Regulation and mechanistic basis of macrolide resistance by the ABC-F ATPase MsrD

Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors currently proliferating among human pathogens that provide resistance against clinically important ribosome-targeting antibiotics. Here, we combine genetic and structural approaches to determine the activity of the streptococcal ARE ABC-F protein MsrD on the ribosome and its regulation in response to macrolide exposure. We show that cladinose-containing macrolides lead to insertion of MsrDL leader peptide into a conserved crevice of the ribosomal exit tunnel, which remained thus far undocumented, concomitantly with 23S rRNA rearrangements that preclude proper accommodation of release factors and inhibits termination. The stalled ribosome obstructs formation of a Rho-independent terminator which prevents msrD transcriptional attenuation. This stalled ribosome is rescued by MsrD powered by its two functionally asymmetric ATPase sites, but not by MsrD mutants which do not provide antibiotic resistance, showing evidence of equivalence between MsrD function in antibiotic resistance and its action on this complex.

Mammalian HEMK1 Methylates Glutamine Residue of the GGQ Motif of Mitochondrial Release Factors

Despite limited reports on glutamine methylation, methylated glutamine is found to be highly conserved in a "GGQ" motif in both prokaryotes and eukaryotes. In bacteria, glutamine methylation of peptide chain release factors 1/2 (RF1/2) by the enzyme PrmC is essential for translational termination and transcript recycling. Two PrmC homologs, HEMK1 and HEMK2, are found in mammals. In contrast to those of HEMK2, the biochemical properties and biological significance of HEMK1 remain largely unknown. In this study, we demonstrated that HEMK1 is an active methyltransferase for the glutamine residue of the GGQ motif of all four putative mitochondrial release factors (mtRFs)—MTRF1, MTRF1L, MRPL58, and MTRFR. In HEMK1-deficient HeLa cells, GGQ motif glutamine methylation was absent in all the mtRFs. We examined cell growth and mitochondrial properties, but disruption of the HEMK1 gene had no considerable impact on the overall cell growth, mtDNA copy number, and mitochondrial membrane potential. Furthermore, mitochondrial protein synthesis was not affected in HEMK1 KO cells. Our results suggest that HEMK1 mediates the GGQ methylation of all four mtRFs in human cells; however, this specific modification seems mostly dispensable in cell growth and mitochondrial protein homeostasis under standard culture conditions.

Repurposing tRNAs for nonsense suppression

Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.

Mechanisms that ensure speed and fidelity in eukaryotic translation termination

Translation termination, which liberates a nascent polypeptide from the ribosome specifically at stop codons, must occur accurately and rapidly. We established single molecule fluorescence assays to track the dynamics of ribosomes and two requisite release factors (eRF1 and eRF3) throughout termination using an in vitro reconstituted yeast translation system. We found that the two eukaryotic release factors bind together to recognize stop codons rapidly and elicit termination via a tightly regulated, multi-step process that resembles tRNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis.

In silico analysis of bacterial translation factors reveal distinct translation event specific pI values

Background Protein synthesis is a cellular process that takes place through the successive translation events within the ribosome by the event-specific protein factors, namely, initiation, elongation, release, and recycling factors. In this regard, we asked the question about how similar are those translation factors to each other from a wide variety of bacteria? Hence, we did a thorough in silico study of the translation factors from 495 bacterial sp., and 4262 amino acid sequences by theoretically measuring their pI and MW values that are two determining factors for distinguishing individual proteins in 2D gel electrophoresis in experimental procedures. Then we analyzed the output from various angles. Results Our study revealed the fact that it’s not all same, or all random, but there are distinct orders and the pI values of translation factors are translation event specific. We found that the translation initiation factors are mainly basic, whereas, elongation and release factors that interact with the inter-subunit space of the intact 70S ribosome during translation are strictly acidic across bacterial sp. These acidic elongation factors and release factors contain higher frequencies of glutamic acids. However, among all the translation factors, the translation initiation factor 2 (IF2) and ribosome recycling factor (RRF) showed variable pI values that are linked to the order of phylogeny. Conclusions From the results of our study, we conclude that among all the bacterial translation factors, elongation and release factors are more conserved in terms of their pI values in comparison to initiation and recycling factors. Acidic properties of these factors are independent of habitat, nature, and phylogeny of the bacterial species. Furthermore, irrespective of the different shapes, sizes, and functions of the elongation and release factors, possession of the strictly acidic pI values of these translation factors all over the domain Bacteria indicates that the acidic nature of these factors is a necessary criterion, perhaps to interact into the partially enclosed rRNA rich inter-subunit space of the translating 70S ribosome.

Charting the sequence-activity landscape of peptide inhibitors of translation termination

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api’s activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.

New Fractional Release Factors, Ozone Depletion Potentials, and Lifetimes for Four Long-Lived CFCs: CFC-13, CFC-114, CFC-114a, and CFC-115

<p>Knowing the stratospheric lifetime of an Ozone Depleting Substance (ODS), and its potential depletion of ozone during that time, is vital to reliably monitor and control the use of ODSs. Here, we present improved policy-relevant parameters: Fractional Release Factors (FRFs), Ozone Depletion Potentials (ODPs), and stratospheric lifetimes, for four understudied long-lived CFCs: CFC-13 (CClF<sub>3</sub>), CFC-114 (CClF<sub>2</sub>CCCLF<sub>2</sub>), CFC-114a (CCl<sub>2</sub>FCF<sub>3</sub>), and CFC-115 (C<sub>2</sub>ClF<sub>5</sub>). Previously derived lifetime estimates for CFC-114 and CFC-115 have substantial uncertainties, while lifetime uncertainties for CFC-13 and CFC-114a are absent from the peer-reviewed literature (Carpenter & Danie <em>et al</em>, 2018).</p><p>This study used both observational and model data to investigate these compounds and this work derives, for the first time, observation-based lifetimes utilising measurements of air samples collected in the stratosphere. We also used a version of the NASA Goddard Space Flight Center (GSFC) 2-D atmospheric model driven by temperature and transport fields derived from MERRA/MERRA-2 reanalysis.</p><p>FRFs for these compounds, which had been lacking until now, were derived using stratospheric air samples collected from several research flights with the high-altitude aircraft M55-Geophysica, and the background trend from archived surface air samples from Cape Grim, Tasmania.</p><p> By using a previously-published correlation between lifetime and FRF for seven well-characterised compounds (CF<sub>4</sub>, C<sub>2</sub>F<sub>6</sub>, C<sub>3</sub>F<sub>8</sub>, CHF<sub>3</sub>, HFC-125, HFC-227ea and SF<sub>6</sub>), we were able to derive lifetimes for these four new species. Lifetime estimates for CFC-114a agreed within the uncertainties (agreement to one sigma) with the lifetime estimates compiled in Burkholder <em>et al</em>. (2018), while the one for CFC-114 agreed within 2 sigma (measurement-related uncertainties) with those cited in Burkholder <em>et al</em>. (2018). However, observation-based lifetimes for CFC-13 and CFC-115 only agreed with those in Burkholder <em>et al</em>. (2018) within 3 sigma. The lifetime uncertainties in this study were reduced compared to those in Carpenter & Danie <em>et al</em> (2018).</p><p>As our lifetime estimates for these latter two compounds are notably lower than previous estimates, this suggests that these two compounds may have had greater emissions than previously thought, in order to account for their abundance. It also implies that they will be removed from the atmosphere more quickly than previously thought.</p><p>New ODPs were derived for these compounds from their new lifetimes and FRFs. Since for two of these compounds (CFC-13 and CFC-114a), there is an absence of observation-derived ODPs in the peer-reviewed literature, this is new and relevant information. The ODPs for CFC-114 and CFC-115 are comparable with estimates from the most recent Scientific Assessment of Ozone Depletion (Burkholder <em>et al</em>., 2018). Providing new and updated lifetimes, FRFs and ODPs for these compounds will help improve future estimates of their tropospheric emissions and their resulting damage to the stratospheric ozone layer.</p><p>             </p><p><strong>References</strong></p><p>Burkholder <em>et al</em>. (2018). Appendix A, Table A-1 in <em>Scientific Assessment of Ozone Depletion: 2018, Global Ozone Research and Monitoring Project</em>, Report No. 58, World Meteorological Organization, Geneva, Switzerland,  http://ozone.unep.org/science/assessment/sap.</p><p>Carpenter, L.J., Danie, J.S.<em> et al </em>(2018). Scenarios and Information for Policymakers Chapter 6, Table 6-1 in <em>Scientific Assessment of Ozone Depletion: 2018, Global Ozone Research and Monitoring Project</em>, Report No. 58, World Meteorological Organization, Geneva, Switzerland.</p>