Calculation of pKas in RNA: on the structural origins and functional roles of protonated nucleotides

pK(a) calculations based on the Poisson-Boltzmann equation have been widely used to study proteins and, more recently, DNA. However, much less attention has been paid to the calculation of pK(a) shifts in RNA. There is accumulating evidence that protonated nucleotides can stabilize RNA structure and participate in enzyme catalysis within ribozymes. Here, we calculate the pK(a) shifts of nucleotides in RNA structures using numerical solutions to the Poisson-Boltzmann equation. We find that significant shifts are predicted for several nucleotides in two catalytic RNAs, the hairpin ribozyme and the hepatitis delta virus ribozyme, and that the shifts are likely to be related to their functions. We explore how different structural environments shift the pK(a)s of nucleotides from their solution values. RNA structures appear to use two basic strategies to shift pK(a)s: (a) the formation of compact structural motifs with structurally-conserved, electrostatic interactions; and (b) the arrangement of the phosphodiester backbone to focus negative electrostatic potential in specific regions.     

Protection from chemical modification of nucleotides in complexes of M1 RNA, the catalytic subunit of RNase P from E coli, and tRNA precursors.

Certain nucleotides in M1 RNA, the catalytic RNA subunit of RNase P from E coli, are protected from chemical modification when M1 RNA forms complexes with tRNA precursor molecules (ES complexes). Many of these nucleotides are important in the formation of the Michaelis complex. In the presence of tRNA precursor molecules, the pattern of protection from chemical modification of a region in M1 RNA that resembles the E site in 23S rRNA is similar to the pattern of protection of the E site in the presence of deacylated tRNA. In the complex with the RNA enzyme, more nucleotides in the substrate become accessible to modification, an indication that the substrate is in an unfolded conformation under these conditions.     

Structure of a protein L23-RNA complex located at the A-site domain of the ribosomal peptidyl transferase centre

Protein L23 from the ribosome of Escherichia coli is the primary ribosomal product cross-linked to affinity-labelled puromycin; it lies, therefore, within the A-site domain of the peptidyl transferase centre on the 50 S subunit. We have characterized this functional domain by isolating and sequencing the RNA binding site of protein L23; it consists of two main fragments of 25 and 105 nucleotides that strongly interact and are separated by 172 nucleotides in the primary sequence. The higher-order structure of the RNA moiety was probed by chemical reagents, and by single-strand and double-strand-specific ribonucleases; a secondary structural model and a tertiary structural interaction are proposed on the basis of these data that are compatible with phylogenetic sequence comparisons.Several nucleotides exhibited altered chemical reactivity, both lower and higher, in the presence of protein L23, thereby implicating a large proportion of the RNA structure in the protein binding. The sites were located mainly at the extremities of the helices and at nucleotides that were putatively bulged out from the helices.The RNA moiety and an adjacent excised fragment contain several highly conserved sequences and a modified adenosine. Such sequences constitute important functional domains of the RNA and may contribute to the putative role of this RNA region in the peptidyl transferase centre.     

Is Boron a Prebiotic Element? A Mini-review of the Essentiality of Boron for the Appearance of Life on Earth

Boron is probably a prebiotic element with special importance in the so-called "sugars world". Boron is not present on Earth in its elemental form. It is found only in compounds, e.g., borax, boric acid, kernite, ulexite, colemanite and other borates. Volcanic spring waters sometimes contain boron-based acids (e.g., boric, metaboric, tetraboric and pyroboric acid). Borates influence the formation of ribofuranose from formaldehyde that feeds the "prebiotic metabolic cycle". The importance of boron in the living world is strongly related to its implications in the prebiotic origins of genetic material; consequently, we believe that throughout the evolution of life, the primary role of boron has been to provide thermal and chemical stability in hostile environments. The complexation of boric acid and borates with organic cis-diols remains the most probable chemical mechanism for the role of this element in the evolution of the living world. Because borates can stabilize ribose and form borate ester nucleotides, boron may have provided an essential contribution to the "pre-RNA world".     

RNA-ligand chemistry: a testable source for the genetic code

In the genetic code, triplet codons and amino acids can be shown to be related by chemical principles. Such chemical regularities could be created either during the code's origin or during later evolution. One such chemical principle can now be shown experimentally. Natural or particularly selected RNA binding sites for at least three disparate amino acids (arginine, isoleucine, and tyrosine) are enriched in codons for the cognate amino acid. Currently, in 517 total nucleotides, binding sites contain 2.4-fold more codon sequences than surrounding nucleotides. The aggregate probability of this enrichment is 10(-7) to 10(-8), had codons and binding site sequences been independent. Thus, at least some primordial coding assignments appear to have exploited triplets from amino acid binding sites as codons.     

Does 5S RNA from E. coli have a pseudoknotted structure?

Chemical modification and limited enzymatic hydrolysis on isolated E. coli 5S RNA have provided informations on the secondary- and tertiary structure compatible with pseudoknotted structures for the A- and B-conformers of the molecule. Changes in the accessibility and reactivity of nucleotides in loop C and at the stem of helix IV in two different 5S RNA conformers are highly suggestive for interactions between bases C35 to C37 with G105 to G107 for the A-form and C38 to U40 and A94 to G96 with additional interactions of C35, C37 with G98 and G100 for the B-form. In both cases the molecules are folded forming pseudoknots and two quasi--continuous double stranded helices with coaxial stacking. The two structures are in perfect agreement with the biochemical data concerning the stability of the molecule and the chemical reactivities of individual nucleotides of the 5S RNA A- and B-conformers.     

An additional dimer linkage structure in Moloney murine leukemia virus RNA.

We have identified an additional dimerization linkage structure in the genome of Moloney murine leukemia virus (MoMLV). Retroviral genomes have long been known to be linked at their 5' ends to form dimers. In MoMLV, a hairpin loop functioning as a dimer linkage structure (DLS) has previously been identified at nucleotides 278-303. Here, we describe RNA dimers formed from sections of the MoMLV 5' untranslated region that do not contain the previously described MoMLV DLS. These dimers exhibit the distinctive characteristics previously described for whole genome dimers. We have mapped this novel region to nucleotides 199-243. This sequence contains a stem-loop structure (nucleotides 204-227) much like the 278-303 region. We describe the chemical and thermal stability of dimers containing the 204-227 stem-loop as well as kinetics and salt-dependence of dimer formation. Our results show that dimerization of MoMLV RNA can be nucleated at multiple sites and suggest that the 5' untranslated region may contain separately folding and dimerizing domains.     

Formation of low molecular weight RNA species in HeLa cells

It has been previously shown that newly synthesized nuclear low molecular weight RNA species C and D are first detected in the cytoplasm for a few minutes before they are finally found in the nucleus. The following are some of the observations made in the present study, regarding the formation of C and D RNA: (1) The 5′ end cap ribose methylation of the C RNA precursor is complete in its cytoplasmic stage; the internal ribose methylation of the precursor seems to be completed about the time of its apparent transition from cytoplasm to nucleus. (2) The few nucleotides lost from the D RNA precursor during maturation seem to be excised sometime near its apparent cytoplasmic → nuclear transition. Newly synthesized C RNA also appears to lose some of its non-conserved nucleotides about the time of that transition, while the other extra nucleotides are lost later, in the nucleus. (3) The maturation of C and D RNA is inhibited early during suppression of protein synthesis by cycloheximide, while their synthesis is not. (4) The cytoplasmic precursors of C and D RNA are not associated with ribonucleoprotein particles as large as those reported for mature C and D RNA, although they do not appear to be free in the cytoplasm. (5) When the cellular UTP pool is depleted by exposure of the cells to amino sugars, and the synthesis of C, D, and other RNA species decreases, the level of[³H]uridine labeling of C and D RNA increases, while that of 4 S and 5 S RNA does not. These data are compatible with the existence of more than one nuclear UTP pool.     

Prebiotic chemistry: a new modus operandi

A variety of macromolecules and small molecules-(oligo)nucleotides, proteins, lipids and metabolites-are collectively considered essential to early life. However, previous schemes for the origin of life-e.g. the 'RNA world' hypothesis-have tended to assume the initial emergence of life based on one such molecular class followed by the sequential addition of the others, rather than the emergence of life based on a mixture of all the classes of molecules. This view is in part due to the perceived implausibility of multi-component reaction chemistry producing such a mixture. The concept of systems chemistry challenges such preconceptions by suggesting the possibility of molecular synergism in complex mixtures. If a systems chemistry method to make mixtures of all the classes of molecules considered essential for early life were to be discovered, the significant conceptual difficulties associated with pure RNA, protein, lipid or metabolism 'worlds' would be alleviated. Knowledge of the geochemical conditions conducive to the chemical origins of life is crucial, but cannot be inferred from a planetary sciences approach alone. Instead, insights from the organic reactivity of analytically accessible chemical subsystems can inform the search for the relevant geochemical conditions. If the common set of conditions under which these subsystems work productively, and compatibly, matches plausible geochemistry, an origins of life scenario can be inferred. Using chemical clues from multiple subsystems in this way is akin to triangulation, and constitutes a novel approach to discover the circumstances surrounding the transition from chemistry to biology. Here, we exemplify this strategy by finding common conditions under which chemical subsystems generate nucleotides and lipids in a compatible and potentially synergistic way. The conditions hint at a post-meteoritic impact origin of life scenario.     

Nucleotide Selectivity in Abiotic RNA Polymerization Reactions

In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.