Goldilocks and RNA: where Mg2+ concentration is just right.

Magnesium, the most abundant divalent cation in cells, catalyzes RNA cleavage but also promotes RNA folding. Because folding can protect RNA from cleavage, we predicted a 'Goldilocks landscape', with local maximum in RNA lifetime at Mg2+ concentrations required for folding. Here, we use simulation and experiment to discover an innate and sophisticated mechanism of control of RNA lifetime.

G-Quadruplexes in Human Ribosomal RNA.

rRNA is the single most abundant polymer in most cells. Mammalian rRNAs are nearly twice as large as those of prokaryotes. Differences in rRNA size are due to expansion segments, which contain extended tentacles in metazoans.

Yeast rRNA Expansion Segments: Folding and Function.

Divergence between prokaryotic and eukaryotic ribosomal RNA (rRNA) and among eukaryotic ribosomal RNAs is focused in expansion segments (ESs). Eukaryotic ribosomes are significantly larger than prokaryotic ribosomes partly because of their ESs. We hypothesize that larger rRNAs of complex organisms could confer increased functionality to the ribosome.

Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution.

The interstitial liquid phase within frozen aqueous solutions is an environment that minimizes RNA degradation and facilitates reactions that may have relevance to the RNA World hypothesis. Previous work has shown that frozen solutions support condensation of activated nucleotides into RNA oligomers, RNA ligation by the hairpin ribozyme, and RNA synthesis by a RNA polymerase ribozyme. In the current study, we examined the activity of a hammerhead ribozyme (HHR) in frozen solution.

History of the ribosome and the origin of translation.

We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome.

Evolution of the ribosome at atomic resolution.

The origins and evolution of the ribosome, 3-4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be "observed" by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity.

Secondary structures of rRNAs from all three domains of life.

Accurate secondary structures are important for understanding ribosomes, which are extremely large and highly complex. Using 3D structures of ribosomes as input, we have revised and corrected traditional secondary (2°) structures of rRNAs. We identify helices by specific geometric and molecular interaction criteria, not by co-variation.

Secondary structure and domain architecture of the 23S and 5S rRNAs.

We present a de novo re-determination of the secondary (2°) structure and domain architecture of the 23S and 5S rRNAs, using 3D structures, determined by X-ray diffraction, as input. In the traditional 2° structure, the center of the 23S rRNA is an extended single strand, which in 3D is seen to be compact and double helical. Accurately assigning nucleotides to helices compels a revision of the 23S rRNA 2° structure.

RNA with iron(II) as a cofactor catalyses electron transfer.

Mg(2+) is essential for RNA folding and catalysis. However, for the first 1.5 billion years of life on Earth RNA inhabited an anoxic Earth with abundant and benign Fe(2+).

Molecular paleontology: a biochemical model of the ancestral ribosome.

Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments.

RNA folding and catalysis mediated by iron (II).

Mg²⁺ shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe²⁺ in the absence of free oxygen as a replacement for Mg²⁺ in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg²⁺ in RNA folding and function can indeed be served by Fe²⁺.

Domain III of the T. thermophilus 23S rRNA folds independently to a near-native state.

The three-dimensional structure of the ribosomal large subunit (LSU) reveals a single morphological element, although the 23S rRNA is contained in six secondary structure domains. Based upon maps of inter- and intra-domain interactions and proposed evolutionary pathways of development, we hypothesize that Domain III is a truly independent structural domain of the LSU. Domain III is primarily stabilized by intra-domain interactions, negligibly perturbed by inter-domain interactions, and is not penetrated by ribosomal proteins or other rRNA.

The influence of Escherichia coli Hfq mutations on RNA binding and sRNA•mRNA duplex formation in rpoS riboregulation.

The Escherichia coli RNA binding protein Hfq plays an important role in regulating mRNA translation through its interactions with small non-coding RNAs (sRNAs) and specific mRNAs sites. The rpoS mRNA, which codes for a transcription factor, is regulated by several sRNAs. DsrA and RprA enhance translation by pairing to a site on this mRNA, while OxyS represses rpoS mRNA translation.

E. coli DNA associated with isolated Hfq interacts with Hfq's distal surface and C-terminal domain.

The RNA-binding protein Hfq has been studied extensively for its function as a modulator of gene expression at the post-transcriptional level. While most Hfq studies have focused on the protein's interaction with sRNAs and mRNAs, Hfq binding to DNA has been observed but is less explored. During the isolation of Hfq from Escherichiacoli, we found genomic DNA fragments associated with the protein after multiple steps of purification.

Mechanism of RNA double helix-propagation at atomic resolution.

The conversion of a nucleic acid from single strands to double strands is thought to involve slow nucleation followed by fast double-strand propagation. Here, for RNA double-strand propagation, we propose an atomic resolution reaction mechanism. This mechanism, called the stack-ratchet, is based on data-mining of three-dimensional structures and on available thermodynamic information.

Effect of Hfq on RprA-rpoS mRNA pairing: Hfq-RNA binding and the influence of the 5' rpoS mRNA leader region.

The rpoS mRNA encodes a stress response transcription factor in Escherichia coli. It is one of a growing number of mRNAs found to be regulated by small RNAs (sRNA). Translation initiation of rpoS mRNA is enhanced by two sRNAs, DsrA and RprA, that pair to the same site near the rpoS start codon in the presence of the Hfq protein.

Evidence for stage-specific modulation of specific microRNAs (miRNAs) and miRNA processing components in zygotic embryo and female gametophyte of loblolly pine (Pinus taeda).

MicroRNAs (miRNAs) are known to regulate plant development, but have not been studied in gymnosperm seed tissues. The presence and characteristics of several miRNAs were examined in zygotic embryos (ZEs) and female gametophytes (FGs) of Pinus taeda (loblolly pine). Evidence for miRNAs was obtained using northern analyses and quantitative reverse transcription polymerase chain reaction (qRT-PCR) mediated with poly(A) polymerase.

Conversion of stable RNA hairpin to a metastable dimer in frozen solution.

Previous studies employing a 79-nucleotide (nt) RNA indicated that this RNA could form two bands in a native polyacrylamide gel while one band was observed in a denaturing gel. This report describes an investigation on the nature of the two corresponding structures and the segment responsible for forming the slower mobility band. Sedimentation equilibrium of the 79-nt RNA was consistent with the two gel bands corresponding to monomer and dimer forms.

Escherichia coli Hfq binds A18 and DsrA domain II with similar 2:1 Hfq6/RNA stoichiometry using different surface sites.

Hfq is a RNA-binding protein in Escherichia coli that plays an essential role in post-transcriptional regulation of mRNAs by facilitating pairing of noncoding RNAs (ncRNAs) to mRNA target sites. Recent work has provided evidence that E. coli Hfq has two distinct RNA-binding surfaces.

Thermodynamics of RNA duplexes with tandem mismatches containing a uracil-uracil pair flanked by C.G/G.C or G.C/A.U closing base pairs.

The thermodynamics governing the denaturation of RNA duplexes containing 8 bp and a central tandem mismatch or 10 bp were evaluated using UV absorbance melting curves. Each of the eight tandem mismatches that were examined had one U-U pair adjacent to another noncanonical base pair. They were examined in two different RNA duplex environments, one with the tandem mismatch closed by G.

Thermodynamic stability of DNA tandem mismatches.

The thermodynamics of nine hairpin DNAs were evaluated using UV-monitored melting curves and differential scanning calorimetry (DSC). Each DNA has the same five-base loop and a stem with 8-10 base pairs. Five of the DNAs have a tandem mismatch in the stem, while four have all base pairs.