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

Chulluncuy, Roberto; Espiche, Carlos; Nakamoto, Jose A.; Fabbretti, Attilio; Milón, Pohl

doi:10.3390/antibiotics5040038

Cited by 20 publications

(22 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An increase in time of donor fluorescence indicates that IF3 domains are moving apart and, vice versa , a decrease of the observed fluorescence indicates that the domains are getting close to each other (Supp. Fig 1) [23]. Binding of IF3 DL to the 30S ribosomal subunit resulted in an increase of fluorescence, consistent with the factor transiting from a closed conformation in solution to a more opened one while bound to the 30S subunit (Fig 2A).…”

Section: Resultssupporting

confidence: 70%

“…To monitor the conformational rearrangements of IF3, we used a fluorescent derivative of the factor harboring donor and silent acceptor dyes specifically linked to the N and C terminal domains, respectively (Fig1, inset) [23]. Double-labeled IF3 (IF3 DL ) allows to monitor intramolecular FRET changes resulting from the movement of IF3 domains with respect to each other.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The complete, functional and dynamic cycle of the bacterial Initiation Factor 3

Nakamoto

Spurio

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

31Initiation factor 3 (IF3) is an essential protein that enhances the fidelity and speed of 32 bacterial initiation of mRNA translation. The dynamic interplay between the two 33 independent IF3 domains, their alternative binding sites, and the mechanism that ensures 34 translation initiation fidelity remains elusive. Here, we show that the functional positioning 35 of IF3 domains occurs at velocities ranging over two orders of magnitude, driven by each 36 30S initiation ligand. IF1 and IF2 rapidly promote the accommodation of IF3 on the 30S 37 platform with the C-terminal domain moving towards the P site. Reversion of this 38 movement is triggered by decoding the mRNA start codon and rate limits translation 39 initiation. Binding of the tRNA results in the concomitant accommodation of the N-terminal 40 domain of IF3, largely dependent on the mRNA and initiator tRNA. 70S initiation complex 41 formation promotes the closing and dissociation of IF3, recycling the factor for a new round 42 of translation initiation. Altogether our results unveil the kinetic spectrum of IF3 43 conformations and highlight fundamental movements of the factor that ensure accurate 44 translation initiation. 45 46 47 48 49 IF3 is an essential bacterial protein, consisting of two domains (IF3C and IF3N) separated 50 by a hydrophilic, lysine-rich, linker [1-3]. IF3 is involved in all steps of the bacterial 51 translation initiation driving the 30S ribosomal subunit trough the transition from the 30S 52 initiation complex (30S IC) to a productive 70S initiation complex (70S IC) [4-6]. When 53bound to the 30S subunit, IF3 prevents the premature 50S subunit association and 54 increases the rate of the P site codon-anticodon interaction between fMet-tRNA fMet and the 55 initiation triplet of the mRNA [5][6][7][8]. As a direct consequence, IF3 acts as an initiation fidelity 56 factor by increasing the dissociation rate of non-canonical and pseudo-30S initiation 57 complexes [8][9][10]. 58Although IF3 functions during the initiation phase of protein synthesis are well established, 59 dynamic aspects of IF3 binding on ribosomal subunits and the release of the factor after 60 the formation of productive 30S IC are still debated [11,12]. A model where IF3C and IF3N 61 domains independently bind the 30S ribosomal subunit was initially proposed in the early 62 '80s [13] and then confirmed by NMR analysis and time-resolved chemical probing 63 experiments [14,15]. Several studies have dealt with the assignment of a topographical 64 localization of IF3 on the 30S ribosomal subunit, producing conflicting conclusions [16][17][18][19]. 65Recently, CryoEM experiments demonstrated that IF3 undergoes large conformational 66 changes to facilitate the accommodation of the initiator tRNA (fMet-tRNA fMet ) into the P site 67for start codon recognition [20]. These experiments, together with other recent structural 68 reconstructions, contributed to the identification of a minimum of four different IF3 69 arrangements on the 30S ribosomal subunit and two alternative...

show abstract

Section: Resultssupporting

confidence: 70%

Section: Resultsmentioning

confidence: 99%

The complete, functional and dynamic cycle of the bacterial Initiation Factor 3

Nakamoto

Spurio

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, IF-3 was found to be underrepresented on the mutant’s 30S compared to the wild-type strain, and this protein is known to stabilize the transiently recycled 30S subunits by preventing reassociation into 70S in E. coli ( Hirokawa et al, 2005 ). Studies in M. tuberculosis showed that kanamycin and streptomycin both affect the positioning of the IF-3 on the 30S platform and the overall dynamics of 30S association with the IF-3, thus perturbing the translation initiation ( Chulluncuy et al, 2016 ). Low abundance of IF-3 on the mutant’s ribosomes may indicate that these 30S ribosomal subunits remain in conformation, thus favoring the inhibition of translation by aminoglycosides.…”

Section: Discussionmentioning

confidence: 99%

PdtaS Deficiency Affects Resistance of Mycobacteria to Ribosome Targeting Antibiotics

et al. 2017

View full text Add to dashboard Cite

Two-component regulatory systems (TCSSs) are key regulatory elements responsible for the adaptation of bacteria to environmental stresses. A classical TCSS is typically comprised of a sensory histidine kinase and a corresponding response regulator. Here, we used homologous recombination to construct a Mycobacterium smegmatis mutant defective in the synthesis of cytosolic histidine kinase PdtaS (Msmeg_1918). The resulting ΔpdtaS mutant strain was tested in the Phenotype Microarray screening system, which allowed us to identify aminoglycoside antibiotic sensitivity, tetracyclines antibiotic resistance as well as membrane transport and respiration, as the main processes affected by removal of pdtaS. The antibiotic sensitivity profiles were confirmed by survival assessment and complementation studies. To gain insight into the molecular mechanisms responsible for the observed phenotype, we compared ribosomal RNA and protein profiles of the mutant and wild-type strains. We carried out Northern blotting and qRT-PCR to compare rRNA levels and analyzed ribosome sedimentation patterns of the wild-type and mutant strains on sucrose gradients. Isolated ribosomes were further used to estimate relative abundance of individual proteins in the ribosomal subunits using label free mass spectrometry analysis. Additionally, the ΔpdtaS mutant revealed lower activity of the respiratory chain as measured by the rate of TTC (triphenyltetrazolium chloride) reduction, while at the same time showing only insignificant changes in the uptake of aminoglycosides. We postulate that deficiency of PdtaS affects the oxidative respiration rates and ribosomal composition causing relevant changes to intrinsic resistance or susceptibility to antibiotics targeting ribosomes, which are commonly used to treat mycobacterial infections.

show abstract

“…Fluorescent measurements were carried out at 22 ˚C using a monochromatic light source (470 nm) powered with 10 mA. Emitted fluorescence was measured with photomultipliers set at 320 V after passing a long-pass filter with a cut-off of 515 nm essentially as described in [35]. Equal volumes (60 μL) of each reactant were rapidly mixed and fluorescence changes over time were measured.…”

Section: Methodsmentioning

confidence: 99%

DNA aptamers for the recognition of HMGB1 from Plasmodium falciparum

et al. 2019

Self Cite

View full text Add to dashboard Cite

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.

show abstract

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

Cited by 20 publications

References 57 publications

The complete, functional and dynamic cycle of the bacterial Initiation Factor 3

The complete, functional and dynamic cycle of the bacterial Initiation Factor 3

PdtaS Deficiency Affects Resistance of Mycobacteria to Ribosome Targeting Antibiotics

DNA aptamers for the recognition of HMGB1 from Plasmodium falciparum

Contact Info

Product

Resources

About