Cotranslational folding of spectrin domains via partially structured states.

How do the key features of protein folding, elucidated from studies on native, isolated proteins, manifest in cotranslational folding on the ribosome? Using a well-characterized family of homologous α-helical proteins with a range of biophysical properties, we show that spectrin domains can fold vectorially on the ribosome and may do so via a pathway different from that of the isolated domain. We use cryo-EM to reveal a folded or partially folded structure, formed in the vestibule of the ribosome. Our results reveal that it is not possible to predict which domains will fold within the ribosome on the basis of the folding behavior of isolated domains; instead, we propose that a complex balance of the rate of folding, the rate of translation and the lifetime of folded or partly folded states will determine whether folding occurs cotranslationally on actively translating ribosomes.Supported by grants from the Swedish Cancer Foundation, the Swedish Research Council and the Knut and Alice Wallenberg Foundation (to G.v.H.); the Wellcome Trust (WT095195 to J.C.) and the European Research Council (ERC-2008-AdG 232648, to R.B.). J.C. is a Wellcome Trust Senior Research Fellow

Structural Constraints Identified with Covariation Analysis in Ribosomal RNA

Covariation analysis is used to identify those positions with similar patterns of sequence variation in an alignment of RNA sequences. These constraints on the evolution of two positions are usually associated with a base pair in a helix. While mutual information (MI) has been used to accurately predict an RNA secondary structure and a few of its tertiary interactions, early studies revealed that phylogenetic event counting methods are more sensitive and provide extra confidence in the prediction of base pairs. We developed a novel and powerful phylogenetic events counting method (PEC) for quantifying positional covariation with the Gutell lab’s new RNA Comparative Analysis Database (rCAD). The PEC and MI-based methods each identify unique base pairs, and jointly identify many other base pairs. In total, both methods in combination with an N-best and helix-extension strategy identify the maximal number of base pairs. While covariation methods have effectively and accurately predicted RNAs secondary structure, only a few tertiary structure base pairs have been identified. Analysis presented herein and at the Gutell lab’s Comparative RNA Web (CRW) Site reveal that the majority of these latter base pairs do not covary with one another. However, covariation analysis does reveal a weaker although significant covariation between sets of nucleotides that are in proximity in the three-dimensional RNA structure. This reveals that covariation analysis identifies other types of structural constraints beyond the two nucleotides that form a base pair

Physical inactivity and smoking after myocardial infarction as predictors for readmission and survival : results from the SWEDEHEART-registry.

BACKGROUND: Physical activity (PA) and smoking cessation are included in the secondary prevention guidelines after myocardial infarction (MI), but they are still underutilised. This study aims to explore how PA level and smoking status (6-10 weeks post-MI) were associated with 1-year readmission and mortality during full follow-up time, and with the cumulative 5-year mortality. METHODS: A population-based cohort of all hospitals providing MI-care in Sweden (SWEDEHEART-registry) in 2004-2014. PA was expressed as the number of exercise sessions of ≥ 30 min in the last 7 days: 0-1 (low), 2-4 (medium) and 5-7 (high) sessions/week. Individuals were categorised as smokers, former smokers or never-smokers. The associations were analysed by unadjusted and adjusted logistic and Cox regressions. RESULTS: During follow-up (M = 3.58 years), a total of 1702 deaths occurred among 30 644 individuals (14.1 cases per 1000 person-years). For medium and high PA, the hazard ratios (HRs) for mortality were 0.39 and 0.36, respectively, compared with low PA. For never-smokers, the HR was 0.45 and former smokers 0.56 compared with smokers. Compared with low PA, the odds ratios (ORs) for readmission in medium PA were 0.65 and 0.59 for CVD and non-CVD causes, respectively. For high PA, the corresponding ORs were 0.63 and 0.55. The association remained in adjusted models. There were no associations between smoking status and readmission. CONCLUSIONS: The PA level and smoking status are strong predictors of mortality post-MI and the PA level also predicts readmission, highlighting the importance of adherence to the secondary prevention guidelines.Ischemisk hjärtsjukdo

Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography

Riboswitches are structural RNA elements that are generally located in the 5′ untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time4. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of ‘mix-and-inject’ time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.Subject terms: Nanocrystallography RNA Riboswitches RNA foldin