Long-Residency Hydration, Cation Binding, and Dynamics of Loop E/Helix IV rRNA-L25 Protein Complex

Molecular dynamics simulations of RNA-protein complex between Escherichia coli loop E/helix IV (LE/HeIV) rRNA and L25 protein reveal a qualitative agreement between the experimental and simulated structures. The major groove of LE is a prominent rRNA cation-binding site. Divalent cations rigidify the LE major groove geometry whereas in the absence of divalent cations LE extensively interacts with monovalent cations via inner-shell binding. The HeIV region shows bistability of its major groove explaining the observed differences between x-ray and NMR structures. In agreement with the experiments, the simulations suggest that helix-alpha1 of L25 is the least stable part of the protein. Inclusion of Mg2+ cations into the simulations causes perturbation of basepairing at the LE/HeIV junction, which does not, however, affect the protein binding. The rRNA-protein complex is mediated by a number of highly specific hydration sites with long-residing water molecules and two of them are bound throughout the entire 24-ns simulation. Long-residing water molecules are seen also outside the RNA-protein contact areas with water-binding times substantially enhanced compared to simulations of free RNA. Long-residency hydration sites thus represent important elements of the three-dimensional structure of rRNA

Conserved Geometrical Base-Pairing Patterns in RNA

RNA molecules fold into a bewildering variety of complex 3D structures. Almost every new RNA structure obtained at high resolution reveals new, unanticipated structural motifs, which we are rarely able to predict at the current stage of our theoretical understanding. Even at the most basic level of specific RNA interactions – base-to-base pairing – new interactions continue to be uncovered as new structures appear. Compilations of possible non-canonical base-pairing geometries have been presented in previous reviews and monographs (Saenger, 1984; Tinoco, 1993). In these compilations, the guiding principle applied was the optimization of hydrogen-bonding. All possible pairs with two standard H-bonds were presented and these were organized according to symmetry or base type. However, many of the features of RNA base-pairing interactions that have been revealed by high-resolution crystallographic analysis could not have been anticipated and, therefore were not incorporated into these compilations. These will be described and classified in the present review. A recently presented approach for inferring basepair geometry from patterns of sequence variation (Gautheret & Gutell, 1997) relied on the 1984 compilation of basepairs (Saenger, 1984), and was extended to include all possible single H-bond combinations not subject to steric clashes. Another recent review may be consulted for a discussion of the NMR spectroscopy and thermodynamic effects of non-canonical (‘mismatched’) RNA basepairs on duplex stability (Limmer, 1997)

Cationic 5,10,15,20-tetrakis(n-methylpyridinium-4-yl)porphyrin Fully Intercalates At 5 \u27-cg-3 \u27 Steps Of Duplex Dna In Solution

The interaction of 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)p (T4MPyP(4+)) with the oligonucleotide DNA duplex [d(GCACGTGC)](2) was studied by two-dimensional (1)H NMR spectroscopy, optical absorbance, circular dichroism, and molecular dynamics simulation employing particle mesh Ewald methods. T4MPyP(4+) is one of the largest aromatic molecules for which intercalative binding to DNA has been proposed, although this has been called into question by recent X-ray crystallographic work [Lipscomb et al. (1996) Biochemistry 35, 2818-2823]. T4MPyP(4+) binding to [d(GCACGTGC)](2) produced a single set of (mostly) upfield-shifted DNA resonances in slow exchange with the resonances of the free DNA. Intra- and intermolecular NOEs observed in the complex showed that the porphyrin intercalates at the central 5\u27-CG-3\u27 step of the DNA duplex without disrupting the flanking base pairs. Absorption and circular dichroism spectra of the complex also support intercalative binding. Molecular dynamics simulations (using explicit solvent and PME methods), carried out for fully and partially intercalated complexes, yielded stable trajectories and plausible structures, but only the symmetrical, fully intercalated model agreed with NOESY data. Stable hydrogen bonding was observed during 600 ps of MD simulation for the base pairs flanking the binding site

Generating New Specific Rna Interaction Interfaces Using C-loops

New RNA interaction interfaces are reported for designing RNA modules for directional supramolecular self-assembly. The new interfaces are generated from existing ones by inserting C-loops between the interaction motifs that mediate supramolecular assembly. C-Loops are new modular motifs recently identified in crystal structures that increase the helical twist of RNA helices in which they are inserted and thus reduce the distance between pairs of loop or loop-receptor motifs from 11 to 9 base-stacking layers while maintaining correct orientation for binding to cognate interaction interfaces. Binding specificities of C-loop-containing molecules for cognate molecules that also have inserted C-loops were found to range up to 20-fold. Binding affinities for most C-loop-containing molecules were generally equal or higher than those for the parent molecules lacking C-loops

Lysogeny and Use of Mycobacteriophage Pita2

The CDC has classified antibiotic resistance as the biggest health challenge of our era; every year 2 million lives are impacted and even lost due to resistant bacteria. Bacteriophages provide an alternative route to fighting infections that does not further the development of antibiotic resistance among bacterial species. A bacteriophage replicates inside a bacterial cell and then causes that cell to lyse, an event that kills the bacterial host. However, some phage can integrate their genomes into the host chromosome without causing lysis. The HHMI SEA-PHAGES program has generated a collection of bacteriophage that infect Actinobacteria species. Over 13,000 phages have been collected thus far, but fewer than 3,000 have been sequenced and genetically analyzed. The purpose of research into the lysogeny of discovered, but unsequenced, bacteriophage is to classify them by immunity range; that is, closely related bacteriophage are unable to infect the lysogenic host, while more distantly related phage are. Initially, a lysogen had to be isolated. For Pita2, a phage isolated and analyzed at Bowling Green State University, the host is Mycobacterium smegmatis. A purified lysogen of Pita2 was analyzed against bacteriophage with known DNA sequences to confirm that it is immune to infection by closely related phage. The lysogeny was then examined for its ability to identify close vs. more distantly related phage among a set unknown bacteriophage. Another virus, namely SARS-CoV2, prevented the completion of this study. However, it was possible to generate preliminary data that established the ability to use immunity as a means to classify newly isolated phage based upon the degree to which they can successfully infect the Pita2 lysogen

Computational Simulation of the Docking of Prochlorothrix Hollandica Plastocyanin to Photosystem I: Modeling the Electron Transfer Complex

We have used several docking algorithms (GRAMM, FTDOCK, DOT, AUTODOCK) to examine protein-protein interactions between plastocyanin (Pc)/photosystem I (PSI) in the electron transfer reaction. Because of the large size and complexity of this system, it is faster and easier to use computer simulations than conduct x-ray crystallography or nuclear magnetic resonance experiments. The main criterion for complex selection was the distance between the copper ion of Pc and the P700 chlorophyll special pair. Additionally, the unique tyrosine residue (Tyr(12)) of the hydrophobic docking surface of Prochlorothrix hollandica Pc yields a specific interaction with the lumenal surface of PSI, thus providing the second constraint for the complex. The structure that corresponded best to our criteria was obtained by the GRAMM algorithm. In this structure, the solvent-exposed histidine that coordinates copper in Pc is at the van der Waals distance from the pair of stacked tryptophans that separate the chlorophylls from the solvent, yielding the shortest possible metal-to-metal distance. The unique tyrosine on the surface of the Prochlorothrix Pc hydrophobic patch also participates in a hydrogen bond with the conserved Asn(633) of the PSI PsaB polypeptide (numbering from the Synechococcus elongatus crystal structure). Free energy calculations for complex formation with wild-type Pc, as well as the hydrophobic patch Tyr(12)Gly and Pro(14)Leu Pc mutants, were carried out using a molecular mechanics Poisson-Boltzman, surface area approach (MM/PBSA). The results are in reasonable agreement with our experimental studies, suggesting that the obtained structure can serve as an adequate model for P. hollandica Pc-PSI complex that can be extended for the study of other cyanobacterial Pc/PSI reaction pairs

Assay of the Reverse Osmosis Purified Water in the Life Science Building at Bowling Green State University, Ohio

Contaminated water sources can cause problems for scientific research and result in costly delays and failures of experiments. At Bowling Green State University, the reverse osmosis supply circulating in the Life Sciences Building has been measurably contaminated for nearly three years, corresponding to a change in servicing of the system. While servicing has been accelerated, the contamination in the system remains. The focus of this research was to identify the species of bacteria and fungi growing inside of the water system so that it might alert those servicing the system, and to begin to eliminate the contamination. Reverse osmosis water samples were collected from various floors of the building and spread onto LB (Luria-Bertani) and MEA (Malt Extract) culture plates. Microbial growth was evident on 93% of the plates. Each separate colony was isolated, followed by DNA extraction and sequencing. Genus and species were determined and the information was presented to the firm that services the reverse osmosis system in order to determine a plan to decontaminate or replace the current plumbing system, and to determine the best way to assure the delivery of water that can be used in scientific research projects

Relative Stabilities of DNA 3-Way, 4-Way and 5-Way Junctions (Multi-Helix Junction Loops) - Unpaired Nucleotides Can Be Stabilizing or Destabilizing

Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled I[Wang,Y.L., Mueller,J.E., Kemper,B. and Seeman,N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions

Specific Rna Self-assembly With Minimal Paranemic Motifs

The paranemic crossover (PX) is a motif for assembling two nucleic acid molecules using Watson-Crick (WC) basepairing without unfolding preformed secondary structure in the individual molecules. Once formed, the paranemic assembly motif comprises adjacent parallel double helices that crossover at every possible point over the length of the motif. The interaction is reversible as it does not require denaturation of basepairs internal to each interacting molecular unit. Paranemic assembly has been demonstrated for DNA but not for RNA and only for motifs with four or more crossover points and lengths of five or more helical half-turns. Here we report the design of RNA molecules that paranemically assemble with the minimum number of two crossovers spanning the major groove to form paranemic motifs with a length of three half turns (3HT). Dissociation constants (K-d\u27s) were measured for a series of molecules in which the number of basepairs; between the crossover points was varied from five to eight basepairs. The paranemic 3HT complex with six basepairs (3HT_6M) was found to be the most stable with K-d = 1 x 10(-8) M. The half-time for kinetic exchange of the 3HT_6M complex was determined to be similar to 100 min, from which we calculated association and dissociation rate constants k(a) = 5.11 x 10(3) M(-1)s(-1) and k(d) = 5.11 x 10(-5) s(-1). RNA paranemic assembly of 3HT and 5HT complexes is blocked by single-base substitutions that disrupt individual intermolecular Watson-Crick basepairs; and is restored by compensatory substitutions that,restore those basepairs. The 3HT motif appears suitable for specific, programmable, and reversible tecto-RNA self-assembly for constructing artificial RNA molecular machines

The Annotation of RNA Motifs

The recent deluge of new RNA structures, including complete atomic-resolution views of both subunits of the ribosome, has on the one hand literally overwhelmed our individual abilities to comprehend the diversity of RNA structure, and on the other hand presented us with new opportunities for comprehensive use of RNA sequences for comparative genetic, evolutionary and phylogenetic studies. Two concepts are key to understanding RNA structure: hierarchical organization of global structure and isostericity of local interactions. Global structure changes extremely slowly, as it relies on conserved long-range tertiary interactions. Tertiary RNA–RNA and quaternary RNA–protein interactions are mediated by RNA motifs, defined as recurrent and ordered arrays of non-Watson–Crick base-pairs. A single RNA motif comprises a family of sequences, all of which can fold into the same three-dimensional structure and can mediate the same interaction(s). The chemistry and geometry of base pairing constrain the evolution of motifs in such a way that random mutations that occur within motifs are accepted or rejected insofar as they can mediate a similar ordered array of interactions. The steps involved in the analysis and annotation of RNA motifs in 3D structures are: (a) decomposition of each motif into non-Watson–Crick base-pairs; (b) geometric classification of each basepair; (c) identification of isosteric substitutions for each basepair by comparison to isostericity matrices; (d) alignment of homologous sequences using the isostericity matrices to identify corresponding positions in the crystal structure; (e) acceptance or rejection of the null hypothesis that the motif is conserved