Through the looking glass: milestones on the road towards mirroring life.

Naturally occurring DNA, RNA, and proteins predominantly exist in only one enantiomeric form (homochirality). Advances in biotechnology and chemical synthesis allow the production of the respective alternate enantiomeric form, enabling access to mirror-image versions of these natural biopolymers. Exploiting the unique properties of such mirror molecules has already led to many applications, such as biostable and nonimmunogenic therapeutics or sensors. However, a 'roadblock' for unlocking the mirror world is the lack of biological systems capable of synthesizing critical building blocks including mirror oligonucleotides and oligopeptides to reducing cost and improve purity. Here, we provide an overview of the current progress, applications, and challenges of the molecular mirror world by identifying milestones towards mirroring life.

In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways.

Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3' fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators.

The prokaryotic activity of the IGR IRESs is mediated by ribosomal protein S1.

Internal ribosome entry sites (IRESs) are RNA elements capable of initiating translation on an internal portion of a messenger RNA. The intergenic region (IGR) IRES of the Dicistroviridae virus family folds into a triple pseudoknot tertiary structure, allowing it to recruit the ribosome and initiate translation in a structure dependent manner. This IRES has also been reported to drive translation in Escherichia coli and to date is the only described translation initiation signal that functions across domains of life. Here we show that unlike in the eukaryotic context the tertiary structure of the IGR IRES is not required for prokaryotic ribosome recruitment. In E. coli IGR IRES translation efficiency is dependent on ribosomal protein S1 in conjunction with an AG-rich Shine-Dalgarno-like element, supporting a model where the translational activity of the IGR IRESs is due to S1-mediated canonical prokaryotic translation.

Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection.

The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

Parenting researchers-an invisible divide.

The corona pandemic is an opportunity to rethink and revamp the academic career and reward system that consistently disadvantages parenting scientists and women.

Proceedings of the Dual Use Research of Concern Panel Discussion: challenges and perspectives.

To address real and perceived emerging risks originating from the ever-accelerating breakthroughs in life science research, the Dual Use Research of Concern (DURC) Panel Discussion, organized by Synbio Canada and the Alberta RNA Research and Training Institute (ARRTI), took place on June 23rd, 2021. It brought together six stakeholders from different levels of academic research, administration, governance, and science publishing to explore the current and future challenges in addressing DURC. Technological advancements within the life sciences, especially within the field of omics technology, make it difficult to apply a simple checklist for dual-use assessment and require continuous and integrated effort. Bottom-up approaches from within the scientific community are suggested by all stakeholders to enable efficient governance and address the true risks resulting from DURC, not just the alleged risks. To address such alleged risks, open and broadscale communication of DURC and its oversight policies may be required. At the same time, any form of open communication also contains the risk of information hazards, defined as potentially creating public fear or informing malicious actors. Here, an overview of the DURC panel and its outcomes is provided.

Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform.

Fluorescently labeled, solute-binding proteins that change their fluorescent output in response to ligand binding are frequently used as biosensors for a wide range of applications. We have previously developed a "Computational Identification of Non-disruptive Conjugation sites" (CINC) approach, an in silico pipeline utilizing molecular dynamics simulations for the rapid design and construction of novel protein-fluorophore conjugate-type biosensors. Here, we report an improved in silico scoring algorithm for use in CINC and its use in the construction of an oligogalacturonide-detecting biosensor set. Using both 4,5-unsaturated and saturated oligogalacturonides, we demonstrate that signal transmission from the ligand-binding pocket of the starting protein scaffold to the CINC-selected reporter positions is effective for multiple different ligands. The utility of an oligogalacturonide-detecting biosensor is shown in Carbohydrate Active Enzyme (CAZyme) activity assays, where the biosensor is used to follow product release upon polygalacturonic acid (PGA) depolymerization in real time. The oligogalacturonide-detecting biosensor set represents a novel enabling tool integral to our rapidly expanding platform for biosensor-based carbohydrate detection, and moving forward, the CINC pipeline will continue to enable the rational design of biomolecular tools to detect additional chemically distinct oligosaccharides and other solutes.

Emerging regulatory challenges of next-generation synthetic biology.

Cell-free synthetic biology is a rapidly developing biotechnology with the potential to solve the world's biggest problems; however, this promise also has implications for global biosecurity and biosafety. Given the current situation of COVID-19 and its economic impact, capitalizing on the potential of cell-free synthetic biology from an economic, biosafety, and biosecurity perspective contributes to our preparedness for the next pandemic, and urges the development of appropriate policies and regulations, together with the necessary mitigation technologies. Proactive involvement from scientists is necessary to avoid misconceptions and assist in the policymaking process.

Proceedings of the 14th annual RiboWest conference: perspectives and outcome.

The RiboWest Conference brings together RNA researchers in Canada with the 2-fold goals of fostering internationally competitive RNA research and of training the next generation of scientists. The 14th Annual RiboWest conference (RiboWest 2018) was held at the University of Lethbridge (Lethbridge, Alberta) from June 10th to 13th, 2018. This meeting was focused on all major aspects of RNA research, ranging from understanding the cellular role of RNA, studying RNA interactions and structures, and employing them as a therapeutic tool. The invited keynote speakers (5) provided insights into the wide-range of RNA-based research. One of the unique features of this conference was that the majority of the oral presentations were given by the trainees (undergraduate/graduate students and postdoctoral researchers). Hosted by the Alberta RNA Research and Training Institute (ARRTI) at the University of Lethbridge as the leading center of RNA research in Western Canada, the RiboWest 2018 was well attended by researchers from across the country (>110 attendees in total). This conference proceedings editorial presents the overview of the conference, and briefly introduces articles published in this special issue of Biochemistry and Cell Biology.

Non-coding RNAs: what are we missing?

Over the past two decades, the importance of small non-coding RNAs (sncRNAs) as regulatory molecules has become apparent in all three domains of life (archaea, bacteria, eukaryotes). In fact, sncRNAs play an important role in the control of gene expression at both the transcriptional and the post-transcriptional level, with crucial roles in fine-tuning cell responses during internal and external stress. Multiple pathways for sncRNA biogenesis and diverse mechanisms of regulation have been reported, and although biogenesis and mechanisms of sncRNAs in prokaryotes and eukaryotes are different, remarkable similarities exist. Here, we briefly review and compare the major sncRNA classes that act post-transcriptionally, and focus on recent discoveries regarding the ribosome as a target of regulation and the conservation of these mechanisms between prokaryotes and eukaryotes.

Cellular roles of the human Obg-like ATPase 1 (hOLA1) and its YchF homologs.

P-loop NTPases comprise one of the major superfamilies of nucleotide binding proteins, which mediate a variety of cellular processes, such as mRNA translation, signal transduction, cell motility, and growth regulation. In this review, we discuss the structure and function of two members of the ancient Obg-related family of P-loop GTPases: human Obg-like ATPase 1 (hOLA1), and its bacterial/plant homolog, YchF. After a brief discussion of nucleotide binding proteins in general and the classification of the Obg-related family in particular, we discuss the sequence and structural features of YchF and hOLA1. We then explore the various functional roles of hOLA1 in mammalian cells during stress response and cancer progression, and of YchF in bacterial cells. Finally, we directly compare and contrast the structure and function of hOLA1 with YchF before summarizing the future perspectives of hOLA1 research. This review is timely, given the variety of recent studies aimed at understanding the roles of hOLA1 and YchF in such critical processes as cellular-stress response, oncogenesis, and protein synthesis.

The C-terminal helix of ribosomal P stalk recognizes a hydrophobic groove of elongation factor 2 in a novel fashion.

Archaea and eukaryotes have ribosomal P stalks composed of anchor protein P0 and aP1 homodimers (archaea) or P1•P2 heterodimers (eukaryotes). These P stalks recruit translational GTPases to the GTPase-associated center in ribosomes to provide energy during translation. The C-terminus of the P stalk is known to selectively recognize GTPases. Here we investigated the interaction between the P stalk and elongation factor 2 by determining the structures of Pyrococcus horikoshii EF-2 (PhoEF-2) in the Apo-form, GDP-form, GMPPCP-form (GTP-form), and GMPPCP-form bound with 11 C-terminal residues of P1 (P1C11). Helical structured P1C11 binds to a hydrophobic groove between domain G and subdomain G' of PhoEF-2, where is completely different from that of aEF-1α in terms of both position and sequence, implying that such interaction characteristic may be requested by how GTPases perform their functions on the ribosome. Combining PhoEF-2 P1-binding assays with a structural comparison of current PhoEF-2 structures and molecular dynamics model of a P1C11-bound GDP form, the conformational changes of the P1C11-binding groove in each form suggest that in response to the translation process, the groove has three states: closed, open, and release for recruiting and releasing GTPases.

Human DDX17 Unwinds Rift Valley Fever Virus Non-Coding RNAs.

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5' non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17135-555) and RVFV non-coding RNAs, IGR and 5' NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17135-555, IGR, and 5' NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5' NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17135-555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17135-555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17135-555 interacting with and unwinding RVFV non-coding regions.

Elongation Factor Tu's Nucleotide Binding Is Governed by a Thermodynamic Landscape Unique among Bacterial Translation Factors.

Molecular switches such as GTPases are powerful devices turning "on" or "off" biomolecular processes at the core of critical biological pathways. To develop molecular switches de novo, an intimate understanding of how they function is required. Here we investigate the thermodynamic parameters that define the nucleotide-dependent switch mechanism of elongation factor (EF) Tu as a prototypical molecular switch. EF-Tu alternates between GTP- and GDP-bound conformations during its functional cycle, representing the "on" and "off" states, respectively. We report for the first time that the activation barriers for nucleotide association are the same for both nucleotides, suggesting a guanosine nucleoside or ribose-first mechanism for nucleotide association. Additionally, molecular dynamics (MD) simulations indicate that enthalpic stabilization of GDP binding compared to GTP binding originates in the backbone hydrogen bonding network of EF-Tu. In contrast, binding of GTP to EF-Tu is entropically driven by the liberation of bound water during the GDP- to GTP-bound transition. GDP binding to the apo conformation of EF-Tu is both enthalpically and entropically favored, a feature unique among translational GTPases. This indicates that the apo conformation does not resemble the GDP-bound state. Finally, we show that antibiotics and single amino acid substitutions can be used to target specific structural elements in EF-Tu to redesign the thermodynamic landscape. These findings demonstrate how, through evolution, EF-Tu has fine-tuned the structural and dynamic features that define nucleotide binding, providing insight into how altering these properties could be exploited for protein engineering.

The Emergency Medical Service Microbiome.

Emergency medical services (EMS) personnel are an integral component of the health care framework and function to transport patients from various locations to and between care facilities. In addition to physical injury, EMS personnel are expected to be at high risk to acquire and transmit health care-associated infections (HAIs) in the workplace. However, currently, little is known about EMS biosafety risk factors and the epidemiological contribution of EMS to pathogen transmission within and outside the health care sector. Health care facility microbiomes contain diverse bacterial, fungal, and viral pathogens that cause over 1.7 million HAIs each year in the United States alone. While hospital microbiomes have been relatively well studied, there is scant information about EMS infrastructure and equipment microbiomes or the role(s) they play in HAI transmission between health care facilities. We review recent literature investigating the microbiome of ambulances and other EMS service facilities which consistently identify antibiotic-resistant pathogens causing HAIs, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus, and Klebsiella pneumoniae Our review provides evidence that EMS microbiomes are dynamic and important pathogen reservoirs, and it underscores the need for more widespread and in-depth microbiome studies to elucidate patterns of pathogen transmission. We discuss emerging DNA sequencing technologies and other methods that can be applied to characterize and mitigate EMS biosafety risks in the future. Understanding the complex interplay between EMS and hospital microbiomes will provide key insights into pathogen transmission mechanisms and identify strategies to minimize HAIs and community infection.

The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome.

Using a combination of biochemical, structural probing and rapid kinetics techniques we reveal for the first time that the universally conserved translational GTPase (trGTPase) HflX binds to the E-site of the 70S ribosome and that its GTPase activity is modulated by peptidyl transferase centre (PTC) and peptide exit tunnel (PET) binding antibiotics, suggesting a previously undescribed mode of action for these antibiotics. Our rapid kinetics studies reveal that HflX functions as a ribosome splitting factor that disassembles the 70S ribosomes into its subunits in a nucleotide dependent manner. Furthermore, our probing and hydrolysis studies show that the ribosome is able to activate trGTPases bound to its E-site. This is, to our knowledge, the first case in which the hydrolytic activity of a translational GTPase is not activated by the GTPase activating centre (GAC) in the ribosomal A-site. Furthermore, we provide evidence that the bound state of the PTC is able to regulate the GTPase activity of E-site bound HflX.

The C-terminal Helix of Pseudomonas aeruginosa Elongation Factor Ts Tunes EF-Tu Dynamics to Modulate Nucleotide Exchange.

Little is known about the conservation of critical kinetic parameters and the mechanistic strategies of elongation factor (EF) Ts-catalyzed nucleotide exchange in EF-Tu in bacteria and particularly in clinically relevant pathogens. EF-Tu from the clinically relevant pathogen Pseudomonas aeruginosa shares over 84% sequence identity with the corresponding elongation factor from Escherichia coli Interestingly, the functionally closely linked EF-Ts only shares 55% sequence identity. To identify any differences in the nucleotide binding properties, as well as in the EF-Ts-mediated nucleotide exchange reaction, we performed a comparative rapid kinetics and mutagenesis analysis of the nucleotide exchange mechanism for both the E. coli and P. aeruginosa systems, identifying helix 13 of EF-Ts as a previously unnoticed regulatory element in the nucleotide exchange mechanism with species-specific elements. Our findings support the base side-first entry of the nucleotide into the binding pocket of the EF-Tu·EF-Ts binary complex, followed by displacement of helix 13 and rapid binding of the phosphate side of the nucleotide, ultimately leading to the release of EF-Ts.

Contribution of two conserved histidines to the dual activity of archaeal RNA guide-dependent and -independent pseudouridine synthase Cbf5.

In all organisms, several distinct stand-alone pseudouridine synthase (PUS) family enzymes are expressed to isomerize uridine into pseudouridine (Ψ) by specific recognition of RNAs. In addition, Ψs are generated in Archaea and Eukaryotes by PUS enzymes which are organized as ribonucleoprotein particles (RNP)--the box H/ACA s/snoRNPs. For this modification system, a unique TruB-like catalytic PUS subunit is associated with various RNA guides which specifically target and secure substrate RNAs by base-pairing. The archaeal Cbf5 PUS displays the special feature of exhibiting both RNA guide-dependent and -independent activities. Structures of substrate-bound TruB and H/ACA sRNP revealed the importance of histidines in positioning the target uridine in the active site. To analyze the respective role of H60 and H77, we have generated variants carrying alanine substitutions at these positions. The impact of the mutations was analyzed for unguided modifications U(55) in tRNA and U2603 in 23S rRNA, and for activity of the box H/ACA Pab91 sRNP enzyme. H77 (H43 in TruB), but not H60, appeared to be crucial for the RNA guide-independent activity. In contrast to earlier suggestions, H60 was found to be noncritical for the activity of the H/ACA sRNP, but contributes together with H77 to the full activity of H/ACA sRNPs. The data suggest that a similar catalytic process was conserved in the two divergent pseudouridylation systems.

Streamlined purification of fluorescently labeled Escherichia coli phosphate-binding protein (PhoS) suitable for rapid-kinetics applications.

Fluorescently labeled phosphate-binding proteins can be used as biomolecular tools to measure the release of inorganic phosphate (Pi) from enzymes in real time, enabling the detailed kinetic analysis of dephosphorylating enzymes using rapid-kinetics approaches. Previously reported methods to purify fluorescently labeled phosphate-binding proteins (PhoS) from Escherichia coli are laborious, and a simplified approach is needed. Here, we report the characterization of a cytosol-localized variant (A197C) of PhoS that allows a streamlined purification for subsequent covalent conjugation with a fluorescent dye. We show that export of PhoS into the periplasmic space is not required for the fluorescence-based detection of Pi binding. Furthermore, we report the addition of a C-terminal His-tag, simplifying the purification of PhoS from the cytosol via Ni2+-affinity chromatography, yielding a fully functional fusion protein (HC PhoS A197C). We demonstrate the utility of fluorescently labeled HC PhoS A197C for rapid-kinetics applications by measuring, using stopped-flow, the Pi release kinetics from LepA/EF4 following 70S ribosome-stimulated GTP hydrolysis. Altogether, the approach developed here allows for the high-yield and simplified in-house production of a Pi detection system suitable for rapid-kinetics approaches with comparable sensitivity to the commercially available Phosphate Sensor.