Evolutionary repair reveals an unexpected role of the tRNA modification m1G37 in aminoacylation

The tRNA modification m1G37, which is introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria due to its role in suppressing translational frameshift errors at proline codons. However, because bacteria can tolerate high levels of mistranslation, it is unclear why loss of m1G37 is not tolerated. Here, we addressed this question by performing experimental evolution of trmD mutant strains of E. coli. Surprisingly, trmD mutant strains were viable even if the m1G37 modification was completely abolished, and showed rapid recovery of growth rate, mainly via tandem duplication or coding mutations in the proline-tRNA ligase gene proS. Growth assays and in vitro aminoacylation assays showed that G37-unmodified tRNAPro is aminoacylated less efficiently than m1G37-modified tRNAPro, and that growth of trmD mutant strains can be largely restored by single mutations in proS that restore aminoacylation of G37-unmodified tRNAPro. These results show that inefficient aminoacylation of tRNAPro is the main reason for growth defects observed in trmD mutant strains and that the ProRS enzyme may act as a gatekeeper of translational accuracy, preventing the use of error-prone unmodified tRNAPro in protein translation. Our work shows the utility of experimental evolution for uncovering the hidden functions of essential genes and has implications for the development of antibiotics targeting TrmD.

The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3' end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-EM analysis of late human pre-40S particles purified using a catalytically-inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3' end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

Disruption of evolutionarily correlated tRNA elements impairs accurate decoding

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem−loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-typetRNAGGCAlaortRNAGGCAlacontaining a reversed 32–38 pair on cognate and near-cognate codons. Structures of wild-typetRNAGGCAlabound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon−anticodon interaction is cognate or near cognate. Further, the 32–38 pair is destabilized in the context of a near-cognate codon−anticodon pair. Reversal of the pairing intRNAGGCAlaablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32–38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.

Molecular basis for t6A modification in human mitochondria

N 6-Threonylcarbamoyladenosine (t6A) is a universal tRNA modification essential for translational accuracy and fidelity. In human mitochondria, YrdC synthesises an l-threonylcarbamoyl adenylate (TC-AMP) intermediate, and OSGEPL1 transfers the TC-moiety to five tRNAs, including human mitochondrial tRNAThr (hmtRNAThr). Mutation of hmtRNAs, YrdC and OSGEPL1, affecting efficient t6A modification, has been implicated in various human diseases. However, little is known about the tRNA recognition mechanism in t6A formation in human mitochondria. Herein, we showed that OSGEPL1 is a monomer and is unique in utilising C34 as an anti-determinant by studying the contributions of individual bases in the anticodon loop of hmtRNAThr to t6A modification. OSGEPL1 activity was greatly enhanced by introducing G38A in hmtRNAIle or the A28:U42 base pair in a chimeric tRNA containing the anticodon stem of hmtRNASer(AGY), suggesting that sequences of specific hmtRNAs are fine-tuned for different modification levels. Moreover, using purified OSGEPL1, we identified multiple acetylation sites, and OSGEPL1 activity was readily affected by acetylation via multiple mechanisms in vitro and in vivo. Collectively, we systematically elucidated the nucleotide requirement in the anticodon loop of hmtRNAs, and revealed mechanisms involving tRNA sequence optimisation and post-translational protein modification that determine t6A modification levels.

Adaptive Partitioning of the tRNA Interaction Interface by Aminoacyl-tRNA-Synthetases

We introduce rugged fitness landscapes called match landscapes for the coevolution of feature-based assortative interactions between P ≥ 2 cognate pairs of tRNAs and aminoacyl-tRNA synthetases (aaRSs) in aaRS-tRNA interaction networks. Our genotype-phenotype-fitness maps assume additive feature-matching energies, a macroscopic theory of aminoacylation kinetics including proofreading, and selection for translational accuracy in multiple, perfectly encoded site-types. We compute the stationary genotype distributions of finite panmictic, asexual populations of haploid aaRs-tRNA interaction networks evolving under mutation, genetic drift, and selection for cognate matching and non-cognate mismatching of aaRS-tRNA pairs. We compared expected genotype frequencies under different matching rules and fitness functions, both with and without linked site-specific modifiers of interaction. Under selection for translational accuracy alone, our model predicts no selection on modifiers to eliminate non-cognate interactions, so long as they are compensated by tighter cognate interactions. Only under combined selection for both translational accuracy and rate do modifiers adaptively eliminate cross-matching in non-cognate aaRS/tRNA pairs. We theorize that the encoding of macromolecular interaction networks is a genetic language that symbolically maps identifying structural and dynamic features of genes and gene-products to functions within cells. Our theory helps explain 1) the remarkable divergence in how aaRSs bind tRNAs, 2) why interaction-informative features are phylogenetically informative, 3) why the Statistical Tree of Life became more tree-like after the Darwinian Transition, and 4) an approach towards computing the probability of the random origin of an interaction network.

Codon-Dependent Translational Accuracy Controls Protein Quality in Escherichia coli but not in Saccharomyces cerevisiae

In order to generate a functional proteome, gene expression pathways must assemble proteins accurately according to the rules of the genetic code. General gene expression accuracy is known to be high, but errors nevertheless occur with measurable frequencies. Here we develop a mass-spectrometry (MS) based assay for the detection of a particular type of gene expression error, amino acid misincorporation. This assay allows assessing a much broader range of misincorporation events compared to current, very sensitive but also very specific enzyme reporter assays. Our assay uncovers a remarkably rich pool of error products for a model protein expressed in E. coli, which depend quantitatively on codon usage in the expression construct. This codon usage dependence can be explained in part as a function of the composition of the tRNA pool in this organism. We further show that codon-dependent differences in error levels correlate with measurable changes in specific protein activity. In contrast to E. coli, error levels are lower, and appear not to be codon usage dependent, when the same model protein is expressed in S. cerevisiae.

An evaluation of the BrainLAB 6D ExacTrac/Novalis Tx System for image-guided intracranial radiotherapy

PurposeStereotactic-fractionated radiotherapy and radiosurgery (RS) for benign and malignant intracranial lesions relies on a very high degree of accuracy in dose alignment due to the ablative dose delivered, and therefore requires a high-precision image guidance modality. The aim of this review is to investigate the localisation and verification accuracy performance of ExacTrac (ET) and Novalis Tx System.Materials and methodsA systematic review of the database Science Direct was carried out using search terms ‘stereotactic radiotherapy (SRT)’ and ‘ET’. All articles before 2000 were excluded. Only articles that involved intracranial lesions, with the exception of one article, were included in the final review.ResultsResults from gold standard Hidden Target Tests and patient data show that patient position can be reproduced within 1·0 mm with the use of ET imaging. In addition, the 6 degrees of freedom algorithm function of ET allows for better translational accuracy as well optimal positioning when rotations are corrected for. Studies showed excellent correlation (p<0·01) between bony ET images and cone beam computed tomography (CBCT) soft tissue registration, evidencing the safe reliance of bony anatomy for image guidance via ET. Shifts were found to be comparable between CBCT and ET.ConclusionThere is the need for regular calibration to prevent systematic errors and potential geographic miss. However, due to ET’s additional benefits, including reduced concomitant dose and faster imaging time, ET is the superior image guidance modality for RS/SRT in the treatment of intracranial lesions.