Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1

Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities

Prevalence of RT-qPCR-detected SARS-CoV-2 infection at schools: First results from the Austrian School-SARS-CoV-2 prospective cohort study.

BACKGROUND: The role of schools in the SARS-CoV-2 pandemic is much debated. We aimed to quantify reliably the prevalence of SARS-CoV-2 infections at schools detected with reverse-transcription quantitative polymerase-chain-reaction (RT-qPCR). METHODS: This nationwide prospective cohort study monitors a representative sample of pupils (grade 1-8) and teachers at Austrian schools throughout the school year 2020/2021. We repeatedly test participants for SARS-CoV-2 infection using a gargling solution and RT-qPCR. We herein report on the first two rounds of examinations. We used mixed-effects logistic regression to estimate odds ratios and robust 95% confidence intervals (95% CI). FINDINGS: We analysed data on 10,734 participants from 245 schools (9465 pupils, 1269 teachers). Prevalence of SARS-CoV-2 infection increased from 0·39% at round 1 (95% CI 028-0·55%, 28 September-22 October 2020) to 1·39% at round 2 (95% CI 1·04-1·85%, 10-16 November). Odds ratios for SARS-CoV-2 infection were 2·26 (95% CI 1·25-4·12, P = 0·007) in regions with >500 vs. ≤500 inhabitants/km2, 1·67 (95% CI 1·42-1·97, P<0·001) per two-fold higher regional 7-day community incidence, and 2·78 (95% CI 1·73-4·48, P<0·001) in pupils at schools with high/very high vs. low/moderate social deprivation. Associations of regional community incidence and social deprivation persisted in a multivariable adjusted model. Prevalence did not differ by average number of pupils per class nor between age groups, sexes, pupils vs. teachers, or primary (grade 1-4) vs. secondary schools (grade 5-8). INTERPRETATION: This monitoring study in Austrian schools revealed SARS-CoV-2 infection in 0·39%-1·39% of participants and identified associations of regional community incidence and social deprivation with higher prevalence. FUNDING: BMBWF Austria

Sensitivity and specificity of the antigen-based anterior nasal self-testing programme for detecting SARS-CoV-2 infection in schools, Austria, March 2021.

This study evaluates the performance of the antigen-based anterior nasal screening programme implemented in all Austrian schools to detect SARS-CoV-2 infections. We combined nationwide antigen-based screening data obtained in March 2021 from 5,370 schools (Grade 1-8) with an RT-qPCR-based prospective cohort study comprising a representative sample of 244 schools. Considering a range of assumptions, only a subset of infected individuals are detected with the programme (low to moderate sensitivity) and non-infected individuals mainly tested negative (very high specificity)

Reducing disease burden and health inequalities arising from chronic disease among Indigenous children: an early childhood caries intervention

Background: This study seeks to determine if implementing a culturally-appropriate early childhood caries (ECC) intervention reduces dental disease burden and oral health inequalities among Indigenous children living in South Australia, Australia. Methods/Design: This paper describes the study protocol for a randomised controlled trial conducted among Indigenous children living in South Australia with an anticipated sample of 400. The ECC intervention consists of four components: (1) provision of dental care; (2) fluoride varnish application to the teeth of children; (3) motivational interviewing and (4) anticipatory guidance. Participants are randomly assigned to two intervention groups, immediate (n = 200) or delayed (n = 200). Provision of dental care (1) occurs during pregnancy in the immediate intervention group or when children are 24-months in the delayed intervention group. Interventions (2), (3) and (4) occur when children are 6-, 12- and 18-months in the immediate intervention group or 24-, 30- and 36-months in the delayed intervention group. Hence, all participants receive the ECC intervention, though it is delayed 24 months for participants who are randomised to the control-delayed arm. In both groups, self-reported data will be collected at baseline (pregnancy) and when children are 24- and 36-months; and child clinical oral health status will be determined during standardised examinations conducted at 24- and 36-months by two calibrated dental professionals. Discussion: Expected outcomes will address whether exposure to a culturally-appropriate ECC intervention is effective in reducing dental disease burden and oral health inequalities among Indigenous children living in South Australia.Jessica Merrick, Alwin Chong, Eleanor Parker, Kaye Roberts-Thomson, Gary Misan, John Spencer, John Broughton, Herenia Lawrence and Lisa Jamieso

Protein compactness and interaction valency define the architecture of a biomolecular condensate across scales

Non-membrane-bound biomolecular condensates have been proposed to represent an important mode of subcellular organization in diverse biological settings. However, the fundamental principles governing the spatial organization and dynamics of condensates at the atomistic level remain unclear. The Saccharomyces cerevisiae Lge1 protein is required for histone H2B ubiquitination and its N-terminal intrinsically disordered fragment (Lge11-80) undergoes robust phase separation. This study connects single- and multi-chain all-atom molecular dynamics simulations of Lge11-80 with the in vitro behavior of Lge11-80 condensates. Analysis of modeled protein-protein interactions elucidates the key determinants of Lge11-80 condensate formation and links configurational entropy, valency, and compactness of proteins inside the condensates. A newly derived analytical formalism, related to colloid fractal cluster formation, describes condensate architecture across length scales as a function of protein valency and compactness. In particular, the formalism provides an atomistically resolved model of Lge11-80 condensates on the scale of hundreds of nanometers starting from individual protein conformers captured in simulations. The simulation-derived fractal dimensions of condensates of Lge11-80 and its mutants agree with their in vitro morphologies. The presented framework enables a multiscale description of biomolecular condensates and embeds their study in a wider context of colloid self-organization

A gated channel into the proteasome core particle.

The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure. We report that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits. Crystallographic analysis showed that deletion of the tail of the alpha 3-subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis. In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP. Inhibition by the alpha-subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme

The substrate translocation channel of the proteasome.

The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure. We have found that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits. Crystallographic analysis showed that deletion of the tail of the alpha3 subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis. In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP. Inhibition by the alpha subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme. Opening of the CP channel by assembly of the holoenzyme is regulated by the ATPase domain of Rpt2, one of 17 subunits in the RP. Thus, open-channel mutations in CP subunits suppress the closed-channel phenotype of an rpt2 mutant. These results identify a specific mechanism for allosteric regulation of the CP by the RP