Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot

The functions of RNA pseudoknots (PKs), which are minimal tertiary structural motifs and an integral part of several ribozymes and ribonucleoprotein complexes, are determined by their structure, stability and dynamics. Therefore, it is important to elucidate the general principles governing their thermodynamics/folding mechanisms. Here, we combine experiments and simulations to examine the folding/unfolding pathways of the VPK pseudoknot, a variant of the Mouse Mammary Tumor Virus (MMTV) PK involved in ribosomal frameshifting. Fluorescent nucleotide analogs (2-aminopurine and pyrrolocytidine) placed at different stem/loop positions in the PK, and laser temperature-jump approaches serve as local probes allowing us to monitor the order of assembly of VPK with two helices with different intrinsic stabilities. The experiments and molecular simulations show that at 50 mM KCl the dominant folding pathway populates only the more stable partially folded hairpin. As the salt concentration is increased a parallel folding pathway emerges, involving the less stable hairpin structure as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. The findings support the principle that the order of PK structure formation is determined by the relative stabilities of the hairpins, which can be altered by sequence variations or salt concentrations. Our study not only unambiguously demonstrates that PK folds by parallel pathways, but also establishes that quantitative description of RNA self-assembly requires a synergistic combination of experiments and simulations.Comment: Supporting Information include

Image Processing and Simulation Toolboxes of Microscopy Images of Bacterial Cells

Recent advances in microscopy imaging technology have allowed the characterization of the dynamics of cellular processes at the single-cell and single-molecule level. Particularly in bacterial cell studies, and using the E. coli as a case study, these techniques have been used to detect and track internal cell structures such as the Nucleoid and the Cell Wall and fluorescently tagged molecular aggregates such as FtsZ proteins, Min system proteins, inclusion bodies and all the different types of RNA molecules. These studies have been performed with using multi-modal, multi-process, time-lapse microscopy, producing both morphological and functional images. To facilitate the finding of relationships between cellular processes, from small-scale, such as gene expression, to large-scale, such as cell division, an image processing toolbox was implemented with several automatic and/or manual features such as, cell segmentation and tracking, intra-modal and intra-modal image registration, as well as the detection, counting and characterization of several cellular components. Two segmentation algorithms of cellular component were implemented, the first one based on the Gaussian Distribution and the second based on Thresholding and morphological structuring functions. These algorithms were used to perform the segmentation of Nucleoids and to identify the different stages of FtsZ Ring formation (allied with the use of machine learning algorithms), which allowed to understand how the temperature influences the physical properties of the Nucleoid and correlated those properties with the exclusion of protein aggregates from the center of the cell. Another study used the segmentation algorithms to study how the temperature affects the formation of the FtsZ Ring. The validation of the developed image processing methods and techniques has been based on benchmark databases manually produced and curated by experts. When dealing with thousands of cells and hundreds of images, these manually generated datasets can become the biggest cost in a research project. To expedite these studies in terms of time and lower the cost of the manual labour, an image simulation was implemented to generate realistic artificial images. The proposed image simulation toolbox can generate biologically inspired objects that mimic the spatial and temporal organization of bacterial cells and their processes, such as cell growth and division and cell motility, and cell morphology (shape, size and cluster organization). The image simulation toolbox was shown to be useful in the validation of three cell tracking algorithms: Simple Nearest-Neighbour, Nearest-Neighbour with Morphology and DBSCAN cluster identification algorithm. It was shown that the Simple Nearest-Neighbour still performed with great reliability when simulating objects with small velocities, while the other algorithms performed better for higher velocities and when there were larger clusters present

Aerospace medicine and biology: A continuing bibliography with indexes, supplement 125

This special bibliography lists 323 reports, articles, and other documents introduced into the NASA scientific and technical information system in January 1974

Probing the Assembly of the Ribosome: Insights from Computational Studies on Ribosomal Proteins

Ribosomes are complex cellular machines that synthesize new proteins in the cell. The accurate and efficient assembly of ribosomal proteins (r-proteins) and ribosomal RNA (rRNA) to form a functional ribosome is important for cell growth, metabolic reactions, and other cellular processes. Ribosomal assembly has been an active research topic for many years because understanding the assembly mechansims can provide insight into protein/RNA recognitions that are important in many other cellular processes, as well as help optimize the development of antibacterial therapeutics. Experimental and computational sutdies thus far have greatly improved our understanding of assembly, yet many questions remain unanswered regarding the complex behaviors of r-proteins and rRNA during the process. To further understand ribosome assembly, we have computationally studied the sequences, structures, and dynamic properties of r-proteins from the 30S subunit and their relationships to RNA binding. We discuss the statistically greater amount of positively charged residues in r-proteins compared to other housekeeping proteins and observe a high level of charged interactions between r-proteins and rRNA in the assembled structure. We also detect a significant correlation between the overall flexibility of a protein and the number of contact points it makes with its rRNA binding site. Protein residues contacting with rRNA are observed to be more mobile in solution when compared to the non-contacting residues. We also describe common modes of structural dynamics, revealing likely conformational changes the proteins make prior to binding, how they relate to possible binding mechanisms used during the assembly and to the location of the protein in the fully assembled ribosome

The 1st Symposium on Chemical Evolution and the Origin and Evolution of Life

This symposium provided an opportunity for all NASA Exobiology principal investigators to present their most recent research in a scientific meeting forum. Papers were presented in the following exobiology areas: extraterrestrial chemistry primitive earth, information transfer, solar system exploration, planetary protection, geological record, and early biological evolution