolysis. These findings and the unusual ribosomal localization of GE82832/dityromycin near protein S13 suggest that the mechanism of inhibition entails an interference with the rotation of the 30S subunit "head" which accompanies the ribosome-unlocking step of translocation.

Impact of methylations of m2G966/m5C967 in 16S rRNA on bacterial fitness and translation initiation.

The functional centers of the ribosome in all organisms contain ribosomal RNA (rRNA) modifications, which are introduced by specialized enzymes and come at an energy cost for the cell. Surprisingly, none of the modifications tested so far was essential for growth and hence the functional role of modifications is largely unknown. Here, we show that the methyl groups of nucleosides m(2)G966 and m(5)C967 of 16S rRNA in Escherichia coli are important for bacterial fitness. In vitro analysis of all phases of translation suggests that the m(2)G966/m(5)C967 modifications are dispensable for elongation, termination and ribosome recycling. Rather, the modifications modulate the early stages of initiation by stabilizing the binding of fMet-tRNA(fMet) to the 30S pre-initiation complex prior to start-codon recognition. We propose that the m(2)G966 and m(5)C967 modifications help shaping the bacterial proteome, most likely by fine-tuning the rates that determine the fate of a given messenger RNA (mRNA) at early checkpoints of mRNA selection.