Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system.

In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of RUNX transcription factor paralogs with apparent functional redundancy. Here we asked what cell-type-specific biologies might be supported by the selective expression of RUNX paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional nonequivalence between RUNX paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain and evolutionary reconstruction suggested convergence of RUNT domain residues toward submaximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.

DNA-directed termination of RNA polymerase II transcription.

RNA polymerase II (RNAPII) transcription involves initiation from a promoter, transcriptional elongation through the gene, and termination in the terminator region. In bacteria, terminators often contain specific DNA elements provoking polymerase dissociation, but RNAPII transcription termination is thought to be driven entirely by protein co-factors. We used biochemical reconstitution, single-molecule studies, and genome-wide analysis in yeast to study RNAPII termination. Transcription into natural terminators by pure RNAPII results in spontaneous termination at specific sequences containing T-tracts. Single-molecule analysis indicates that termination involves pausing without backtracking. The "torpedo" Rat1-Rai1 exonuclease (XRN2 in humans) greatly stimulates spontaneous termination but is ineffectual on other paused RNAPIIs. By contrast, elongation factor Spt4-Spt5 (DSIF) suppresses termination. Genome-wide analysis further indicates that termination occurs by transcript cleavage at the poly(A) site exposing a new 5' RNA-end that allows Rat1-Rai1 loading, which then catches up with destabilized RNAPII at specific termination sites to end transcription.

Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein.

Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.

Negative DNA supercoiling induces genome-wide Cas9 off-target activity.

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.

Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination.

Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, "open," and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex.

POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells.

POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.

Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins.

Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a "chaperone" to promote 3' to 5' filament growth via highly dynamic engagement with 5' filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.

HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51.

DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3' to 5' polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2-4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.

The transcriptome of HTLV-1-infected primary cells following reactivation reveals changes to host gene expression central to the proviral life cycle.

Infections by Human T cell Leukaemia Virus type 1 (HTLV-1) persist for the lifetime of the host by integrating into the genome of CD4+ T cells. Proviral gene expression is essential for proviral survival and the maintenance of the proviral load, through the pro-proliferative changes it induces in infected cells. Despite their role in HTLV-1 infection and a persistent cytotoxic T lymphocyte response raised against the virus, proviral transcripts from the sense-strand are rarely detected in fresh cells extracted from the peripheral blood, and have recently been found to be expressed intermittently by a small subset of cells at a given time. Ex vivo culture of infected cells prompts synchronised proviral expression in infected cells from peripheral blood, allowing the study of factors involved in reactivation in primary cells. Here, we used bulk RNA-seq to examine the host transcriptome over six days in vitro, following proviral reactivation in primary peripheral CD4+ T cells isolated from subjects with non-malignant HTLV-1 infection. Infected cells displayed a conserved response to reactivation, characterised by discrete stages of gene expression, cell division and subsequently horizontal transmission of the virus. We observed widespread changes in Polycomb gene expression following reactivation, including an increase in PRC2 transcript levels and diverse changes in the expression of PRC1 components. We hypothesize that these transcriptional changes constitute a negative feedback loop that maintains proviral latency by re-deposition of H2AK119ub1 following the end of proviral expression. Using RNAi, we found that certain deubiquitinases, BAP1, USP14 and OTUD5 each promote proviral transcription. These data demonstrate the detailed trajectory of HTLV-1 proviral reactivation in primary HTLV-1-carrier lymphocytes and the impact on the host cell.

Advancing Health Equity through 15-min Cities and Chrono-urbanism.

Implementing the 15-min city and chrono-urbanism aims to improve sustainability and quality of life by ensuring residents' proximity to essential services. The 15-min city model is gaining global traction, with localized adaptations to suit communities' needs. Beyond environmental motivations, 15-min cities can benefit public health through enhanced walkability, social cohesion, and universal accessibility. However, research examining the intersection of health and equity among chrono-urbanism and the 15-min city remains limited. This study aims to develop a framework to integrate health and equity into chrono-urbanism and 15-min city plans. We describe the potential benefits and risks of the 15-min approach for urban planning, daily behaviors, and health outcomes. Potential benefits of 15-min cities for health equity include proximity to destinations, increased physical activity, strengthened social capital, reduced emissions, and traffic calming. Risks that must be mitigated include gentrification, variable proximity definitions, infrastructure upgrades, and inadequate cultural sensitivity. Recommendations to integrate 15-min cities into planning activities include conducting comprehensive baseline assessments, aligning goals with sustainability, economic development, flexible zoning, inclusive public spaces, and diverse community engagement tactics. We recommend interventions targeting marginalized communities and developing standardized measurement tools for comparison, monitoring, and evaluation. A nuanced, equitable approach to implementing 15-min cities can help urban plans support health equity across diverse populations.

Health implications of urban tree canopy policy scenarios in Denver and Phoenix: A quantitative health impact assessment.

BACKGROUND: Urban tree canopy (UTC) goals are a popular policy to increase urban vegetation, support climate strategies, and encourage a healthy environment. Health studies related to UTC are needed across cities to support evidence-based decision-making. METHODS: We used a quantitative Health Impact Assessment (HIA) to model the annual number of premature deaths prevented, and the number of stroke and dementia cases, under UTC goals in Denver, Colorado, and Phoenix, Arizona, USA, using standing policy goals (20% and 25% UTC, respectively) and 50% ("half-way") attainment scenarios from current levels (16.5% and 13% UTC, respectively), using publicly accessible national datasets, and a proportional representation of UTC change to standardize across methodologies. We estimated UTC health impacts by relating UTC with scenario-based changes in the Normalized Difference Vegetation Index (NDVI) and considered health equity in UTC distributions and benefits. RESULTS: We projected that at 2020 populations, uniform 20% UTC attainment across Denver block groups would avert 200 (95% uncertainty interval: (UI) 100, 306) annual premature deaths among adults 18 and older, along with 4.1 (95% UI: 2.2, 6.7) annual cases of stroke (adults ≥35), and 2.6 (95% UI: 1.5, 4.1) cases of dementia (adults ≥65), with "halfway" attainment from current levels (16.5% UTC) capturing ∼64% of these benefits. In Phoenix, uniform 25% UTC would annually prevent 368 (95% UI: 181, 558) premature deaths, 8.7 (95% UI: 4.7, 13.9) cases of stroke, and 5,1 (95% UI: 2.9, 8.0) of dementia, with the "halfway" scenario (17% UTC) achieving ∼44% of these results. Both cities saw significantly different greenspace exposures and health outcomes by socioeconomic vulnerability. Denver had more spatially and socioeconomically heterogeneous projected health benefits than Phoenix. CONCLUSIONS: Implementing UTC goals can prevent excess mortality and chronic diseases among urban residents. UTC goals can be used as a health promotion and prevention tool.

Health science students' preparedness for climate change: a scoping review on knowledge, attitudes, and practices.

INTRODUCTION: Climate change (CC) is a global public health issue, and the role of health professionals in addressing its impact is crucial. However, to what extent health professionals are prepared to deal with CC-related health problems is unclear. We aimed to evaluate the knowledge, attitudes, and practices of health students about the CC. METHODS: We conducted a scoping review through systematic searches in PubMed, Scopus, Web of Science, Proquest, and EBSCO. We included original scientific research with no language or time restrictions. Two authors independently reviewed and decided on the eligibility of the studies, then performed data extraction. RESULTS: 21 studies were included, with a total of 9205 undergraduate nursing, medical, pharmacy, and public health students mainly. Most health science students (> 75%) recognized human activities as the main cause of CC. However, they perceived a lack of knowledge on how to address CC. Moreover, we found inadequate coverage or limited development of CC in related curricula that may contribute to incomplete learning or low confidence in the theoretical and practical concepts of students. CONCLUSION: The findings of our scoping review suggest that while health sciences students possess a general understanding of CC, there is a significant gap in their knowledge regarding its specific health impacts. To address this gap, there is a need for targeted education and training for future health care professionals that emphasizes the health effects of CC.

3D modelling of cavity-free lasing in nitrogen plasma filaments.

In this article we show results on cavity-free lasing in nitrogen filaments using our 3D, time-dependent Maxwell-Bloch code, Dagon. This code was previously used to model plasma-based soft X-ray lasers and it has been adapted to model lasing in nitrogen plasma filaments. In order to assess the predictive capabilities of the code, we have conducted several benchmarks against experimental and 1D modelling results. Afterwards, we study the amplification of an externally seeded UV beam in nitrogen plasma filaments. Our results show that the phase of the amplified beam carries information about the temporal dynamics of amplification and collisional processes inside the plasma, along with information about the spatial structure of the amplified beam and the active region of the filament. We thus conclude that measuring the phase of an UV probe beam, in combination with 3D Maxwell-Bloch modelling, might be an excellent method for diagnosing electron density value and gradients, mean ionization, density of N2+ ions and the magnitude of collisional processes inside these filaments.

Health and economic benefits of meeting WHO air quality guidelines, Western Pacific Region.

Objective: To quantify the number of avoidable annual deaths and associated economic benefits from meeting the World Health Organization (WHO) air quality guidelines for ambient concentrations for fine particulate matter (PM2.5) for Member States of the WHO Western Pacific Region. Methods: Using the AirQ+ software, we performed a quantitative health impact assessment comparing country-level PM2.5 concentrations with the 2005 and 2021 air quality guidelines recommended maximum concentrations of 10 and 5 µg/m3, respectively. We obtained PM2.5 data from the WHO Global Health Observatory (latest available year 2016), and population and mortality estimates from the United Nations World Population Prospects database for the latest 5-year period available (2015-2019), which we averaged to 1-year estimates. A risk estimate for all-cause mortality, based on a meta-analysis, was embedded within AirQ+ software. Our economic assessment used World Bank value of a statistical life adjusted to country-specific gross domestic product (latest available year 2014). Findings: Data were complete for 21 of 27 Member States. If these countries achieved the 2021 guidelines for PM2.5, an estimated 3.1 million deaths would be avoided annually, which are 0.4 million more deaths avoided than meeting the 2005 guidelines. China would avoid the most deaths per 100 000 population (303 deaths) and Brunei Darussalam the least (5 deaths). The annual economic benefit per capita ranged from 5781 United States dollars (US$) in Singapore to US$ 143 in Solomon Islands. Conclusion: Implementing effective measures to reduce PM2.5 emissions would save a substantial number of lives and money across the Region.

Genetic screening for anticancer genes highlights FBLN5 as a synthetic lethal partner of MYC.

BACKGROUND: When ectopically overexpressed, anticancer genes, such as TRAIL, PAR4 and ORCTL3, specifically destroy tumour cells without harming untransformed cells. Anticancer genes can not only serve as powerful tumour specific therapy tools but studying their mode of action can reveal mechanisms underlying the neoplastic transformation, sustenance and spread. METHODS: Anticancer gene discovery is normally accidental. Here we describe a systematic, gain of function, forward genetic screen in mammalian cells to isolate novel anticancer genes of human origin. Continuing with over 30,000 transcripts from our previous study, 377 cell death inducing genes were subjected to screening. FBLN5 was chosen, as a proof of principle, for mechanistic gene expression profiling, comparison pathways analyses and functional studies. RESULTS: Sixteen novel anticancer genes were isolated; these included non-coding RNAs, protein-coding genes and novel transcripts, such as ZNF436-AS1, SMLR1, TMEFF2, LINC01529, HYAL2, NEIL2, FBLN5, YPEL4 and PHKA2-processed transcript. FBLN5 selectively caused inhibition of MYC in COS-7 (transformed) cells but not in CV-1 (normal) cells. MYC was identified as synthetic lethality partner of FBLN5 where MYC transformed CV-1 cells experienced cell death upon FBLN5 transfection, whereas FBLN5 lost cell death induction in MCF-7 cells upon MYC knockdown. CONCLUSIONS: Sixteen novel anticancer genes are present in human genome including FBLN5. MYC is a synthetic lethality partner of FBLN5. Video Abstract.

Toxoplasma gondii Infection and Threatened Abortion in Women from Northern Peru.

Introduction: Toxoplasma gondii infection can cause important complications during pregnancy. Threatened abortion may be a late indicator for infection in settings with high prevalence of toxoplasmosis. We aimed to determine the association between T. gondii infection and threatened abortion in women from northern Peru. Methods: We conducted a secondary analysis of a cross-sectional study in pregnant women from a hospital and a rural community in Lambayeque, Peru. Exposure variable was serological diagnosis of toxoplasmosis, defined as the demonstration of either IgM or IgG antibodies against T. gondii. Outcome variable was threatened abortion, defined as the diagnosis of bloody vaginal discharge or bleeding during the first half of pregnancy. Prevalence ratios were estimated in simple and multiple regression analyses. Results: Of 218 pregnant women, 35.8% presented positive serology for T. gondii and 14.7% had threatened abortion in their current pregnancy. Pregnant women with positive T. gondii infection had 2.45-fold higher frequency of threatened abortion (PR: 2.45, 95% CI: 1.15-5.21). In addition, the frequency of threatened abortion decreased by 9% for each additional year of age (PR: 0.91, 95% CI: 0.86-0.97). A previous history of threatened abortion also showed a higher frequency of threatened abortion (PR: 5.22, 95% CI: 2.45-11.12). Conclusions: T. gondii infection is associated with threatened abortion. An early age of pregnancy and a previous history of abortion are also associated with this condition.

Kinetics of HTLV-1 reactivation from latency quantified by single-molecule RNA FISH and stochastic modelling.

The human T cell leukemia virus HTLV-1 establishes a persistent infection in vivo in which the viral sense-strand transcription is usually silent at a given time in each cell. However, cellular stress responses trigger the reactivation of HTLV-1, enabling the virus to transmit to a new host cell. Using single-molecule RNA FISH, we measured the kinetics of the HTLV-1 transcriptional reactivation in peripheral blood mononuclear cells (PBMCs) isolated from HTLV-1+ individuals. The abundance of the HTLV-1 sense and antisense transcripts was quantified hourly during incubation of the HTLV-1-infected PBMCs ex vivo. We found that, in each cell, the sense-strand transcription occurs in two distinct phases: the initial low-rate transcription is followed by a phase of rapid transcription. The onset of transcription peaked between 1 and 3 hours after the start of in vitro incubation. The variance in the transcription intensity was similar in polyclonal HTLV-1+ PBMCs (with tens of thousands of distinct provirus insertion sites), and in samples with a single dominant HTLV-1+ clone. A stochastic simulation model was developed to estimate the parameters of HTLV-1 proviral transcription kinetics. In PBMCs from a leukemic subject with one dominant T-cell clone, the model indicated that the average duration of HTLV-1 sense-strand activation by Tax (i.e. the rapid transcription) was less than one hour. HTLV-1 antisense transcription was stable during reactivation of the sense-strand. The antisense transcript HBZ was produced at an average rate of ~0.1 molecules per hour per HTLV-1+ cell; however, between 20% and 70% of HTLV-1-infected cells were HBZ-negative at a given time, the percentage depending on the individual subject. HTLV-1-infected cells are exposed to a range of stresses when they are drawn from the host, which initiate the viral reactivation. We conclude that whereas antisense-strand transcription is stable throughout the stress response, the HTLV-1 sense-strand reactivation is highly heterogeneous and occurs in short, self-terminating bursts.

Cas12a target search and cleavage on force-stretched DNA.

Using optical tweezers, we investigate target search and cleavage by CRISPR-Cas12a on force-stretched λ-DNA. Cas12a uses fast, one-dimensional hopping to locate its target. Binding and cleavage occur rapidly and specifically at low forces (≤5 pN), with a 1.8 nm rate-limiting conformational change. Mechanical distortion slows diffusion, increases off-target binding but hinders cleavage.

Health impacts of bike-sharing systems in the U.S.

BACKGROUND: Bike-sharing systems (BSS) are short-term bike rentals that can be borrowed from one location and retired to another at the conclusion of the trip. In 2019, 109,589 BSS trips were made each day in the U.S, and half of those in New York City (NYC). AIM: This study aims to quantify the health risks and benefits of BSS in the U.S. and NYC. METHODS: This study followed a quantitative health impact assessment approach to estimate the risks and benefits of BSS. Specifically, we quantified the health impacts of physical activity, air pollution, and traffic incidents. We analyzed all the trips made by BSS in the U.S. and NYC. Input data on transport, traffic safety, air quality, and physical activity were collated from public records and scientific publications. We modeled the health impacts on adult users related to mortality, disease incidence, disability-adjusted life years (DALYs), and health economic impacts (related to morbidity and mortality). RESULTS: We estimated that in the U.S. BSS trips resulted in an annual reduction of 4.7 premature deaths, 737 DALYs, and 36 million $USD in health economic impacts, mainly derived from the increment in physical activity. In NYC, we estimated an annual reduction of 2 premature deaths, 355 DALYs, and 15 million $USD in health economic impacts. CONCLUSION: BSS in the U.S. and NYC provide a health benefit for bicyclists. Improvements in air quality and traffic safety across U.S. cities will maximize the health benefits of BSS.

Optimal molecular crowding accelerates group II intron folding and maximizes catalysis.

Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg2+]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg2+] required, and we found an "optimum" [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions.