scholarly journals Evolution of a virus-like architecture and packaging mechanism in a repurposed bacterial protein

Science ◽  
2021 ◽  
Vol 372 (6547) ◽  
pp. 1220-1224
Author(s):  
Stephan Tetter ◽  
Naohiro Terasaka ◽  
Angela Steinauer ◽  
Richard J. Bingham ◽  
Sam Clark ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Building Blocks ◽  
Bacterial Protein ◽  
Host Proteins ◽  
Stem Loop ◽  
Evolutionary Pathway ◽  
Bacterial Enzyme ◽  
Multiple Copies ◽  
Virion Formation ◽  
Icosahedral Capsid

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme that lacks affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding messenger RNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid that is impermeable to nucleases, and emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing nonviral carriers for diverse vaccine and delivery applications.

scholarly journals Evolution of a virus-like architecture and packaging mechanism in a repurposed bacterial protein

2020 ◽  
Author(s):  
Stephan Tetter ◽  
Naohiro Terasaka ◽  
Angela Steinauer ◽  
Richard J. Bingham ◽  
Sam Clark ◽  
...  
Keyword(s):  
Building Blocks ◽  
Bacterial Protein ◽  
Host Proteins ◽  
Stem Loop ◽  
Evolutionary Pathway ◽  
Global Impact ◽  
Bacterial Enzyme ◽  
Multiple Copies ◽  
Virion Formation ◽  
Icosahedral Capsid

AbstractViruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme lacking affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding mRNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid impermeable to nucleases, while emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing non-viral carriers for diverse vaccine and delivery applications.

scholarly journals Dynamism and Cooperativity Essential for Efficient Catalysis in Aspergillus Niger Monoamine Oxidase

2019 ◽  
Author(s):  
Christian Curado-Carballada ◽  
Ferran Feixas ◽  
Sílvia Osuna
Keyword(s):  
Aspergillus Niger ◽  
Monoamine Oxidase ◽  
Active Site ◽  
Conformational Dynamics ◽  
Catalytic Efficiency ◽  
Building Blocks ◽  
Chiral Amine ◽  
Evolutionary Pathway ◽  
Accelerated Molecular Dynamics ◽  
Open States

<p><b> </b><i>Aspergillus niger </i>Monoamine Oxidase (MAO-N) is a homodimeric enzyme responsible for the oxidation of amines into the corresponding imine. Laboratory evolved variants of MAO-N in combination with a non-selective chemical reductant represents a powerful strategy for the deracemisation of chiral amine mixtures and, thus, is of interest for obtaining chiral amine building blocks. MAO-N presents a rich conformational dynamics with a flexible ß-hairpin region that can adopt closed, partially closed and open states. Despite the ß-hairpin conformational dynamics is altered along the laboratory evolutionary pathway of MAO-N, the connection between the ß-hairpin conformational dynamics and active site catalysis still remains unclear. In this work, we use accelerated molecular dynamics to elucidate the potential interplay between the ß-hairpin conformational dynamics and catalytic activity in MAO-N wild type and its evolved D5 variant. Our study reveals a delicate communication between both MAO-N subunits that impacts the active site architecture, and thus its catalytic efficiency. In both MAO-N WT and the laboratory evolved D5 variant, the ß-hairpin conformation in one of the monomers affects the productive binding of the substrate in the active site of the other subunit. However, both MAO-N WT and D5 variants show a quite different behaviour due to the distal mutations introduced experimentally with Directed Evolution. </p>

scholarly journals The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

2019 ◽  
Vol 116 (49) ◽  
pp. 24542-24550 ◽  
Author(s):  
Jiarui Song ◽  
Dhenugen Logeswaran ◽  
Claudia Castillo-González ◽  
Yang Li ◽  
Sreyashree Bose ◽  
...  
Keyword(s):  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Structural Element ◽  
Stem Loop ◽  
Evolutionary Pathway ◽  
Pol Iii

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3′ long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

scholarly journals EF-G–induced ribosome sliding along the noncoding mRNA

2019 ◽  
Vol 5 (6) ◽  
pp. eaaw9049 ◽  
Author(s):  
M. Klimova ◽  
T. Senyushkina ◽  
E. Samatova ◽  
B. Z. Peng ◽  
M. Pearson ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Elongation Factor ◽  
Transfer Rna ◽  
Noncoding Region ◽  
Forward Movement ◽  
Stem Loop ◽  
A Site ◽  
Hydrolysis Of ◽  
Reading Frames ◽  
Factor G

Translational bypassing is a recoding event during which ribosomes slide over a noncoding region of the messenger RNA (mRNA) to synthesize one protein from two discontinuous reading frames. Structures in the mRNA orchestrate forward movement of the ribosome, but what causes ribosomes to start sliding remains unclear. Here, we show that elongation factor G (EF-G) triggers ribosome take-off by a pseudotranslocation event using a small mRNA stem-loop as an A-site transfer RNA mimic and requires hydrolysis of about two molecules of guanosine 5′-triphosphate per nucleotide of the noncoding gap. Bypassing ribosomes adopt a hyper-rotated conformation, also observed with ribosomes stalled by the SecM sequence, suggesting common ribosome dynamics during translation stalling. Our results demonstrate a new function of EF-G in promoting ribosome sliding along the mRNA, in contrast to codon-wise ribosome movement during canonical translation, and suggest a mechanism by which ribosomes could traverse untranslated parts of mRNAs.

scholarly journals Consequences of Genomic DNA Mono-Ribonucleotides for Chromosomal Stability

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. 1006-1006
Author(s):  
Tavia Roache
Keyword(s):  
Messenger Rna ◽  
Genomic Dna ◽  
Cellular Response ◽  
Excision Repair ◽  
Access Point ◽  
Building Blocks ◽  
Negative Consequences ◽  
Negative Effects ◽  
Topoisomerase 1 ◽  
Rnase H2

Abstract Mono-ribonucleotides are building blocks for polynucleotide RNA chains (e.g., messenger RNA), but if mis-incorporated into duplex DNA can cause mutagenesis and chromosomal instability. During DNA synthesis by Pol γ, remnants of unremoved RNA primers contribute to elevated mono-ribonucleotide triphosphates resulting in nucleotide pool imbalance, ultimately favoring mis-incorporated ribonucleotides during replication. Moreover, although polymerases generally replicate DNA with high fidelity, the steric gate occasionally allows a mis-incorporated ribonucleotide. Thus, a mono-ribonucleotide is one of the most abundant lesions in genomic DNA of eukaryotes. If unremoved from double-stranded DNA, the ribonucleotide exerts negative effects on replication, transcription, and genomic maintenance, with lasting effects on cellular homeostasis. Even a single ribonucleotide in telomeric DNA comprises shelterin binding and telomere capping causing vulnerability to spontaneous hydrolysis which potentiates telomere shortening. Consistent with this, a ribonucleotide positioned in double-helical DNA alters its structure by torsinally distorting the sugar-phosphate backbone. Fortunately, cellular response and repair pathways exist to help cells cope with mis-incorporated mono-ribonucleotides. The Ribonucleotide Excision Repair (RER) or a Topoisomerase 1 (Top1)-mediated pathway remove embedded ribonucleotides. For RER, RNase H2 incises 5’ of a mono-ribonucleotide, creating an access point for its removal. If cells are deficient in RNase H2, Top1 initiates removal of the ribonucleotide. However, Top1 is less accurate than RNase H2, which can lead to mutagenesis. Studying the mechanisms in which ribonucleotides are incorporated into DNA or further metabolized should provide insight to their negative consequences for chromosomal integrity, cancer, and auto-immune disease attributed to a genetic deficiency of RNase H2.

EXTRACTION OF ENZYMES AND ASSESSMENT OF METABOLISM IN BOVINE RUMEN EPITHELIUM

1982 ◽  
Vol 62 (2) ◽  
pp. 429-438 ◽  
Author(s):  
ROY S. BUSH
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Total Activity ◽  
Small Contribution ◽  
Bacterial Protein ◽  
Total Enzyme Activity ◽  
Rumen Epithelium ◽  
Bacterial Enzyme ◽  
Protein Contamination ◽  
Total Enzyme

Papillae collected from the rumens of freshly killed cows were used to estimate the most appropriate methods for enzyme extraction from rumen epithelium and the amount of enzymes in extracts which might be of bacterial origin. Extractions of enzymes from fresh and frozen papillae were compared for the Polytron homogenizer (PT), the Potter-Elvehjem homogenizer (PE), the Waring blender, sonication and acetone powdering plus PE. PE extraction yielded solutions with the highest specific activity for each enzyme. PT extraction released the most protein and total enzyme activity into solution. PT extraction was chosen for the remaining tests because of the high total activity released. Mixed rumen bacteria were homogenized by sonication. Electrophoretic examination of epithelial and bacterial extracts showed differential migration for malate dehydrogenase. Lactate dehydrogenase from the epithelium showed four distinct isozymes whereas the bacterial enzyme showed little distinct band development. Contamination of epithelial extracts by bacterial protein was estimated to be less than 5%. The specific activities of 10 enzymes were found to be similar in epithelial and bacterial extracts so that a small amount of protein contamination would result in only a small contribution to total enzyme activity. The presence of the enzymes assayed in this study plus a number reported in the literature showed that rumen epithelial metabolism is more diverse than previously recognized. Key words: Rumen epithelium, enzymes, extraction

scholarly journals Regulators of Viral Frameshifting: More Than RNA Influences Translation Events

2020 ◽  
Vol 7 (1) ◽  
pp. 219-238
Author(s):  
Wesley D. Penn ◽  
Haley R. Harrington ◽  
Jonathan P. Schlebach ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Messenger Rna ◽  
Protein Production ◽  
Productive Infection ◽  
Cis Elements ◽  
Stem Loop ◽  
Viral Translation ◽  
Ribosomal Frameshifting ◽  
Evolutionarily Conserved ◽  
Rna Pseudoknot ◽  
New Protein

Programmed ribosomal frameshifting (PRF) is a conserved translational recoding mechanism found in all branches of life and viruses. In bacteria, archaea, and eukaryotes PRF is used to downregulate protein production by inducing a premature termination of translation, which triggers messenger RNA (mRNA) decay. In viruses, PRF is used to drive the production of a new protein while downregulating the production of another protein, thus maintaining a stoichiometry optimal for productive infection. Traditionally, PRF motifs have been defined by the characteristics of two cis elements: a slippery heptanucleotide sequence followed by an RNA pseudoknot or stem-loop within the mRNA. Recently, additional cis and new trans elements have been identified that regulate PRF in both host and viral translation. These additional factors suggest PRF is an evolutionarily conserved process whose function and regulation we are just beginning to understand.

scholarly journals Annotating RNA motifs in sequences and alignments

2014 ◽  
Author(s):  
Paul P Gardner ◽  
Hisham Eldai
Keyword(s):  
Rna Structure ◽  
Messenger Rna ◽  
Building Blocks ◽  
Regulatory Elements ◽  
Rna Motifs ◽  
Regulate Gene Expression ◽  
Translational Machinery ◽  
Cellular Life ◽  
Functional Characterisation ◽  
Initial Discovery

RNA performs a diverse array of important functions across all cellular life. These functions include important roles in translation, building translational machinery and maturing messenger RNA. More recent discoveries include the miRNAs and bacterial sRNAs that regulate gene expression, the thermosensors, riboswitches and other cis-regulatory elements that help prokaryotes sense their environment and eukaryotic piRNAs that suppress transposition. However, there can be a long period between the initial discovery of a RNA and determining its function. We present a bioinformatic approach to characterise RNA motifs, which are the central building blocks of RNA structure. These motifs can, in some instances, provide researchers with functional hypotheses for uncharacterised RNAs. Moreover, we introduce a new profile-based database of RNA motifs - RMfam - and illustrate its application for investigating the evolution and functional characterisation of RNA. All the data and scripts associated with this work is available from: https://github.com/ppgardne/RMfam

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