Primordial aminoacyl-tRNA synthetases preferred minihelices to full-length tRNA.

Aminoacyl-tRNA synthetases (AARS) and tRNAs translate the genetic code in all living cells. Little is known about how their molecular ancestors began to enforce the coding rules for the expression of their own genes. Schimmel et al. proposed in 1993 that AARS catalytic domains began by reading an 'operational' code in the acceptor stems of tRNA minihelices. We show here that the enzymology of an AARS urzyme•TΨC-minihelix cognate pair is a rich in vitro realization of that idea. The TΨC-minihelixLeu is a very poor substrate for full-length Leucyl-tRNA synthetase. It is a superior RNA substrate for the corresponding urzyme, LeuAC. LeuAC active-site mutations shift the choice of both amino acid and RNA substrates. AARS urzyme•minihelix cognate pairs are thus small, pliant models for the ancestral decoding hardware. They are thus an ideal platform for detailed experimental study of the operational RNA code.

HetMM: A Michaelis-Menten model for non-homogeneous enzyme mixtures.

The Michaelis-Menten model requires its reaction velocities to come from a preparation of homogeneous enzymes, with identical or near-identical catalytic activities. However, this condition is not always met. We introduce a kinetic model that relaxes this requirement, by assuming there are an unknown number of enzyme species drawn from a probability distribution whose standard deviation is estimated. Through simulation studies, we demonstrate the method accurately discriminates between homogeneous and heterogeneous data, even with moderate levels of experimental error. We applied this model to three homogeneous and three heterogeneous biological systems, showing that the standard and heterogeneous models outperform respectively. Lastly, we show that heterogeneity is not readily distinguished from negatively cooperative binding under the Hill model. These two distinct attributes-inequality in catalytic ability and interference between binding sites-yield similar Michaelis-Menten curves that are not readily resolved without further experimentation. Our user-friendly software package allows homogeneity testing and parameter estimation.

Genomic database furnishes a spontaneous example of a functional Class II glycyl-tRNA synthetase urzyme.

The chief barrier to studies of how genetic coding emerged is the lack of experimental models for ancestral aminoacyl-tRNA synthetases (AARS). We hypothesized that conserved core catalytic sites could represent such ancestors. That hypothesis enabled engineering functional "urzymes" from TrpRS, LeuRS, and HisRS. We describe here a fourth urzyme, GlyCA, detected in an open reading frame from the genomic record of the arctic fox, Vulpes lagopus. GlyCA is homologous to a bacterial heterotetrameric Class II GlyRS-B. Alphafold2 predicted that the N-terminal 81 amino acids would adopt a 3D structure nearly identical to the HisRS urzyme (HisCA1). We expressed and purified that N-terminal segment. Enzymatic characterization revealed a robust single-turnover burst size and a catalytic rate for ATP consumption well in excess of that previously published for HisCA1. Time-dependent aminoacylation of tRNAGly proceeds at a rate consistent with that observed for amino acid activation. In fact, GlyCA is actually 35 times more active in glycine activation by ATP than the full-length GlyRS-B α-subunit dimer. ATP-dependent activation of the 20 canonical amino acids favors Class II amino acids that complement those favored by HisCA and LeuAC. These properties reinforce the notion that urzymes represent the requisite ancestral catalytic activities to implement a reduced genetic coding alphabet.

Enzymic recognition of amino acids drove the evolution of primordial genetic codes.

How genetic information gained its exquisite control over chemical processes needed to build living cells remains an enigma. Today, the aminoacyl-tRNA synthetases (AARS) execute the genetic codes in all living systems. But how did the AARS that emerged over three billion years ago as low-specificity, protozymic forms then spawn the full range of highly-specific enzymes that distinguish between 22 diverse amino acids? A phylogenetic reconstruction of extant AARS genes, enhanced by analysing modular acquisitions, reveals six AARS with distinct bacterial, archaeal, eukaryotic, or organellar clades, resulting in a total of 36 families of AARS catalytic domains. Small structural modules that differentiate one AARS family from another played pivotal roles in discriminating between amino acid side chains, thereby expanding the genetic code and refining its precision. The resulting model shows a tendency for less elaborate enzymes, with simpler catalytic domains, to activate amino acids that were not synthesised until later in the evolution of the code. The most probable evolutionary route for an emergent amino acid type to establish a place in the code was by recruiting older, less specific AARS, rather than adapting contemporary lineages. This process, retrofunctionalisation, differs from previously described mechanisms through which amino acids would enter the code.

Practical overview of the ICRP Publication 145 phantoms for use with MCNP.

This paper provides a practical overview of the mesh-type reference phantoms (MRCPs) published by the ICRP by providing supplementary guidance on the proper implementation of using the MRCPs in computational codes, such as Monte Carlo N-Particle. Various validation scenarios for photon and neutron exposures were also examined where published values were reproduced using the MRCPs for both external and internal exposures. Generally, the MRCPs performed well in the validation scenarios with the values reproduced being within 10% of the published values.

Domain acquisition by class I aminoacyl-tRNA synthetase urzymes coordinated the catalytic functions of HVGH and KMSKS motifs.

Leucyl-tRNA synthetase (LeuRS) is a Class I aminoacyl-tRNA synthetase (aaRS) that synthesizes leucyl-tRNAleu for codon-directed protein synthesis. Two signature sequences, HxGH and KMSKS help stabilize transition-states for amino acid activation and tRNA aminoacylation by all Class I aaRS. Separate alanine mutants of each signature, together with the double mutant, behave in opposite ways in Pyrococcus horikoshii LeuRS and the 129-residue urzyme ancestral model generated from it (LeuAC). Free energy coupling terms, Δ(ΔG‡), for both reactions are large and favourable for LeuRS, but unfavourable for LeuAC. Single turnover assays with 32Pα-ATP show correspondingly different internal products. These results implicate domain motion in catalysis by full-length LeuRS. The distributed thermodynamic cycle of mutational changes authenticates LeuAC urzyme catalysis far more convincingly than do single point mutations. Most importantly, the evolutionary gain of function induced by acquiring the anticodon-binding (ABD) and multiple insertion modules in the catalytic domain appears to be to coordinate the catalytic function of the HxGH and KMSKS signature sequences. The implication that backbone elements of secondary structures achieve a major portion of the overall transition-state stabilization by LeuAC is also consistent with coevolution of the genetic code and metabolic pathways necessary to produce histidine and lysine sidechains.

Genomic epidemiology of SARS-CoV-2 variants during the first two years of the pandemic in Colombia.

BACKGROUND: The emergence of highly transmissible SARS-CoV-2 variants has led to surges in cases and the need for global genomic surveillance. While some variants rapidly spread worldwide, other variants only persist nationally. There is a need for more fine-scale analysis to understand transmission dynamics at a country scale. For instance, the Mu variant of interest, also known as lineage B.1.621, was first detected in Colombia and was responsible for a large local wave but only a few sporadic cases elsewhere. METHODS: To better understand the epidemiology of SARS-Cov-2 variants in Colombia, we used 14,049 complete SARS-CoV-2 genomes from the 32 states of Colombia. We performed Bayesian phylodynamic analyses to estimate the time of variants' introduction, their respective effective reproductive number, and effective population size, and the impact of disease control measures. RESULTS: Here, we detect a total of 188 SARS-CoV-2 Pango lineages circulating in Colombia since the pandemic's start. We show that the effective reproduction number oscillated drastically throughout the first two years of the pandemic, with Mu showing the highest transmissibility (Re and growth rate estimation). CONCLUSIONS: Our results reinforce that genomic surveillance programs are essential for countries to make evidence-driven interventions toward the emergence and circulation of novel SARS-CoV-2 variants.

Colombia reported its first COVID-19 case on 6th March 2020. By April 2022, the country had reported over 6 million infections and over 135,000 deaths. Here, we aim to understand how SARS-CoV-2, the virus that causes COVID-19, spread through Colombia over this time and how the predominant version of the virus (variant) changed over time. We found that there were multiple introductions of different variants from other countries into Colombia during the first two years of the pandemic. The Gamma variant was dominant earlier in 2021 but was replaced by the Delta variant. The Mu variant had the highest potential to be transmitted. Our findings provide valuable insights into the pandemic in Colombia and highlight the importance of continued surveillance of the virus to guide the public health response.

Tracing the international arrivals of SARS-CoV-2 Omicron variants after Aotearoa New Zealand reopened its border.

In the second quarter of 2022, there was a global surge of emergent SARS-CoV-2 lineages that had a distinct growth advantage over then-dominant Omicron BA.1 and BA.2 lineages. By generating 10,403 Omicron genomes, we show that Aotearoa New Zealand observed an influx of these immune-evasive variants (BA.2.12.1, BA.4, and BA.5) through the border. This is explained by the return to significant levels of international travel following the border's reopening in March 2022. We estimate one Omicron transmission event from the border to the community for every ~5,000 passenger arrivals at the current levels of travel and restriction. Although most of these introductions did not instigate any detected onward transmission, a small minority triggered large outbreaks. Genomic surveillance at the border provides a lens on the rate at which new variants might gain a foothold and trigger new waves of infection.

Quantitatively defining species boundaries with more efficiency and more biological realism.

We introduce a widely applicable species delimitation method based on the multispecies coalescent model that is more efficient and more biologically realistic than existing methods. We extend a threshold-based method to allow the ancestral speciation rate to vary through time as a smooth piecewise function. Furthermore, we introduce the cutting-edge proposal kernels of StarBeast3 to this model, thus enabling rapid species delimitation on large molecular datasets and allowing the use of relaxed molecular clock models. We validate these methods with genomic sequence data and SNP data, and show they are more efficient than existing methods at achieving parameter convergence during Bayesian MCMC. Lastly, we apply these methods to two datasets (Hemidactylus and Galagidae) and find inconsistencies with the published literature. Our methods are powerful for rapid quantitative testing of species boundaries in large multilocus datasets and are implemented as an open source BEAST 2 package called SPEEDEMON.

Genomic epidemiology of Delta SARS-CoV-2 during transition from elimination to suppression in Aotearoa New Zealand.

New Zealand's COVID-19 elimination strategy heavily relied on the use of genomics to inform contact tracing, linking cases to the border and to clusters during community outbreaks. In August 2021, New Zealand entered its second nationwide lockdown after the detection of a single community case with no immediately apparent epidemiological link to the border. This incursion resulted in the largest outbreak seen in New Zealand caused by the Delta Variant of Concern. Here we generated 3806 high quality SARS-CoV-2 genomes from cases reported in New Zealand between 17 August and 1 December 2021, representing 43% of reported cases. We detected wide geographical spread coupled with undetected community transmission, characterised by the apparent extinction and reappearance of genomically linked clusters. We also identified the emergence, and near replacement, of genomes possessing a 10-nucleotide frameshift deletion that caused the likely truncation of accessory protein ORF7a. By early October, New Zealand moved from an elimination strategy to a suppression strategy and the role of genomics changed markedly from being used to track and trace, towards population-level surveillance.

StarBeast3: Adaptive Parallelized Bayesian Inference under the Multispecies Coalescent.

As genomic sequence data become increasingly available, inferring the phylogeny of the species as that of concatenated genomic data can be enticing. However, this approach makes for a biased estimator of branch lengths and substitution rates and an inconsistent estimator of tree topology. Bayesian multispecies coalescent (MSC) methods address these issues. This is achieved by constraining a set of gene trees within a species tree and jointly inferring both under a Bayesian framework. However, this approach comes at the cost of increased computational demand. Here, we introduce StarBeast3-a software package for efficient Bayesian inference under the MSC model via Markov chain Monte Carlo. We gain efficiency by introducing cutting-edge proposal kernels and adaptive operators, and StarBeast3 is particularly efficient when a relaxed clock model is applied. Furthermore, gene-tree inference is parallelized, allowing the software to scale with the size of the problem. We validated our software and benchmarked its performance using three real and two synthetic data sets. Our results indicate that StarBeast3 is up to one-and-a-half orders of magnitude faster than StarBeast2, and therefore more than two orders faster than *BEAST, depending on the data set and on the parameter, and can achieve convergence on large data sets with hundreds of genes. StarBeast3 is open-source and is easy to set up with a friendly graphical user interface. [Adaptive; Bayesian inference; BEAST 2; effective population sizes; high performance; multispecies coalescent; parallelization; phylogenetics.].

Phylodynamics reveals the role of human travel and contact tracing in controlling the first wave of COVID-19 in four island nations.

New Zealand, Australia, Iceland, and Taiwan all saw success in controlling their first waves of Coronavirus Disease 2019 (COVID-19). As islands, they make excellent case studies for exploring the effects of international travel and human movement on the spread of COVID-19. We employed a range of robust phylodynamic methods and genome subsampling strategies to infer the epidemiological history of Severe acute respiratory syndrome coronavirus 2 in these four countries. We compared these results to transmission clusters identified by the New Zealand Ministry of Health by contact tracing strategies. We estimated the effective reproduction number of COVID-19 as 1-1.4 during early stages of the pandemic and show that it declined below 1 as human movement was restricted. We also showed that this disease was introduced many times into each country and that introductions slowed down markedly following the reduction of international travel in mid-March 2020. Finally, we confirmed that New Zealand transmission clusters identified via standard health surveillance strategies largely agree with those defined by genomic data. We have demonstrated how the use of genomic data and computational biology methods can assist health officials in characterising the epidemiology of viral epidemics and for contact tracing.

Real-Time Genomics for Tracking Severe Acute Respiratory Syndrome Coronavirus 2 Border Incursions after Virus Elimination, New Zealand.

Since severe acute respiratory syndrome coronavirus 2 was first eliminated in New Zealand in May 2020, a total of 13 known coronavirus disease (COVID-19) community outbreaks have occurred, 2 of which led health officials to issue stay-at-home orders. These outbreaks originated at the border via isolating returnees, airline workers, and cargo vessels. Because a public health system was informed by real-time viral genomic sequencing and complete genomes typically were available within 12 hours of community-based positive COVID-19 test results, every outbreak was well-contained. A total of 225 community cases resulted in 3 deaths. Real-time genomics were essential for establishing links between cases when epidemiologic data could not do so and for identifying when concurrent outbreaks had different origins.

Evolutionary history of cotranscriptional editing in the paramyxoviral phosphoprotein gene.

The phosphoprotein gene of the paramyxoviruses encodes multiple protein products. The P, V, and W proteins are generated by transcriptional slippage. This process results in the insertion of non-templated guanosine nucleosides into the mRNA at a conserved edit site. The P protein is an essential component of the viral RNA polymerase and is encoded by a faithful copy of the gene in the majority of paramyxoviruses. However, in some cases, the non-essential V protein is encoded by default and guanosines must be inserted into the mRNA in order to encode P. The number of guanosines inserted into the P gene can be described by a probability distribution, which varies between viruses. In this article, we review the nature of these distributions, which can be inferred from mRNA sequencing data, and reconstruct the evolutionary history of cotranscriptional editing in the paramyxovirus family. Our model suggests that, throughout known history of the family, the system has switched from a P default to a V default mode four times; complete loss of the editing system has occurred twice, the canonical zinc finger domain of the V protein has been deleted or heavily mutated a further two times, and the W protein has independently evolved a novel function three times. Finally, we review the physical mechanisms of cotranscriptional editing via slippage of the viral RNA polymerase.

Use of Genomics to Track Coronavirus Disease Outbreaks, New Zealand.

Real-time genomic sequencing has played a major role in tracking the global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), contributing greatly to disease mitigation strategies. In August 2020, after having eliminated the virus, New Zealand experienced a second outbreak. During that outbreak, New Zealand used genomic sequencing in a primary role, leading to a second elimination of the virus. We generated genomes from 78% of the laboratory-confirmed samples of SARS-CoV-2 from the second outbreak and compared them with the available global genomic data. Genomic sequencing rapidly identified that virus causing the second outbreak in New Zealand belonged to a single cluster, thus resulting from a single introduction. However, successful identification of the origin of this outbreak was impeded by substantial biases and gaps in global sequencing data. Access to a broader and more heterogenous sample of global genomic data would strengthen efforts to locate the source of any new outbreaks.

Adaptive dating and fast proposals: Revisiting the phylogenetic relaxed clock model.

Relaxed clock models enable estimation of molecular substitution rates across lineages and are widely used in phylogenetics for dating evolutionary divergence times. Under the (uncorrelated) relaxed clock model, tree branches are associated with molecular substitution rates which are independently and identically distributed. In this article we delved into the internal complexities of the relaxed clock model in order to develop efficient MCMC operators for Bayesian phylogenetic inference. We compared three substitution rate parameterisations, introduced an adaptive operator which learns the weights of other operators during MCMC, and we explored how relaxed clock model estimation can benefit from two cutting-edge proposal kernels: the AVMVN and Bactrian kernels. This work has produced an operator scheme that is up to 65 times more efficient at exploring continuous relaxed clock parameters compared with previous setups, depending on the dataset. Finally, we explored variants of the standard narrow exchange operator which are specifically designed for the relaxed clock model. In the most extreme case, this new operator traversed tree space 40% more efficiently than narrow exchange. The methodologies introduced are adaptive and highly effective on short as well as long alignments. The results are available via the open source optimised relaxed clock (ORC) package for BEAST 2 under a GNU licence (https://github.com/jordandouglas/ORC).

UglyTrees: a browser-based multispecies coalescent tree visualizer.

SUMMARY: Visualization is a vital task in phylogenetics and yet there is a deficit in programs which visualize the multispecies coalescent (MSC) model. UglyTrees (UT) is an easy-to-use program for visualizing multiple gene trees embedded within a single species trees. The mapping between gene and species nodes is automatically detected allowing for ready access to the program. UT can scrape the contents of a website for MSC analyses, enabling the sharing of interactive MSC figures through optional parameters in the URL. If a posterior distribution is uploaded, the transitions between MSC states are animated allowing the visual tracking of trees throughout the sequence. AVAILABILITY AND IMPLEMENTATION: UT runs in all major web browsers including mobile devices, and is hosted at www.uglytrees.nz. The MIT-licensed code is available at https://github.com/UglyTrees/uglytrees.github.io.

Genomic epidemiology reveals transmission patterns and dynamics of SARS-CoV-2 in Aotearoa New Zealand.

New Zealand, a geographically remote Pacific island with easily sealable borders, implemented a nationwide 'lockdown' of all non-essential services to curb the spread of COVID-19. Here, we generate 649 SARS-CoV-2 genome sequences from infected patients in New Zealand with samples collected during the 'first wave', representing 56% of all confirmed cases in this time period. Despite its remoteness, the viruses imported into New Zealand represented nearly all of the genomic diversity sequenced from the global virus population. These data helped to quantify the effectiveness of public health interventions. For example, the effective reproductive number, Re of New Zealand's largest cluster decreased from 7 to 0.2 within the first week of lockdown. Similarly, only 19% of virus introductions into New Zealand resulted in ongoing transmission of more than one additional case. Overall, these results demonstrate the utility of genomic pathogen surveillance to inform public health and disease mitigation.

Bayesian inference and comparison of stochastic transcription elongation models.

Transcription elongation can be modelled as a three step process, involving polymerase translocation, NTP binding, and nucleotide incorporation into the nascent mRNA. This cycle of events can be simulated at the single-molecule level as a continuous-time Markov process using parameters derived from single-molecule experiments. Previously developed models differ in the way they are parameterised, and in their incorporation of partial equilibrium approximations. We have formulated a hierarchical network comprised of 12 sequence-dependent transcription elongation models. The simplest model has two parameters and assumes that both translocation and NTP binding can be modelled as equilibrium processes. The most complex model has six parameters makes no partial equilibrium assumptions. We systematically compared the ability of these models to explain published force-velocity data, using approximate Bayesian computation. This analysis was performed using data for the RNA polymerase complexes of E. coli, S. cerevisiae and Bacteriophage T7. Our analysis indicates that the polymerases differ significantly in their translocation rates, with the rates in T7 pol being fast compared to E. coli RNAP and S. cerevisiae pol II. Different models are applicable in different cases. We also show that all three RNA polymerases have an energetic preference for the posttranslocated state over the pretranslocated state. A Bayesian inference and model selection framework, like the one presented in this publication, should be routinely applicable to the interrogation of single-molecule datasets.

Characterisation of assembly and ubiquitylation by the RBCC motif of Trim5α.

The post-entry restriction factor Trim5α blocks infection of retroviral pathogens shortly after the virus gains entry to the cell, preventing reverse transcription and integration into the host genome. Central to the mechanism of restriction is recognition of the lattice of capsid protein that forms the inner-shell of the retrovirus. To recognise this lattice, Trim5α has been shown to assemble into a large hexagonal array, complementary to the capsid lattice. Structures of the Trim5α coiled-coil region reveal an elongated anti-parallel dimer consistent with the edges of this array placing the Bbox domain at each end of the coiled-coil to facilitate assembly. To investigate the nature of this assembly we have designed and characterised a monomeric version of the TRIM RBCC motif with a truncated coiled-coil. Biophysical characterisation by SEC-MALLS, AUC, and SAXS demonstrate that this construct forms compact folded domain that assembles into a trimer that would support the formation of a hexagonal lattice. Furthermore, the RING domain and elements of the coiled-coil region are shown to contribute to assembly. Ubiquitylation assays demonstrate that this assembly increases ubiquitylation activity providing a link from recognition of the capsid lattice and assembly to the activation of innate immune signalling and restriction.