Structure of a rare non-standard sequence k-turn bound by L7Ae protein

Kt-23 from Thelohania solenopsae is a rare RNA kink turn (k-turn) where an adenine replaces the normal guanine at the 2n position. L7Ae is a member of a strongly conserved family of proteins that bind a range of k-turn structures in the ribosome, box C/D and H/ACA small nucleolar RNAs and U4 small nuclear RNA. We have solved the crystal structure of T. solenopsae Kt-23 RNA bound to Archeoglobus fulgidus L7Ae protein at a resolution of 2.95 Å. The protein binds in the major groove displayed on the outer face of the k-turn, in a manner similar to complexes with standard k-turn structures. The k-turn adopts a standard N3 class conformation, with a single hydrogen bond from A2b N6 to A2n N3. This contrasts with the structure of the same sequence located in the SAM-I riboswitch, where it adopts an N1 structure, showing the inherent plasticity of k-turn structure. This potentially can affect any tertiary interactions in which the RNA participates

How RNA acts as a nuclease:some mechanistic comparisons in the nucleolytic ribozymes

Recent structural and mechanistic studies have shed considerable light on the catalytic mechanisms of nucleolytic ribozymes. The discovery of several new ribozymes in this class has now allowed comparisons to be made, and the beginnings of mechanistic groupings to emerge.</jats:p

The oretical and experimental investigations of structure, reactivity and bonding in some organic systems

A theoretical study has been made of some aspects of prototype potential energy surfaces for some simple organic reactions. Addition of prototype electrophiles to simple alkenes has been investigated by means of non-empirical and semi-empirical calculations, within the Hartree-Fock formalism, and the resulting carbonium ions studied. The systems under investigation may be formally considered as being derived from electrophilic addition of H(^+) to ethylene, fluoroethylene and chloroethylene, or of X(^+) (X=F,Cl) to ethylene, and may thus be represented as (C(_2)H(_4)X)(^+), X=H,F and Cl. For the simplest system, C(_2)H(_5)(^+), two basic structures have been considered, the classical ethylcation and the bridge-protonated ethylene. The energies of these species have been minimised with respect to the C-C bond lengths and also, in the case of the latter ion, with respect to the distance of the bridging H from the CC bond centre. Examination of conformational processes in the classical ion has shown a virtual absence of any barrier to rigid rotation about the cc bond. The calculated relative energies of the species has indicated, subject to limitations imposed by the basis set size and partial geometry optimisation, that in the gas phase the classical ion should be ~5.2k cal mole(^-1) more stable than the bridge protonated ethylene. Furthermore calculations along an idealised reaction coordinate representing trans-formation between the two species have indicated the absence of an activation barrier thus suggesting the bridged ion to be the transition state for the scrambling of the hydrogen atoms of the ethyl cation. These results have been compared with mass spectrometric data. The approach of a prototype nucleophile (H(^-)) to ethyl cation has been examined, results suggesting a preferential cis attack. Conformational processes in the 1- and 2- fluoroethyl chlorethyl cations have been examined. The rotational barrier in the 2- fluoroethyl cation has been shown to be very large (10.5k cal mole) and, with the exception of the 2- chloroethyl cation, all the barriers for the substituted ethyl cations have been shown to be dominated by attractive terms. In both the fluoro and chloroethyl systems, predicted ordering of stabilities of cations has been 1- haloethyl > bridge-protonated haloethylene > 2- haloethyl, and idealised reaction coordinates have been constructed relating the ions in the fluoro case, the results predict the total absence of any activation barrier in trans-forming 2- to 1- fluoroethyl cation, whilst, in the analogous chloro case, a small barrier (4.3 k cal mole(^-1)) is predicted. Relative thermochemical stabilities of the ions have been computed, and the stabilising/destabilising effects of halogen substitution in these carbonium ions investigated and compared with experimental data. The halogen bridged 'halonium' ions have been studied, and their total energies minimised with respect to the distance of the halogen atom from the CC bond centre. The calculations have indicated that the fluoronium ion should be of marginally greater stability than the 2- fluoroethyl cation (3.6k cal mole(^-1)) and this has been discussed in the light of published nmr studies of the ionisation of 2-halo-3fluoro 2,3-dimethyl butanes in SO(_2)/SbF(_5). Results for the chloronium ion have indicated that this ion should be considerably more stable (15.8k cal mole(^-1)) than the corresponding 2- chloroethyl cation. Electron Spectroscopy for Chemical Applications (ESCA) has been employed for the measurement of core binding energies in three series of closely related molecules (i) a series of acetyl compounds of general formula CH(_3)COX, X=H, CH(_3), OH, OCH(_3), NH(_2), NHCH(_3), COCH(_3), CO(_2)H, CN and OCOCH(_3). (ii) a series of five membered ring heterocycles. (iii) a series of pyrimidine bases and related compounds. Assignment of core levels has been accomplished in two ways, (i) Direct correlation of measured binding energies with orbital energies derived from SCF calculations, i.e. assuming Koopmans' theorem, (ii) Correlation of shifts in core binding energies with computed electron distributions within the molecule using the charge potential model. In general, assignments based upon the different methods have been found to be in agreement. Furthermore in the case of some members of the pyrimidine series comparison has been possible between charge potential assignments using both ab initio and CNDO/II populations. Agreement between the two sets has been complete

The k-junction motif in RNA structure

The k-junction is a structural motif in RNA comprising a three-way helical junction based upon kink turn (k-turn) architecture. A computer program written to examine relative helical orientation identified the three-way junction of the Arabidopsis TPP riboswitch as an elaborated k-turn. The Escherichia coli TPP riboswitch contains a related k-junction, and analysis of &gt;11 000 sequences shows that the structure is common to these riboswitches. The k-junction exhibits all the key features of an N1-class k-turn, including the standard cross-strand hydrogen bonds. The third helix of the junction is coaxially aligned with the C (canonical) helix, while the k-turn loop forms the turn into the NC (non-canonical) helix. Analysis of ligand binding by ITC and global folding by gel electrophoresis demonstrates the importance of the k-turn nucleotides. Clearly the basic elements of k-turn structure are structurally well suited to generate a three-way helical junction, retaining all the key features and interactions of the k-turn

A critical base pair in k-turns determines the conformational class adopted, and correlates with biological function

k-turns are commonly-occurring motifs that introduce sharp kinks into duplex RNA, thereby facilitating tertiary contacts. Both the folding and conformation of k-turns are determined by their local sequence. k-turns fall into two conformational classes, called N3 and N1, that differ in the pattern of hydrogen bonding in the core. We show here that this is determined by the basepair adjacent to the critical G•A pairs. We determined crystal structures of a series of Kt-7 variants in which this 3b,3n position has been systematically varied, showing that this leads to a switch in the conformation. We have previously shown that the 3b,3n position also determines the folding characteristics of the k-turn, i.e. whether or not the k-turn can fold in the presence of metal ions alone. We have analyzed the distribution of 3b,3n sequences from four classes of k-turns from ribosomes, riboswitches and U4 snRNA, finding a strong conservation of properties for a given k-turn type. We thus demonstrate a strong association between biological function, 3b,3n sequence and k-turn folding and conformation. This has strong predictive power, and can be applied to the modeling of large RNA architectures

Wrapping Transition and Wrapping-Mediated Interactions for Discrete Binding along an Elastic Filament: An Exact Solution

The wrapping equilibria of one and two adsorbing cylinders are studied along a semi-flexible filament (polymer) due to the interplay between elastic rigidity and short-range adhesive energy between the cylinder and the filament. We show that statistical mechanics of the system can be solved exactly using a path integral formalism which gives access to the full effect of thermal fluctuations, going thus beyond the usual Gaussian approximations which take into account only the contributions from the minimal energy configuration and small fluctuations about this minimal energy solution. We obtain the free energy of the wrapping-unwrapping transition of the filament around the cylinders as well as the effective interaction between two wrapped cylinders due to thermal fluctuations of the elastic filament. A change of entropy due to wrapping of the filament around the adsorbing cylinders as they move closer together is identified as an additional source of interactions between them. Such entropic wrapping effects should be distinguished from the usual entropic configuration effects in semi-flexible polymers. Our results may be applicable to the problem of adsorption of proteins as well as synthetic nano-particles on semi-flexible polymers such as DNA.Comment: 24 pages, 12 figure