"Acceptor-donor-acceptor" motifs recognize the Watson-Crick, Hoogsteen and Sugar "donor-acceptor-donor" edges of adenine and adenosine-containing ligands

Nucleotides are among the most extensively exploited chemical moieties in nature and, as a part of a handful of different protein ligands, nucleotides play key roles in energy transduction, enzymatic catalysis and regulation of protein function. We have previously reported that in many proteins with different folds and functions a distinctive adenine-binding motif is involved in the recognition of the Watson-Crick edge of adenine. Here, we show that many proteins do have clear structural motifs that recognize adenosine (and some other nucleotides and nucleotide analogs) not only through the Watson-Crick edge, but also through the sugar and Hoogsteen edges. Each of the three edges of adenosine has a donor-acceptor-donor (DAD) pattern that is often recognized by proteins via a complementary acceptor-donor-acceptor (ADA) motif, whereby three distinct hydrogen bonds are formed: two conventional N-H...O and N-H...N hydrogen bonds, and one weak C-H...O hydrogen bond. The local conformation of the adenine-binding loop is betabetabeta or betabetaalpha and reflects the mode of nucleotide binding. Additionally, we report 21 proteins from five different folds that simultaneously recognize both the sugar edge and the Watson-Crick edge of adenine. In these proteins a unique beta-loop-beta supersecondary structure grasps an adenine-containing ligand between two identical adenine-binding motifs as part of the betaalphabeta-loop-beta fold.     

Importance of conserved residues for the conformation of the T-loop in tRNAs

The conformation of the T-loop of yeast tRNA(Asp) was studied by structural mapping techniques using chemical and enzymatic probes and by three-dimensional graphics modeling with the known crystallographic structures of tRNAs as references. The structural importance of C61 (conserved in the T-stem of all tRNAs) for the loop conformation was directly checked by ethylnitrosourea phosphate alkylation, either on the 3'-half tRNAAsp molecule or on a variant in which C61 was replaced by U61. The reactivity of P60 against ethylnitrosourea alkylation in the variant emphasizes the role of the hydrogen bond between this phosphate and position N4 of C61 for stabilizing the conformation of the T-loop. Experiments on several tRNA variants, containing C61 but altered in the sequence or in the length of the T-loop, indicate that other structural features help to stabilize the hydrogen bond network around P60. Evidence is presented that the reverse Hoogsteen base pair T54-A58 contributes to this stabilization by maintaining the hydrogen bonding between the 2'OH of ribose 58 and P60. Using graphics modeling and based on the chemical data. T-loops of several variants were constructed. It appears that both the constant length of the T-loop and the presence of psi 55 are crucial for the correct interaction between the T- and D-loops. The conclusion of this study is that the T-loop in tRNA possesses an intrinsic conformation (mainly governed by the constant residues) existing primarily without the structural context of the entire tRNA molecule.     

Isolation of novel tRNA(Ala) mutants by library selection in a tRNA(Ala) knockout strain

The relationship between tRNA structure and function has been widely investigated by site-directed mutagenesis. This method has been a very useful tool to reveal the critical bases in tRNAs that are important for recognition and aminoacylation, but has been limited by the large number of possible base combinations in tRNA molecules. We have devised a new method that uses tRNA knockout cells for selection of functional tRNAs from a mutant tRNA gene library to overcome this limitation. To explore the mechanism of tRNA(Ala) recognition, the bases of the acceptor-stem region were randomized and active mutants were selected in a tRNA(Ala) knockout strain. Mutants of tRNA(Ala) having diverse sequence combinations in the acceptor-stem region and a broad range of functional activity to support knockout cell growth were isolated. The mutant tRNAs selected by the method included molecules containing novel base substitutions as well as extensively altered base combinations that would not be readily generated by rationally designed site-directed mutagenesis. Our results emphasize the importance of the acceptor stem as a structural unit in which some nucleotides may carry more weight than others, but in summation every nucleotide contributes to the interaction with the enzyme.     

Analysis of tRNA abstract shapes of precursor/derivative amino acids in Archaea

Wong's theory of the genetic code's origin states that because of historical constraints, codon assignment depends on the relation between precursor and derivative amino acids, a result of the coevolutionary process between amino acids' biosynthetic pathways and tRNAs. Based on arguments supporting the assumption that natural selection favors more stable and thus functionally constrained structures, we tested whether precursor and derivative tRNAs are equally evolved by measuring their structural parameters, thermostability and molecular plasticity. We also estimated the extent to which precursor and derivative tRNAs differ within Archaea. We used Archaea sequences of both precursor and derivative tRNAs in order to examine the plastic repertoires or sets of suboptimal structures at a defined free energy interval. We grouped secondary structures according to their helix nesting and adjacency using abstract shapes analysis. This clustering enabled us to infer a consensus sequence for all shapes that fit the clover leaf secondary structure [Giegerich, R., et al., Nucleic Acids Res 2004; 32 (16): 4843-51.]. This consensus sequence was then folded in order to retrieve a set of suboptimal structures. For each pair of precursor and derivative tRNAs, we compared these plastic repertoires based on the number of secondary structures, the thermostability of the minimum free energy structure and two structural parameters (base pair propensity (P) and mean length of helical stem structures (S)), which were measured for every representative secondary structure [Schultes, E.A., et al., J Mol Evol 1999; 49 (1): 76-83.]. We found that derivative tRNAs have fewer numbers of shapes, higher thermostability and more stable parameters than precursor tRNAs, a fact in full agreement with Wong's coevolution theory of the genetic code.     

Nucleotide sequence of the mitochondrial structural genes for cysteine-tRNA and histidine-tRNA of yeast.

We have determined the nucleotide sequence of a segment of Saccharomyces cerevisiae mtDNA that contains the structural genes for a cysteine-tRNA and a histidine-tRNA. The genes are approximately 85 bp apart, they do not contain intervening sequences or sequences coding for the 3'-CCA terminus and they are surrounded by nearly pure AT segments. The tRNAs deduced are very AT-rich, 74 and 75 nucleotides long, respectively, and contain one or more unusual features not found in tRNAs from other sources.     

Why do water molecules around small hydrophobic solutes form stronger hydrogen bonds than in the bulk?

Molecular solutes are known to have a strong effect on the structural and dynamical properties of the surrounding water. In our recent study (PNAS, 114, 322 (2017)) we have identified the presence of strengthened water hydrogen bonds near hydrophobic solutes by using both IR spectroscopy and ab-initio molecular dynamics simulations. The water molecules involved in the enhanced hydrogen bonding have been shown to display extensive structural ordering and restricted mobility. We observed that an individual pair of water molecules can make stronger hydrogen bond to each other if it is not surrounded by intercalating water molecules. Here we present compelling simulation results which unravel a simple mechanistic picture of the emergence of the hydrogen bond (HB) strengthening around solvated methane. We show explicitly that actual absence of water molecules within the excluded volume due to the hydrophobic molecule assures smaller residual torque on neighboring water molecules enabling the formation of stronger HBs between them.     

Analysis of conformational parameters in nucleic acid fragments. I. Single crystals of nucleosides and nucleotides

A study has been undertaken of conformational parameters in single crystal structures of nucleosides and nucleotides using the techniques of helical conformational analysis. A "quasi-helix" was generated from the geometry of base-paired structures, using published data extracted from the Cambridge structural database. A total of 54 base-pairs were found in these structures, for each of which were calculated hydrogen bond parameters, propeller twist, buckle and C1'-C1' separations. These were analysed according to various classifications. Propeller twists are found to show a wide range of values and the magnitude of twist appears to be unrelated to hydrogen bond parameters or C1'-C1' separation. The values of the buckle parameter vary over a smaller range of values and are unrelated to propeller twist magnitude. There is found to be a greater tendency to form homo-base-pairs among compounds containing adenine bases.     

Dynamic behavior of the telomerase RNA hairpin structure and its relationship to dyskeratosis congenita

In this paper, we present the results from a comprehensive study of nanosecond-scale implicit and explicit solvent molecular dynamics simulations of the wild-type telomerase RNA hairpin. The effects of various mutations on telomerase RNA dynamics are also investigated. Overall, we found that the human telomerase hairpin is a very flexible molecule. In particular, periodically the molecule exhibits dramatic structural fluctuations represented by the opening and closing of a non-canonical base-pair region. These structural deviations correspond to significant disruptions of the direct hydrogen bonding network in the helix, widening of the major groove of the hairpin structure, and causing several U and C nucleotides to protrude into the major groove from the helix permitting them to hydrogen bond with, for example, the P3 domain of the telomerase RNA. We suggest that these structural fluctuations expose a nucleation point for pseudoknot formation. We also found that mutations in the pentaloop and non-canonical region stabilize the hairpin. Moreover, our results show that the hairpin with dyskeratosis congenita mutations is more stable and less flexible than the wild-type hairpin due to base stacking in the pentaloop. The results from our molecular dynamics simulations are in agreement with experimental observations. In addition, they suggest a possible mechanism for pseudoknot formation based on the dynamics of the hairpin structure and also may explain the mutational aspects of dyskeratosis congenita.     

Nucleotide correlations that suggest tertiary interactions in the TV-replacement loop-containing mitochondrial tRNAs of the nematodes, Caenorhabditis elegans and Ascaris suum.

In the predicted secondary structures of 20 of the 22 tRNAs encoded in mitochondrial DNA (mtDNA) molecules of the nematodes, Caenorhabditis elegans and Ascaris suum, the T psi C arm and variable loop are replaced with a loop of 6 to 12 nucleotides: the TV-replacement loop. From considerations of patterns of nucleotide correlations in the central regions of these tRNAs, it seems highly likely that tertiary interactions occur within five sets of binary and ternary combinations of nucleotides that correspond in location to nucleotides known to be involved in tertiary interactions in yeast tRNA(Phe) and other standard tRNAs. These observations are consistent with the nematode TV-replacement loop-containing mt-tRNAs being folded into a similar L-shaped functional form to that demonstrated for standard tRNAs, and for the bovine DHU (dihydrouridine) arm replacement-loop-containing mt-tRNA(Ser(AGY)). However, the apparent occurrence in nematode mt-tRNAs of tertiary bonds common to standard tRNAs contrasts with the situation in bovine mt-tRNA(Ser(AGY)) where the functional form is dependent on an almost unique set of tertiary interactions. Because three of the proposed conserved tertiary interactions in the nematode mt-tRNAs involve nucleotides that occur in the variable loop in standard tRNAs, it seems more likely that in nematode mt-tRNAs it is the T psi C arm rather than the variable loop that has undergone the greatest proportional decrease in nucleotide number.     

Cytosolic yeast tRNA(His) is covalently modified when imported into mitochondria of Trypanosoma brucei.

The mitochondrial genome of Trypanosoma brucei does not encode any tRNAs. Instead, mitochondrial tRNAs are synthesized in the nucleus and subsequently imported into mitochondria. The great majority of mitochondrial tRNAs have cytosolic counterparts showing identical primary sequences. The only difference found between mitochondrial and cytosolic isotypes of the tRNAs are mitochondria-specific nucleotide modifications which appear to be a common feature of imported tRNAs in trypanosomes. In this study, a mutated yeast cytosolic tRNAHis was expressed in trypanosomes and its import phenotype was analyzed by cell fractionation and nuclease treatment of intact mitochondria. Furthermore, cytosolic and mitochondrial isotypes of the yeast tRNA(His) were specifically labeled and analyzed by limited alkaline hydrolysis. These experiments revealed the presence of mitochondria-specific nucleotide modifications in the yeast tRNA(His). The positions of the modifications were determined by direct enzymatic sequencing of the tRNA(His) and shown to correspond to the ultimate and penultimate nucleotides before the anticodon, the same relative positions which are modified in the mitochondrial isotype of trypanosomal tRNA(Tyr). The results demonstrate that covalent modification of tRNAs; in trypanosomal mitochondria can be used, in analogy to processing of precursor proteins during mitochondrial protein import, as a marker for import of both endogenous and heterologous tRNAs.     

Discovering the interaction propensities of amino acids and nucleotides from protein-RNA complexes

With the availability of many genome sequences, the mining of biological data is attracting much attention, most of it limited to the sequences of macromolecules. Sequence data are easy to analyze as they can be treated as strings of characters, whereas the structure of a macromolecule is much more complex. We developed a set of algorithms to analyze the structures of protein-RNA complexes at the atomic level and used them to analyze protein-RNA interactions using structural data on 51 protein-RNA complexes. The analysis revealed, among other things, that: (1) polar and charged amino acids have a strong tendency to interact with nucleotides, (2) arginine and asparagine tend to hydrogen bond with uracil, and (3) histidine favors uracil in water-mediated bonding with RNA. We analyzed a large set of structural data of protein-RNA complexes involving water-mediated hydrogen bonds as well as direct hydrogen bonds. The interaction patterns discovered from the analysis provide useful information for predicting the structure of RNA that binds proteins, and of proteins that bind RNA.     

DSSPcont: Continuous secondary structure assignments for proteins

The DSSP program automatically assigns the secondary structure for each residue from the three-dimensional co-ordinates of a protein structure to one of eight states. However, discrete assignments are incomplete in that they cannot capture the continuum of thermal fluctuations. Therefore, DSSPcont (http://cubic.bioc.columbia.edu/services/DSSPcont) introduces a continuous assignment of secondary structure that replaces 'static' by 'dynamic' states. Technically, the continuum results from calculating weighted averages over 10 discrete DSSP assignments with different hydrogen bond thresholds. A DSSPcont assignment for a particular residue is a percentage likelihood of eight secondary structure states, derived from a weighted average of the ten DSSP assignments. The continuous assignments have two important features: (i) they reflect the structural variations due to thermal fluctuations as detected by NMR spectroscopy; and (ii) they reproduce the structural variation between many NMR models from one single model. Therefore, functionally important variation can be extracted from a single X-ray structure using the continuous assignment procedure.     

Ribosomal elongation cycle: energetic, kinetic and stereochemical aspects

As a preface to an analysis of the ribosomal elongation cycle, we examine the energetics of macromolecular structural transformations. We show that the kinetic barriers and changes of the energetic levels during these transformations are essentially determined by disruption of hydrogen and cation-ligand bonds, and by uncompensated losses of these bonds (ULBs). The disruption of a hydrogen or cation-ligand bond increases the heights of kinetic barriers by the energy of these bonds. The association and dissociation of macromolecules, and conformational transitions within macromolecules, can change the numbers of ULBs but cannot completely eliminate them. Two important general conclusions are drawn from this analysis. First, occupation of enzyme active centers by substrates should be accompanied by a reduction in the number of ULBs. This reduction decreases the activation barriers in enzyme reactions, and is a major contributor to catalysis. Second, the enzymic reactions of the ribosomal cycle (structural changes caused by transpeptidation and by GTP hydrolyses in EF-Tu and EF-G) disrupt kinetic traps that prevent tRNAs from dissociating into solution during their motion within the ribosome and are necessary for progression of the cycle. These results are general purpose structural-functional blocks for building a molecular model of the ribosomal elongation cycle. Here, we demonstrate the utility of these blocks for analysis of acceptance of cognate tRNAs into the ribosomal elongation cycle.     

Site-directed in vitro replacement of nucleosides in the anticodon loop of tRNA: application to the study of structural requirements for queuine insertase activity.

We have investigated the specificity of the enzymes Q-insertase and mannosyl-Q transferase that replace the guanosine at position 34 (wobble base) in the anticodon of several tRNAs by Q or mannosyl-Q derivatives. We have restructured in vitro the normal anticodon of yeast tRNA-Asp-GUC, yeast tRNAArgICG and yeast tRNALeuUAG. With yeast tRNA-Asp-GUC, we have replaced one or several nucleotides in the vicinity of G34 by one of the four canonical nucleotides or by pseudouridylic acid; we have also constructed a tRNAAsp with eight bases instead of seven in the anticodon loop. With yeast tRNAArgICG and yeast tRNALeuUAG, we have replaced their anticodon by the trinucleotide GUC, coding for aspartic acid. The chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and after 72 h the amount of Q34 and mannosyl-Q34 incorporated was measured. Our results show that the U33G34U35 sequence, within an anticodon loop of seven bases in chimerical yeast tRNA-Asp-GUC, tRNAArgGUC or tRNALeuGUC, is the main determinant for Q-insertase activity at position 34; the rest of the tRNA sequence has only a slight influence. For mannosyl-Q transferase, however, a much broader structural feature of the tRNA than just the U33G34U35 sequence is important for the efficiency of Q34 transformation into mannosyl-Q34.     

Search for characteristic structural features of mammalian mitochondrial tRNAs

A number of mitochondrial (mt) tRNAs have strong structural deviations from the classical tRNA cloverleaf secondary structure and from the conventional L-shaped tertiary structure. As a consequence, there is a general trend to consider all mitochondrial tRNAs as "bizarre" tRNAs. Here, a large sequence comparison of the 22 tRNA genes within 31 fully sequenced mammalian mt genomes has been performed to define the structural characteristics of this specific group of tRNAs. Vertical alignments define the degree of conservation/variability of primary sequences and secondary structures and search for potential tertiary interactions within each of the 22 families. Further horizontal alignments ascertain that, with the exception of serine-specific tRNAs, mammalian mt tRNAs do fold into cloverleaf structures with mostly classical features. However, deviations exist and concern large variations in size of the D- and T-loops. The predominant absence of the conserved nucleotides G18G19 and T54T55C56, respectively in these loops, suggests that classical tertiary interactions between both domains do not take place. Classification of the tRNA sequences according to their genomic origin (G-rich or G-poor DNA strand) highlight specific features such as richness/poorness in mismatches or G-T pairs in stems and extremely low G-content or C-content in the D- and T-loops. The resulting 22 "typical" mammalian mitochondrial sequences built up a phylogenetic basis for experimental structural and functional investigations. Moreover, they are expected to help in the evaluation of the possible impacts of those point mutations detected in human mitochondrial tRNA genes and correlated with pathologies.     

Stable tRNA precursors in HeLa cells.

Two tRNA precursors were isolated from 32P-labeled or unlabeled HeLa cells by two dimensional polyacrylamide gel electrophoresis, and were sequenced. These were the precursors of tRNAMet and tRNALeu, and both contained four extra nucleotides including 5'-triphosphates at their 5'-end and nine extra nucleotides including oligo U at their 3'-end. These RNAs are the first naturally occurring tRNA precursors from higher eukaryotes whose sequences have been determined. In these molecules, several modified nucleosides such as m2G, t6A and ac4C in mature tRNAs were undermodified. Two additional hydrogen bonds were formed in the clover leaf structures of these tRNA precursors. These extra hydrogen bonds may be responsible for the stabilities of these tRNA precursors.     

Bioinformatics analysis of plant orthologous introns: identification of an intronic tRNA-like sequence

Orthologous introns have identical positions relative to the coding sequence in orthologous genes of different species. By analyzing the complete genomes of five plants we generated a database of 40,512 orthologous intron groups of dicotyledonous plants, 28,519 orthologous intron groups of angiosperms, and 15,726 of land plants (moss and angiosperms). Multiple sequence alignments of each orthologous intron group were obtained using the Mafft algorithm. The number of conserved regions in plant introns appeared to be hundreds of times fewer than that in mammals or vertebrates. Approximately three quarters of conserved intronic regions among angiosperms and dicots, in particular, correspond to alternatively-spliced exonic sequences. We registered only a handful of conserved intronic ncRNAs of flowering plants. However, the most evolutionarily conserved intronic region, which is ubiquitous for all plants examined in this study, including moss, possessed multiple structural features of tRNAs, which caused us to classify it as a putative tRNA-like ncRNA. Intronic sequences encoding tRNA-like structures are not unique to plants. Bioinformatics examination of the presence of tRNA inside introns revealed an unusually long-term association of four glycine tRNAs inside the Vac14 gene of fish, amniotes, and mammals.     

Interactions of caffeine with DNA double helix fragments. Molecular mechanics simulation

Calculations of the energy of interaction between the caffeine molecule and DNA double helix fragment of four complementary pairs have been performed by the molecular mechanics method. The calculations demonstrate the existence of energy minima corresponding to the caffeine molecule position in both wide and narrow grooves. Each of three proton acceptor atoms of caffeine is able to form hydrogen bond with each of three amino groups of DNA bases. The interactions of caffeine with both hydrogen bonded nucleotide and other nucleotides of the two strands contribute considerably to the total energy. The substantial contribution of interactions of caffeine with other than H-bonded nucleotides results in a rather close packing of atom groups in possible DNA-caffeine complexes. The mechanisms of influence of caffeine on interactions of DNA with other biologically active compounds are discussed.     

Building a stable RNA U-turn with a protonated cytidine

The U-turn is a classical three-dimensional RNA folding motif first identified in the anticodon and T-loops of tRNAs. It also occurs frequently as a building block in other functional RNA structures in many different sequence and structural contexts. U-turns induce sharp changes in the direction of the RNA backbone and often conform to the 3-nt consensus sequence 5′-UNR-3′ (N = any nucleotide, R = purine). The canonical U-turn motif is stabilized by a hydrogen bond between the N3 imino group of the U residue and the 3′ phosphate group of the R residue as well as a hydrogen bond between the 2′-hydroxyl group of the uridine and the N7 nitrogen of the R residue. Here, we demonstrate that a protonated cytidine can functionally and structurally replace the uridine at the first position of the canonical U-turn motif in the apical loop of the neomycin riboswitch. Using NMR spectroscopy, we directly show that the N3 imino group of the protonated cytidine forms a hydrogen bond with the backbone phosphate 3′ from the third nucleotide of the U-turn analogously to the imino group of the uridine in the canonical motif. In addition, we compare the stability of the hydrogen bonds in the mutant U-turn motif to the wild type and describe the NMR signature of the C+-phosphate interaction. Our results have implications for the prediction of RNA structural motifs and suggest simple approaches for the experimental identification of hydrogen bonds between protonated C-imino groups and the phosphate backbone.