Non-canonical Structures in Promoter Modulate Gene Expression in Escherichia coli

Herein we show how sequences that can form different non-canonical structures affect gene expression levels when inserted in the core of σ70-dependent promoter, between the −35 and −10 elements recognized by RNA polymerase, in E. coli. We note that influence on level of GFP expression varies considerably depending on introduction of non-canonical structural elements in the antisense and sense strands as well as with their propensities to form G-triplex, G-hairpin, hairpin or G-quadruplex structures. Moreover, the extent of repression of expression does not relate to the in vitro thermal stability in a simple manner. Repression is most likely caused by steric interference rather than improper distance between the −35 and −10 elements. Although properties like thermal stability and topology can be somewhat different under in vivo and in vitro conditions, our results suggest that the extent of expression suppression cannot be dependent solely on thermal stabilities of G-rich structures alone. This work is licensed under a Creative Commons Attribution 4.0 International License

Towards Understanding of Polymorphism of the G-rich Region of Human Papillomavirus Type 52

The potential to affect gene expression via G-quadruplex stabilization has been extended to all domains of life, including viruses. Here, we investigate the polymorphism and structures of G-quadruplexes of the human papillomavirus type 52 with UV, CD and NMR spectroscopy and gel electrophoresis. We show that oligonucleotide with five G-tracts folds into several structures and that naturally occurring single nucleotide polymorphisms (SNPs) have profound effects on the structural polymorphism in the context of G-quadruplex forming propensity, conformational heterogeneity and folding stability. With help of SNP analysis, we were able to select one of the predominant forms, formed by G-rich sequence d(G3TAG3CAG4ACACAG3T). This oligonucleotide termed HPV52(1–4) adopts a three G-quartet snap back (3 + 1) type scaffold with four syn guanine residues, two edgewise loops spanning the same groove, a no-residue V loop and a propeller type loop. The first guanine residue is incorporated in the central G-quartet and all four-guanine residues from G4 stretch are included in the three quartet G-quadruplex core. Modification studies identified several structural elements that are important for stabilization of the described G-quadruplex fold. Our results expand set of G-rich targets in viral genomes and address the fundamental questions regarding folding of G-rich sequences

Does standing up enhance performance on the stroop task in healthy young adults?

Understanding the changes in cognitive processing that accompany changes in posture can expand our understanding of embodied cognition and open new avenues for applications in (neuro)ergonomics. Recent studies have challenged the question of whether standing up alters cognitive performance. An electronic database search for randomized controlled trials was performed using Academic Search Complete, CINAHL Ultimate, MEDLINE, PubMed, and Web of Science following PRISMA guidelines, PICOS framework, and standard quality assessment criteria (SQAC). We pooled data from a total of 603 healthy young adults for incongruent and 578 for congruent stimuli and Stroop effect (mean age = 24 years). Using random-effects results, no difference was found between sitting and standing for the Stroop effect (Hedges’ g = 0.13, 95% CI = −0.04 to 0.29, p = 0.134), even when comparing congruent (Hedges’ g = 0.1095% CI: −0.132 to 0.339Z = 0.86p = 0.389) and incongruent (Hedges’ g = 0.1895% CI: −0.072 to 0.422Z = 1.39p = 0.164) stimuli separately. Importantly, these results imply that changing from a seated to a standing posture in healthy young adults is unlikely to have detrimental effects on selective attention and cognitive control. To gain a full understanding of this phenomenon, further research should examine this effect in a population of healthy older adults, as well as in a population with pathology

Stroop in motion

There is conflicting evidence about how interference control in healthy adults is affected by walking as compared to standing or sitting. Although the Stroop paradigm is one of the best-studied paradigms to investigate interference control, the neurodynamics associated with the Stroop task during walking have never been studied. We investigated three Stroop tasks using variants with increasing interference levels – word-reading, ink-naming, and the switching of the two tasks, combined in a systematic dual-tasking fashion with three motor conditions – sitting, standing, and treadmill walking. Neurodynamics underlying interference control were recorded using the electroencephalogram. Worsened performance was observed for the incongruent compared to congruent trials and for the switching Stroop compared to the other two variants. The early frontocentral event-related potentials (ERPs) associated with executive functions (P2, N2) differentially signaled posture-related workloads, while the later stages of information processing indexed faster interference suppression and response selection in walking compared to static conditions. The early P2 and N2 components as well as frontocentral Theta and parietal Alpha power were sensitive to increasing workloads on the motor and cognitive systems. The distinction between the type of load (motor and cognitive) became evident only in the later posterior ERP components in which the amplitude non-uniformly reflected the relative attentional demand of a task. Our data suggest that walking might facilitate selective attention and interference control in healthy adults. Existing interpretations of ERP components recorded in stationary settings should be considered with care as they might not be directly transferable to mobile settings

Fused in Liposarcoma Protein, a New Player in the Regulation of HIV-1 Transcription, Binds to Known and Newly Identified LTR G-Quadruplexes

[Image: see text] HIV-1 integrated long terminal repeat (LTR) promoter activity is modulated by folding of its G-rich region into non-canonical nucleic acids structures, such as G-quadruplexes (G4s), and their interaction with cellular proteins. Here, by a combined pull-down/mass spectrometry/Western-blot approach, we identified the fused in liposarcoma (FUS) protein and found it to preferentially bind and stabilize the least stable and bulged LTR G4, especially in the cell environment. The outcome of this interaction is the down-regulation of viral transcription, as assessed in a reporter assay with LTR G4 mutants in FUS-silencing conditions. These data indicate that the complexity and dynamics of HIV-1 LTR G4s are much greater than previously envisaged. The G-rich LTR region, with its diverse G4 landscape and multiple cell protein interactions, stands out as prime sensing center for the fine regulation of viral transcription. This region thus represents a rational antiviral target for inhibiting both the actively transcribing and latent viruses

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR.

Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods represent an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5' position, which allows 5'-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods

Human Virus Genomes Are Enriched in Conserved Adenine/Thymine/Uracil Multiple Tracts That Pause Polymerase Progression

The DNA secondary structures that deviate from the classic Watson and Crick base pairing are increasingly being reported to form transiently in the cell and regulate specific cellular mechanisms. Human viruses are cell parasites that have evolved mechanisms shared with the host cell to support their own replication and spreading. Contrary to human host cells, viruses display a diverse array of nucleic acid types, which include DNA or RNA in single-stranded or double-stranded conformations. This heterogeneity improves the possible occurrence of non-canonical nucleic acid structures. We have previously shown that human virus genomes are enriched in G-rich sequences that fold in four-stranded nucleic acid secondary structures, the G-quadruplexes.Here, by extensive bioinformatics analysis on all available genomes, we showed that human viruses are enriched in highly conserved multiple A (and T or U) tracts, with such an array that they could in principle form quadruplex structures. By circular dichroism, NMR, and Taq polymerase stop assays, we proved that, while A/T/U-quadruplexes do not form, these tracts still display biological significance, as they invariably trigger polymerase pausing within two bases from the A/T/U tract. "A" bases display the strongest effect. Most of the identified A-tracts are in the coding strand, both at the DNA and RNA levels, suggesting their possible relevance during viral translation. This study expands on the presence and mechanism of nucleic acid secondary structures in human viruses and provides a new direction for antiviral research