Principles for enhancing virus capsid capacity and stability from a thermophilic virus capsid structure

The capsids of double-stranded DNA viruses protect the viral genome from the harsh extracellular environment, while maintaining stability against the high internal pressure of packaged DNA. To elucidate how capsids maintain stability in an extreme environment, we use cryoelectron microscopy to determine the capsid structure of thermostable phage P74-26 to 2.8-A resolution. We find P74-26 capsids exhibit an overall architecture very similar to those of other tailed bacteriophages, allowing us to directly compare structures to derive the structural basis for enhanced stability. Our structure reveals lasso-like interactions that appear to function like catch bonds. This architecture allows the capsid to expand during genome packaging, yet maintain structural stability. The P74-26 capsid has T = 7 geometry despite being twice as large as mesophilic homologs. Capsid capacity is increased with a larger, flatter major capsid protein. Given these results, we predict decreased icosahedral complexity (i.e. T \u3c /= 7) leads to a more stable capsid assembly

Principles for enhancing virus capsid capacity and stability from a thermophilic virus capsid structure [preprint]

The capsids of double-stranded DNA viruses protect the viral genome from the harsh extracellular environment, while maintaining stability against the high internal pressure of packaged DNA. To elucidate how capsids maintain stability in an extreme environment, we used cryoelectron microscopy to determine the capsid structure of the thermostable phage P74-26. We find the P74-26 capsid exhibits an overall architecture that is very similar to those of other tailed bacteriophages, allowing us to directly compare structures to derive the structural basis for enhanced stability. Our structure reveals lasso-like interactions that appear to function like catch bonds. This architecture allows the capsid to expand during genome packaging, yet maintain structural stability. The P74-26 capsid has T=7 geometry despite being twice as large as mesophilic homologs. Capsid capacity is increased through a novel mechanism with a larger, flatter major capsid protein. Our results suggest that decreased icosahedral complexity (i.e. lower T number) leads to a more stable capsid assembly

ArfB can displace mRNA to rescue stalled ribosomes

Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal alpha-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 A and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths

Liberdade de expressão religiosa: os limites jurídicos entre o proselitismo e o discurso de ódio

O presente estudo objetivo analisar os limites jurídicos da liberdade de expressão, especialmente no âmbito religioso, analisando historicamente o avanço dos direitos fundamentais e sua repercussão atual, a partir da análise de precedentes da Suprema Corte. Além disso, tem como propósito apresentar a especial relevância da liberdade de expressão - conceituada como um direito de posição preferencial - por traduzir a pluralidade de ideias no contexto republicano. Pretende, ainda, oferecer subsídios para a manutenção e convivência harmônica de um ambiente democrático social, favorecendo, de um lado, a livre manifestação do pensamento, com espaço destinado ao proselitismo, mas também, na outra face, protegendo os direitos e garantias individuais, notadamente a dignidade humana

Age-of-Information Dependent Random Access in NOMA-Aided Multiple-Relay Slotted ALOHA

We propose and evaluate the performance of a Non-Orthogonal Multiple Access (NOMA) dual-hop multiple relay (MR) network from an information freshness perspective using the Age of Information (AoI) metric. More specifically, we consider an age dependent (AD) policy, named as AD-NOMA- MR, in which users only transmit, with a given probability, after they reach a certain age threshold. The packets sent by the users are potentially received by the relays, and then forwarded to a common sink in a NOMA fashion by randomly selecting one of the available power levels, and multiple packets are received if all selected levels are unique. We derive analytical expressions for the average AoI of AD-NOMA-MR. Through numerical and simulation results, we show that the proposed policy can improve the average AoI up to 76.6% when compared to a previously proposed AD Orthogonal Multiple Access MR policy.Comment: 6 pages, 5 figures. Paper accepted for presentation at the 2023 Joint European Conference on Networks and Communications & 6G Summit (EuCNC/6G Summit), Gothenburg, Sweden, 202

Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation [preprint]

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. Where and how in the elongation cycle +1-frameshifting occurs remains poorly understood. We captured six ∼3.5-Å-resolution cryo-EM structures of ribosomal elongation complexes formed with the GTPase elongation factor G (EF-G). Three structures with a +1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G, the tRNA shifts to the +1-frame codon near the P site, whereas the freed mRNA base bulges between the P and E sites and stacks on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during mRNA translocation

Mechanism of ribosome rescue by ArfA and RF2

ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2 A resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA*RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA \u27senses\u27 the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled

Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM [preprint]

Toxic dipeptide repeat (DPR) proteins are produced from expanded G4C2 hexanucleotide repeats in the C9ORF72 gene, which cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥ 20 repeats inhibit the ribosome’s peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryo-EM structures reveal that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center. Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with the DPR proteins and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD

Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-A-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G*GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation