Mutations in the conserved P loop perturb the conformation of two structural elements in the peptidyl transferase center of 23 S ribosomal RNA

Evidence is presented for the participation of the P loop (nucleotides G2250-C2254) of 23 S rRNA in establishing the tertiary structure of the peptidyl transferase center. Single base substitutions were introduced into the P loop, which participates in peptide bond formation through direct interaction with the CCA end of P site-bound tRNA. These mutations altered the pattern of reactivity of RNA to chemical probes in a structural subdomain encompassing the P loop and extending roughly from G2238 to A2433. Most of the effects on chemical modification in the P loop subdomain occurred near sites of tertiary interactions inferred from comparative sequence analysis, indicating that these mutations perturb the tertiary structure of this region of RNA. Changes in chemical modification were also seen in a subdomain composed of the 2530 loop (nucleotides G2529-A2534) and the A loop (nucleotides U2552-C2556), the latter a site of interaction with the CCA end of A site-bound tRNA. Mutations in the P loop induced effects on chemical modification that were commensurate with the severity of their characterized functional defects in peptide bond formation, tRNA binding and translational fidelity. These results indicate that, in addition to its direct role in peptide bond formation, the P loop contributes to the tertiary structure of the peptidyl transferase center and influences the conformation of both the acceptor and peptidyl tRNA binding sites.     

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.     

Interaction of structural modules in substrate binding by the ribozyme from Bacillus subtilis RNase P

The ribozyme from bacterial ribonuclease P recognizes two structural modules in a tRNA substrate: the T stem-loop and the acceptor stem. These two modules are connected through a helical linker. The T stem-loop binds at a surface confined in a folding domain away from the active site. Substrates for the Bacillus subtilis RNase P RNA were previously selected in vitro that are shown to bind comparably well or better than a tRNA substrate. Chemical modification of P RNA-substrate complexes with dimethylsulfate and kethoxal was performed to determine how the P RNA recognizes three in vitro selected substrates. All three substrates bind at the surface known to interact with the T stem-loop of tRNA. Similar to a tRNA, the secondary structure of these substrates contains a helix around the cleavage site and a hairpin loop at the corresponding position of the T stem-loop. Unlike a tRNA, these two structural modules are connected through a non-helical linker. The two structural modules in the tRNA and in the selected substrates bind to two different domains in P RNA. The properties of substrate recognition exhibited by this ribozyme may be exploited to isolate new ribozyme-substrate pairs with interactive structural modules.     

An RNA model system for investigation of pseudouridine stabilization of the codon-anticodon interaction in tRNALys, tRNAHis and tRNATyr

The nucleoside conformation of pseudouridine (psi) was investigated in a series of RNA oligonucleotides and compared with the same sequences containing the parent, unmodified uridine nucleoside. 1H NMR spectroscopy was used to determine the glycosyl conformational preference in pseudouridine systems at the nucleoside level; these experiments were extended to trimers, and ultimately to RNA tetraloop hairpins that are models for the codon-anticodon interaction in tRNA. ROESY 1D and 2D NMR experiments were used to measure the nucleoside conformational preference as a function of temperature. The thermodynamic stability of the RNA tetraloops was also analyzed using UV monitored Tm experiments which established that pseudouridine has a very strong stabilizing effect on double-stranded, base pairing interactions when the modification is located within a base-paired region. This was shown for a tetraloop hairpin model of the codon-anticodon interaction in tRNA(Tyr) which contains a psi at position 35. Pseudouridine also stabilizes double-stranded RNA when the psi modification is in a single-stranded region adjacent to a duplex region as occurs for psi at positions 38 or 39 in tRNA(Lys) and tRNA(His). These results establish that pseudouridine modification of RNA is a powerful and versatile mechanism for stabilizing local RNA structure in both single-stranded and double-stranded regions. Previously postulated roles for pseudouridine as a "conformational switch" are unlikely in light of the increased barrier to rotation about the glycosyl bond upon modification of uridine to pseudouridine. The Tm and NMR data show that local RNA stacking stabilization as a result of psi will stabilize adjacent double-stranded RNA regions such as the codon-anticodon interaction in tRNA.     

Structural features of the DNA hairpin d(ATCCTA-GTTA-TAGGAT): formation of a G-A base pair in the loop

The three-dimensional structure of the hairpin formed by d(ATCCTA-GTTA-TAGGAT) has been determined by means of two-dimensional NMR studies, distance geometry and molecular dynamics calculations. The first and the last residues of the tetraloop of this hairpin form a sheared G-A base pair on top of the six Watson-Crick base pairs in the stem. The glycosidic torsion angles of the guanine and adenine residues in the G-A base pair reside in the anti and high- anti domain ( approximately -60 degrees ) respectively. Several dihedral angles in the loop adopt non-standard values to accommodate this base pair. The first and second residue in the loop are stacked in a more or less normal helical fashion; the fourth loop residue also stacks upon the stem, while the third residue is directed away from the loop region. The loop structure can be classified as a so-called type-I loop, in which the bases at the 5'-end of the loop stack in a continuous fashion. In this situation, loop stability is unlikely to depend heavily on the nature of the unpaired bases in the loop. Moreover, the present study indicates that the influence of the polarity of a closing A.T pair is much less significant than that of a closing C.G base pair.     

Differential chemical probing of a group II self-splicing intron identifies bases involved in tertiary interactions and supports an alternative secondary structure model of domain V

Dimethyl sulfate modification was used to probe for tertiary structural elements in the group II intron PI.LSU/2 from the mitochondrial pre-ribosomal RNA of the brown alga Pylaiella littoralis. Modification of the lariat form of the intron under conditions that allow both native folding and conformational homogeneity is found to be generally consistent with secondary and tertiary structural features identified previously for group II ribozymes. A comparison of chemical probing at temperatures just below and above the first melting transition illustrates the cooperative unfolding of tertiary structure and identifies novel candidates for tertiary interactions in addition to defining elements of secondary structure. Substitution of the GAAA terminal loop of domain V is shown to be compatible with retention of conformational homogeneity (despite the loss of an important tertiary interaction), but produces a concise methylation footprint in domain I at the site previously shown to harbor the receptor for that loop. The analysis also identified two nucleotide positions in domain V with novel secondary and potential tertiary structural roles. The proposed refinement of domain V secondary structure is supported by an expanded comparative analysis of group II sequences and bears increased resemblance to U2:U6 snRNA pairing in the spliceosome.     

Investigation of the solution structure of chymotrypsin inhibitor 2 using molecular dynamics: comparison to x-ray crystallographic and NMR data

The native solution structure and dynamics of chymotrypsin inhibitor 2 (CI2) have been studied using a long (5.3 ns) molecular dynamics (MD) simulation without any imposed restraints. The majority of the experimentally observed spin-spin coupling constants, short- and long-range nuclear Overhauser effect (NOE) cross peaks and the amide hydrogen exchange behavior were reproduced by the MD simulation. This good correspondence suggests that the major structural features of the protein during the simulation are representative of the true protein structure in solution. Two water molecules formed hydrogen bond bridges between beta2 and beta3, in agreement with X-ray crystallographic data and a recent reassessment of the solution structure using time-averaged NMR restraints during MD refinement. The active-site loop of the protein displayed the greatest structural changes and the highest mobility. When this loop region was excluded, the average Calpha r.m.s. deviation of the simulated solution structures from the crystal structure was approximately 1.5 Angstrom from 0.5 to 5.3 ns. There is structural heterogeneity in particular regions of the NMR-derived solution structures, which could be a result of imprecision or true internal motion. A study of the distribution of mobility through the protein allows us to distinguish between these two alternatives. In particular, deviations in the active-site loop appear to be a result of heightened mobility, which is also supported by good correspondence between calculated and experimental S2 N-H order parameters. On the other hand, other ill-defined regions of the NMR-derived structures are well defined in the simulation and are probably the result of a lack of structural restraints (i.e. NOEs), as opposed to reflecting the true mobility.     

A cell-free yellow lupin extract containing activities of pseudouridine 35 and 55 synthases

Plant cytoplasmic tyrosine tRNA was pseudouridylated at three different positions: 35, 39 and 55. These pseudouridines were introduced by three different enzymes--pseudouridine synthases. Variants of the Arabidopsis thaliana pre-tRNA(Tyr) were constructed that allow to monitor specifically pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis we have prepared an extract from Lupinus luteus cv. Ventus seeds containing activities of at least psi35 and psi55 synthases. This is the first report describing the preparation of the lupin seed extract that specifically modifies plant pre-tRNA(Tyr) transcribed by T7 RNA polymerase. U35 is converted to psi35 only in an intron-dependent manner, while pseudouridylation of U55 is insensitive to the presence or absence of an intron.     

Aminoacylation of tRNA in the evolution of an aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases catalyze aminoacylation of tRNAs by joining an amino acid to its cognate tRNA. The selection of the cognate tRNA is jointly determined by separate structural domains that examine different regions of the tRNA. The cysteine-tRNA synthetase of Escherichia coli has domains that select for tRNAs containing U73, the GCA anticodon, and a specific tertiary structure at the corner of the tRNA L shape. The E. coli enzyme does not efficiently recognize the yeast or human tRNACys, indicating the evolution of determinants for tRNA aminoacylation from E. coli to yeast to human and the coevolution of synthetase domains that interact with these determinants. By successively modifying the yeast and human tRNACys to ones that are efficiently aminoacylated by the E. coli enzyme, we have identified determinants of the tRNA that are important for aminoacylation but that have diverged in the course of evolution. These determinants provide clues to the divergence of synthetase domains. We propose that the domain for selecting U73 is conserved in evolution. In contrast, we propose that the domain for selecting the corner of the tRNA L shape diverged early, after the separation between E. coli and yeast, while that for selecting the GCA-containing anticodon loop diverged late, after the separation between yeast and human.     

Central sleep apnoea and heart failure (part II)

The three-dimensional (3-D) structure of a RNA pseudoknot that causes the efficient ribosomal frameshifting in the gag-pro region of mouse mammary tumor virus (MMTV) has been determined recently by nuclear magnetic resonance (NMR) studies. But since the structure refinement in the studies did not use metal ions and waters, it is not clear how metal ions participate in the stabilization of the pseudoknot, and what kind of ion-RNA interactions dominate in the tertiary contacts for the RNA pseudoknotting. Based on the reported structure data of the pseudoknot VPK of MMTV, we gradually refined the structure by restrained molecular dynamics (MD) using NMR distance restraints. Restrained MD simulation of the RNA pseudoknot was performed with sodium ions and water molecules. Our results are in good agreement with known NMR data and delineate the importance of the metal ion coordination in the stability of the pseudoknot. In the non-coaxially stacking pseudoknot, stem 1 (S1), stem 2 (S2), and the intervening A14 involves unconventional stacking of base pairs coordinated by Na+ and/or bridging water molecules. A6 and G7 of loop L1 make a perfect base stacking in the major groove and are further stabilized by coordinated Na+ ions and water molecules. The first 4-nucleotide (nt) ACUC of loop L2 form a sharp turn and the following 4-nt AAAA cross the minor groove of S1 and are steadied by interactions with the nucleotides of S , bridging water molecules and coordinated Na+ ions. Our studies suggest that the metal ion plays a crucial role in the RNA pseudoknotting of VPK. In the stacking interior of S1 and S2, the Na+ ion is positioned in the major groove and interacts directly with the carbonyl group O6 of G28 and carbonyl group O4 of U13 in the wobble base pair U13:G28. The ion-RNA interactions in MMTV VPK not only stabilize the RNA pseudoknot but also modify the electrostatic properties of the nucleotides at the critical parts of the pseudoknot VPK.