Biology subjects

Atkinson, G. C.

Publications and source records attributed to Atkinson, G. C..

25 records · Page 2Linked to original sources

bioRxiv2020

Structural basis of resistance to lincosamide, streptogramin A, and pleuromutilin antibiotics by ABCF ATPases in Gram-positive pathogens

Target protection proteins bind to antibiotic targets and confer resistance to the host organism. One class of such proteins, termed antibiotic resistance (ARE) ATP binding cassette (ABC) proteins of the F-subtype (ARE ABCFs), are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition by antibiotics that target the large ribosomal subunit. Using single-particle cryo-EM, we have solved the structure of ARE ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a comparative approach to understanding how these proteins mediate antibiotic resistance on the ribosome. We present evidence of mechanistically diverse allosteric relays converging on a few peptidyltransferase center (PTC) nucleotides, and propose a general model of antibiotic resistance mediated by these ARE ABCFs.

microbiology↗

bioRxiv2020

Structural basis for bacterial ribosome quality control

In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality-control (RQC) pathways. RQC begins with splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal tails. How such tailing can occur without the regular suite of translational components is, however, unclear. Using ex vivo single-particle cryo-EM, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with YabO, a protein homologous to, yet distinct from, Hsp15. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.

biochemistry↗

bioRxiv2019

C-terminal RRM/ACT domain is crucial for fine-tuning the activation of 'long' RelA-SpoT Homolog enzymes by ribosomal complexes

The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain short and multi-domain long RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA) / RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e. capable of both degrading and synthesising the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterised Escherichia coli RelA and Bacillus subtilis Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex - the ultimate inducer of (p)ppGpp production by RelA and Rel - and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome.

biochemistry↗

bioRxiv2019

A role for the Saccharomyces cerevisiae ABCF protein New1 during translation termination

Translation on the ribosome is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related ABCF ATPase, New1, is encoded by a non-essential gene with a cold sensitivity and ribosome assembly defect knock-out phenotype. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide ribosome assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed a combination of yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination.

biochemistry↗

bioRxiv2019

Discovery of small alarmone synthetases and their inhibitors as toxin-antitoxin loci

Under stressful conditions, bacterial RelA-SpoT Homologue (RSH) enzymes synthesise the alarmone (p)ppGpp, a nucleotide messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and at high concentrations inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single domain Small Alarmone Synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA and SpoT. We have discovered that multiple SAS subfamilies can be encoded in broadly distributed conserved bicistronic operon architectures in bacteria and bacteriophages that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralisation by the protein products of six neighbouring antitoxin genes. The toxicity of Cellulomonas marina ToxSAS FaRel is mediated by alarmone accumulation combined with depletion of cellular ATP and GTP pools, and this is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system is a novel TA paradigm comprising multiple different antitoxins that exemplifies how ancient nucleotide-based signalling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.

microbiology↗

bioRxiv2018

Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae

In addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5 part of the open reading frames. We observed no E-site codon-or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.

biochemistry↗

bioRxiv2018

Predicting Functional Associations using Flanking Genes (FlaGs)

Analysis of conservation of gene neighbourhoods over different evolutionary levels is important for understanding operon and gene cluster evolution, and predicting functional associations. Our tool FlaGs (Flanking Genes) takes a list of NCBI protein accessions as in input, clusters neighbourhood-encoded proteins into homologous groups using sensitive sequence searching, and outputs a graphical visualization of the gene neighbourhood and its conservation, along with a phylogenetic tree annotated with flanking gene conservation. FlaGs has demonstrated utility for molecular evolutionary analysis, having uncovered a new toxin-antitoxin system in prokaryotes and bacteriophages. FlaGs can be downloaded from https://github.com/GCA-VH-lab/FlaGs or run at www.webflags.se.

bioinformatics↗