David M J Lilley

..We can identify two classes of k-turns, that differ in the manner of the hydrogen bonding at the adenine of the bulge-distal G•A pair...

..Guanine nucleobases appear to be a common choice of general base, but the general acid is more variable. By contrast, the larger ribozymes such as the self-splicing introns and RNase P act as metalloenzymes...

..The dependence of the hairpin ribozyme reaction rates on pH has been studied. Both cleavage and ligation reactions display a bell-shaped pH dependence, consistent with general acid-base catalysis involving the nucleotide at position 8...

..The hairpin ribozyme is probably closely similar, while the remaining nucleolytic ribozymes provide some interesting contrasts...

..Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes...

..Ultimately, the RNA world would probably have given way to protein-based life quite quickly, and the origins of peptidyl transferase activity are discussed below...

..Recent success in obtaining X-ray crystal structures of resolving enzymes bound to DNA junctions has revealed how the structural selectivity of these enzymes is achieved...

..Lastly, important new folding-enhancing elements have been discovered...

..However, in all such cases the affinity is lowered, and the results underline the importance of both G.A pairs to the stability of the K-turn...

..These interactions are generally more polymorphic, and hydrogen bonds probably form where possible, as permitted by the global structure...

..Our data show that the form of the complex between endonuclease I and a DNA junction depends on the core of the junction and on interactions with the first six base-pairs of the arms containing the 5' ends of the continuous strands...

..7 +/- 0.1 and 7.7 +/- 0.1 for cleavage and 6.1 +/- 0.3 and 6.9 +/- 0.3 for ligation. The data provide good evidence for general acid-base catalysis by the nucleobases...

..The A730 loop is the probable active site of the ribozyme, and A756 within it is a strong candidate to play a direct role in the transesterification chemistry, possibly by general acid-base catalysis...

..This is closely similar to the probable mechanism of the hairpin ribozyme, and the active site arrangements for the two ribozymes appear topologically equivalent. This has probably arisen by convergent evolution...

..This result is in close agreement with our FRET data, supporting the contention that FRET data can be used to provide orientational information...

..Our data unequivocally establish that Förster transfer obeys the orientation dependence as expected for a dipole-dipole interaction...

..We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2'-O nucleophile by G638 and protonation of the 5'-O leaving group by A756...

..This shows how the enzyme is selective for the structure of a four-way junction, such that both continuous strands can be accommodated into the two active sites so that a productive resolution event is possible...

..In the absence of this, we find that a cruciform junction is no longer subject to bilateral cleavage, but instead, just one strand is cleaved. Thus, the N-terminus is required for a productive resolution of the junction...

..It is likely that the stereochemical environment of the methyl group affects the interaction with metal ions in the center of the junction...

..The 2b and 2n adenine nucleobases are not directly hydrogen bonded, but each makes hydrogen bonds to their opposing strands...

..However, no restoration was obtained with exogenous adenine base either, indicating that the cavity that might result from ablation of the base was closed...

..We observe significant rates of cleavage and ligation, and a bell-shaped pH dependence for both...

..These studies reveal that structural changes in both DNA and protein occur in the action of this junction-resolving enzyme...

..This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species...

..The novel ribozyme is active in both cleavage and ligation reactions. The reaction is efficient, although relatively slow. The results are consistent with a role for nucleobase catalysis in the catalytic mechanism of this ribozyme...

..It appears that the two ribozymes are mechanistically equivalent...

..Thus this RNA may act as a relatively flexible hinge during RNA folding, until fixed into its ultimate kinked structure by the binding of L7 or related protein...

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..NAIM experiments indicate that a protonated base is required at this position for the ligation reaction. A756 is thus a strong candidate for nucleobase participation in the catalytic chemistry...

..While it has the N1 structure in the ribosome, on engineering it into the SAM-I riboswitch, it changes to the N3 structure, resulting in a significant alteration in the trajectory of the helical arms...

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..A mechanism of hydrolysis consistent with all of these data is proposed...

..The 10(5)-fold rate enhancement is probably brought about by a combination of metal-ion participation and stereochemical factors in the environment of the folded RNA structure...

..This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure...

..The main players are the RNA nucleobases and bound metal ions. Although most ribozymes carry out phosphoryl transfer, the same considerations appear to apply to peptidyl transfer in the ribosome...

..Calorimetry points to particularly critical role of Asp55, as mutation completely abolishes all binding of both manganese and calcium ions...

..The K-turn is therefore unlikely to be of itself a primary organizing feature in RNA...

..Moreover, analysis of single-molecule FRET data at an 8-msec resolution failed to reveal evidence for structural transitions. It seems probable that this junction adopts a single conformation as a unique and stable fold...

..05 min(-1)) and the cleavage reaction in its protonated state (rate 0.18 min(-1)). A756 is a candidate for the nucleobase undergoing protonation/deprotonation...

..This regaining of cleavage activity by complementation indicates that helix I of one VS RNA can interact with another VS RNA molecule to generate a functional active site in trans...

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..The loops clearly act as important auxiliary elements in the function of the ribozyme, permitting folding to occur efficiently under physiological conditions...

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..Depletion of FAN1 from human cells causes hypersensitivity to ICLs, defects in ICL repair, and genome instability. These data at least partly explain how ubiquitination of FANCD2 promotes DNA repair...

..These dynamic properties may be important in mediating interactions between the IRES and the ribosome and initiation factors...

..This site provides (1) a database of sequence and structure, as a resource for the RNA community, and (2) a tool to enable the manipulation and comparison of 3D structures where known...

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..The FRET approach can also be extended into the time domain to analyze conformational equilibria, and increasingly to the single-molecule level...

..It has had a number of significant successes in RNA, DNA and DNA-protein complexes, and in all cases to date the results have stood the test of time and eventual comparison with crystallographic analysis...

..This is consistent with the dependence of activity on the length of helix III. The scissile phosphate is well placed to interact with the probable active site of the ribozyme, the loop containing A730...

..The data support a passive conformational selection model by which the protein selects a fraction of RNA that is already in the kinked conformation, thereby drawing the equilibrium into this form...

..Depletion of SLX4 causes a decrease in DSB-induced homologous recombination. These data show that SLX4 is a regulator of structure-specific nucleases and that SLX4 and SLX1 are important regulators of genome stability in human cells...

..Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair...

..In addition to the provision of distance information, recent studies have emphasized the orientation dependence of energy transfer...

..This provides a mechanism whereby the sequence-dependent conformational bias could determine the extent of genetic exchange upon junction resolution...

..Thus, riboswitch function should depend on the relative rates of ligand binding and the transcriptional process...

..This transition occurs in response to the noncooperative, site-specific binding of magnesium ions. We observe a good correlation between the extent of ion-induced folding and cleavage activity...

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..Dynamic interplay between structural elements, as we demonstrate for the hairpin ribozyme, may be a general theme for other functional RNA molecules...

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..This strongly indicates that the observed heterogeneity in dynamic properties does not arise as an artifact of surface attachment, but is intrinsic to the nature of the molecules...

..This further supports the notion that electrostatic stabilization of the stacked structures dictates the interconversion rates between different structural forms...

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..58 +/- 0.019 and 14 +/- 1.5 mM. These results are consistent with a two metal ion mechanism for the cleavage reaction, in which the hydrolytic water molecule is contained in the first coordination sphere of the site 1-bound metal ion...

..These interactions effectively measure the relative orientations and positions of the arms of the junction, thereby ensuring that binding is selective for branched DNA that can achieve this geometry...

..Analysis of crystal packing interactions in the crystal structure of Hjc suggests a molecular basis for this autoinhibition. The wider implications of these findings for the quantitative study of DNA-protein interactions is discussed...

..This method of mapping two-dimensional reaction landscapes at low forces is readily applicable to other nucleic acid systems and their interactions with proteins and enzymes...