The conformation of acetylated virginiamycin M1 and virginiamycin M1 in explicit solvents

The three-dimensional structure of acetylated virginiamycin M(1) (acetylated VM1) in chloroform and in a water/acetonitrile mixture (83:17 v/v) have been established through 2D high resolution NMR experiments and molecular dynamics modeling and the results compared with the conformation of the antibiotic VM1 in the same and other solvents. The results indicated that acetylation of the C-14 OH group of VM1 caused it to rotate about 90 degrees from the position it assumed in non-acetylated VM1. The conformation of both VM1 and acetylated VM1 appear to flatten in moving from a nonpolar to polar solvent. However, the acetylated form has a more hydrophobic nature. The acetylated VM1 in chloroform and in water/acetonitrile solution had a similar configuration to that of VM1 bound to 50S ribosomes and to the Vat(D) active sites as previously determined by X-ray crystallography. Docking studies of VM1 to the 50S ribosomal binding site and the Vat(D) gave conformations very similar to those derived from X-ray crystallographic studies. The docking studies with acetylated VM1 suggested the possibility of a hydrogen bond from the acetyl carbonyl group oxygen of acetylated VM1 to the 2' hydroxyl group of ribose of adenosine 2538 at the ribosomal VM1 binding site. No hydrogen bonds between acetylated VM1 and the Vat(D) active sites were found; the loss of this binding interaction partly accounts for the release of the product from the active site.     

Impact of RNA structure on the prediction of donor and acceptor splice sites

Background  

gene identification in genomic DNA sequences by computational methods has become an important task in bioinformatics and computational gene prediction tools are now essential components of every genome sequencing project. Prediction of splice sites is a key step of all gene structural prediction algorithms.     

Prediction of human mRNA donor and acceptor sites from the DNA sequence

Artificial neural networks have been applied to the prediction of splice site location in human pre-mRNA. A joint prediction scheme where prediction of transition regions between introns and exons regulates a cutoff level for splice site assignment was able to predict splice site locations with confidence levels far better than previously reported in the literature. The problem of predicting donor and acceptor sites in human genes is hampered by the presence of numerous amounts of false positives: here, the distribution of these false splice sites is examined and linked to a possible scenario for the splicing mechanism in vivo. When the presented method detects 95% of the true donor and acceptor sites, it makes less than 0.1% false donor site assignments and less than 0.4% false acceptor site assignments. For the large data set used in this study, this means that on average there are one and a half false donor sites per true donor site and six false acceptor sites per true acceptor site. With the joint assignment method, more than a fifth of the true donor sites and around one fourth of the true acceptor sites could be detected without accompaniment of any false positive predictions. Highly confident splice sites could not be isolated with a widely used weight matrix method or by separate splice site networks. A complementary relation between the confidence levels of the coding/non-coding and the separate splice site networks was observed, with many weak splice sites having sharp transitions in the coding/non-coding signal and many stronger splice sites having more ill-defined transitions between coding and non-coding.     

Crystal structures of Pasteurella multocida sialyltransferase complexes with acceptor and donor analogues reveal substrate binding sites and catalytic mechanism

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 A resolutions, respectively. Ternary X-ray structures of the protein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have been determined at 2.0 and 2.27 A resolutions, respectively. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously. The four structures presented here reveal that binding of the nucleotide-activated donor sugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helps define the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysis have been identified. Structural and kinetic data support a direct displacement mechanism involving an oxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate the acceptor hydroxyl group.     

Studies on the catalytic rate constant of ribosomal peptidyltransferase

A detailed kinetic analysis of a model reaction for the ribosomal peptidyltransferase is described, using fMet-tRNA or Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor. The initiation complex (fMet-tRNA X AUG X 70 S ribosome) or (Ac-Phe-tRNA X poly(U) X 70 S ribosome) (complex C) is isolated and then reacted with excess puromycin (S) to give fMet-puromycin or Ac-Phe-puromycin. This reaction (puromycin reaction) is first order at all concentrations of S tested. An important asset of this kinetic analysis is the fact that the relationship between the first order rate constant kobs and [S] shows hyperbolic saturation and that the value of kobs at saturating [S] is a measure of the catalytic rate constant (k cat) of peptidyltransferase in the puromycin reaction. With fMet-tRNA as the donor, this kcat of peptidyltransferase is 8.3 min-1 when the 0.5 M NH4Cl ribosomal wash is present, compared to 3.8 min-1 in its absence. The kcat of peptidyltransferase is 2.0 min-1 when Ac-Phe-tRNA replaces fMet-tRNA in the presence of the ribosomal wash and decreases to 0.8 min-1 in its absence. This kinetic procedure is the best method available for evaluating changes in the activity of peptidyltransferase in vitro. The results suggest that peptidyltransferase is subjected to activation by the binding of fMet-tRNA to the 70 S initiation complex.     

The conformational flexibility of the antibiotic virginiamycin M1

The antibiotic virginiamycin is a combination of two molecules, virginiamycin M₁ (VM1) and virginiamycin S₁ (VS1) or analogues, which function synergistically by binding to bacterial ribosomes and inhibiting bacterial protein synthesis. Both VM1 and VS1 dissolve poorly in water and are soluble in more hydrophobic solvents. We have recently reported that the 3D conformation of VM1 in CDCl₃ solution (Aust. J. Chem. 57:415, 2004; Org. Biomol. Chem. 2:2919, 2004) differs markedly from the conformation bound to a VM1 binding enzyme (Sugantino and Roderick in Biochemistry 41:2209, 2002) and to 50S ribosomes (Hansen et al. in J. Mol. Biol. 330:1061, 2003) as found by X-ray crystallographic studies. We now report the results of further NMR studies and subsequent molecular modeling of VM1 dissolved in CD₃CN/H₂O and compare the structure with that in CD₃OD and CDCl₃. The conformations of VM1 in CD₃CN/H₂O, CD₃OD and CDCl₃ differ substantially from one another and from the bound form, with the aqueous form most like the bound structure. We propose that the flexibility of the VM1 molecule in response to environmental conditions contributes to its effectiveness as an antibiotic.     

The mechanism of action of virginiamycin M on the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu

Mitochondrial DNA from Ustilago cynodontis has been investigated in several of its properties. Its dG + dC content is equal to 33.5%; its buoyant density (1.698 g/cm3) is higher, by 5 mg/cm3, and its melting temperature (82.5 degrees C) is lower than expected for a bacterial DNA having the same base composition; the first derivative of its melting curve indicates a large compositional heterogeneity, its molarity of elution from hydroxyapatite is high, 0.28 M phosphate, and allows its partial separation from nuclear DNA. Degradation by micrococcal nuclease indicates that about 25% of the DNA is formed by stretches having no more than 15% dG + dC. Finally, the unit size of mitochondrial genome is about 50 X 10(6). In most of its properties, the mitochondrial genome of U. cynodontis presents strong analogies with that of Saccharomyces cerevisiae. A parallel investigation on mitochondrial DNA from Acanthamoeba castellanii which has as genome unit size of only 27 X 10(6), has shown that this shares with the former the dG + dC content (32.9%), the melting temperature (82.5 degrees C), a large compositional heterogeneity and a very similar pattern of micrococcal nuclease degradation; its buoyant density (1.692 g/cm3) and its molarity of elution from hydroxyapatite (0.25 M phosphate) are, however, normal, probably because of a different short-sequence pattern and the fact that its dA + dT-rich stretches are shorter, on the average.     

Substrate specificity of Escherichia coli peptidyltransferase at the donor site

The substrate specificity of $\text{E.}\text{coli}$ peptidyltransferase at the donor site was investigated by the “50S reaction”. Seventeen N-acetylated or unacetylated aminoacyl-tRNAs and dipeptidyl-tRNAs were used as the donor substrates and puromycin as the acceptor. Results indicated that the nature of amino acid side chain of the donor tRNA has a predominant effect on the reaction rate of peptidyltransferase. Amino acids or dipeptides with high hydrophobicity were transferred faster than those with low hydrophobicity. Amino acids with alkyl side chains are better donors than those with aromatic side chains. Substrates with C-terminal proline were transferred extremely slowly which can probably be attributed to its unusual α-imino structure in addition to its low hydrophobicity.     

The TaqI 'star' reaction: strand preferences reveal hydrogen-bond donor and acceptor sites in canonical sequence recognition

TaqI endonuclease recognizes and cleaves its canonical sequence, TCGA, with complete fidelity under standard conditions. In the presence of some organic solvents, TaqI endonuclease introduced additional single-strand and double-strand cuts at sequences termed TaqI 'star' sites. Using 'middle-labeled' DNA, the relative rates of cleavage of each strand were simultaneously determined for several star sites. These star recognition sequences differed from the canonical sequence by a single base, and all potential star sites were either nicked or cleaved. Star sites within the middle labeled substrate represented ten of the twelve possible star sequences for each strand. For each group of identical star sites, one strand was consistently preferred for cleavage. Based on these preferences, a model for TaqI recognition of the TCGA sequence is proposed. According to this model, sequence discrimination is mediated by eight hydrogen bonds formed between TaqI and the cognate nucleotides within the major groove.     

The in vitro binding of virginiamycin M to bacterial ribosomes and ribosomal subunits

Summary Virginiamycin M (VM), an antibiotic of type A synergimycin group of antibiotics, binds to bacterial ribosomes and subunits in vitro: the amount of linked drug is linearly dependent on ribosome and VM concentrations. The technique used to measure the association reaction is based on the finding that the unbound drug is adsorbed by norite A: this procedure is twice as sensitive as the sedimentation and filtration methods (for technical reasons, column chromatography and equilibrium dialysis are unsuitable for this study). Saturation curves with 70S and 50S particles overlap, thus indicating a comparable affinity of the inhibitor for ribosomes and large subunits; instead, very small amount of VM, if any, attaches to 30S particles. Kinetics of binding is influenced by the temperature; the 4° C and 25° C saturation curves overlap, however, upon pre-incubation of ribosomes in 10 mM Mg buffer at 37° C (reactivation). This suggests that binding of VM depends on the configura tion of the 50S particles, which is altered at low temperature. Differences in Mg⁺⁺ concentration in the range 1 to 20 mM do not modify the binding curve, nor does the replacement of K⁺ by either NH ₄ ⁺ or Na⁺. Previously bound labelled VM is slowly displaced by an excess of unlabeled VM, and the associa tion curve remains unchanged in the presence of VS. Binding of VM is inhibited (10 to 60%) in the presence of an excess (tenfold to hundredfold) of one of the 50S inhibitors: chloramphenicol, oleandomycin and erythromycin. From the Scatchard plot, an as sociation constant of 3.2 × 10⁵M^–1 has been calculated: this value is about 1/8 of that reported for VS, a component of type B synergimycin group of antibiotics. The v value is 0.85 for both ribosomes and large subunits, indicating a monomolecular association of VM with ribonucleoprotein particles.     

Mutational analysis of the donor substrate binding site of the ribosomal peptidyltransferase center.

Previous experiments have shown that the top of helix 90 of 23S rRNA is highly important for the ribosomal peptidyltransferase activity and might be part of the donor (P) site. Developing on these studies, mutations in the 23S rRNA at the highly conserved positions G2505, G2582, and G2583 were investigated. None of the mutations affected assembly, subunit association, or the capacity of tRNA binding to A and P sites. A "selective transpeptidation assay" revealed that the mutations specifically impaired peptide bond formation. Results with a modified "fragment" assay using the minimal donor substrate pA-fMet are consistent with a model where the nucleotides psiGG2582 form a binding pocket for C75 of the tRNA.     

Synthetic donor and acceptor splice sites function in an RNA polymerase B (II) transcription unit

[This corrects the article on p. 2026 in vol. 3.].