Transition-state stabilization in Escherichia coli ribonuclease P RNA-mediated cleavage of model substrates.

We have used model substrates carrying modified nucleotides at the site immediately 5' of the canonical RNase P cleavage site, the -1 position, to study Escherichia coli RNase P RNA-mediated cleavage. We show that the nucleobase at -1 is not essential but its presence and identity contribute to efficiency, fidelity of cleavage and stabilization of the transition state. When U or C is present at -1, the carbonyl oxygen at C2 on the nucleobase contributes to transition-state stabilization, and thus acts as a positive determinant. For substrates with purines at -1, an exocyclic amine at C2 on the nucleobase promotes cleavage at an alternative site and it has a negative impact on cleavage at the canonical site. We also provide new insights into the interaction between E. coli RNase P RNA and the -1 residue in the substrate. Our findings will be discussed using a model where bacterial RNase P cleavage proceeds through a conformational-assisted mechanism that positions the metal(II)-activated H2O for an in-line attack on the phosphorous atom that leads to breakage of the phosphodiester bond.

pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA.

We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNA(fMet) to the aa-tRNAs Phe-tRNA(Phe), Ala-tRNA(Ala), Gly-tRNA(Gly), Pro-tRNA(Pro), Asn-tRNA(Asn), and Ile-tRNA(Ile), selected to cover a large range of intrinsic pK(a)-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, pK(a)(obs), at which the rate was half maximal. The pK(a)(obs)-values were downshifted relative to the intrinsic pK(a)-value of aa-tRNAs in bulk solution. Gly-tRNA(Gly) had the smallest downshift, while Ile-tRNA(Ile) and Ala-tRNA(Ala) had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pK(a)-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.

Mechanism of the translation termination reaction on the ribosome.

Ribosomal release factors (RFs) catalyze the termination of protein synthesis by triggering hydrolysis of the peptidyl-tRNA ester bond in the peptidyl transferase center of the ribosome. With new medium-resolution crystallographic structures of RF-ribosome complexes available, it has become possible to examine the detailed mechanism of this process to resolve the key factors responsible for catalysis of the termination reaction. Here, we report computer simulations of the termination reaction that utilize both the new RF complex structures and information from a high-resolution complex with a P-site substrate analogue. The calculations yield a consistent reaction mechanism that reproduces experimental rates and allows us to identify key interactions responsible for the catalytic efficiency. The results are also in general agreement with an earlier model based on molecular docking. The methylated glutamine residue of the universally conserved GGQ motif plays a key role in the hydrolysis reaction by orienting the water nucleophile and by stabilizing the transition state, and its side chain makes an entropic contribution to the lowering of the activation barrier. Two additional water molecules interacting with the P-site substrate are also found to be critically important. Furthermore, the 2'-OH group of the peptidyl-tRNA substrate is predicted to act as a proton shuttle for the leaving group in analogy with the consensus mechanism for peptidyl transfer. Thus, the ribosome's ability to catalyze both the termination (hydrolysis) and peptidyl transfer (aminolysis) reactions is largely explained by this type of unified mechanism, with similar transition states occurring in both processes.

RNase P RNA-mediated cleavage.

Metal(II)-induced hydrolysis of RNA produce products with 5'-hydroxyls and 2';3'-cyclic phosphates at the ends. Ribozymes are RNA molecules that act as catalysts. Some ribozymes that cleave RNA also generate 5'-hydroxyls and 2';3'-cyclic phosphates whereas others produces 5'-phosphates and 3'-hydroxyls at the ends of the cleavage products. RNase P is an essential endoribonuclease involved in RNA processing. The catalytic RNA subunit of RNase P is a trans-acting ribozyme that cleaves various RNA substrates in vitro generating 5'-phosphates and 3'-hydroxyls as cleavage products. The activity depends on the presence of metal(II) ions such as Mg(2+). RNase P RNA has therefore to facilitate a nucleophilic attack that generates the correct product ends and prevent metal(II)-induced hydrolysis of the RNA substrate. In this review, we will discuss our current understanding of the interactions between RNase P RNA and its substrate, role of specific residues with respect to catalysis and positioning of functionally important Mg(2+) at and in the vicinity of the cleavage site that ensures that products with correct ends are generated. Moreover, we will discuss the composition of RNase P and its RNA subunit in an evolutionary perspective.

A Role for the 2' OH of peptidyl-tRNA substrate in peptide release on the ribosome revealed through RF-mediated rescue.

The 2' OH of the peptidyl-tRNA substrate is thought to be important for catalysis of both peptide bond formation and peptide release in the ribosomal active site. The release reaction also specifically depends on a release factor protein (RF) to hydrolyze the ester linkage of the peptidyl-tRNA upon recognition of stop codons in the A site. Here, we demonstrate that certain amino acid substitutions (in particular those containing hydroxyl or thiol groups) in the conserved GGQ glutamine of release factor RF1 can rescue defects in the release reaction associated with peptidyl-tRNA substrates lacking a 2' OH. We explored this rescue effect through biochemical and computational approaches that support a model where the 2' OH of the P-site substrate is critical for orienting the nucleophile in a hydrogen-bonding network productive for catalysis.

Improved bio-energy yields via sequential ethanol fermentation and biogas digestion of steam exploded oat straw.

Using standard laboratory equipment, thermochemically pretreated oat straw was enzymatically saccharified and fermented to ethanol, and after removal of ethanol the remaining material was subjected to biogas digestion. A detailed mass balance calculation shows that, for steam explosion pretreatment, this combined ethanol fermentation and biogas digestion converts 85-87% of the higher heating value (HHV) of holocellulose (cellulose and hemicellulose) in the oat straw into biofuel energy. The energy (HHV) yield of the produced ethanol and methane was 9.5-9.8 MJ/(kg dry oat straw), which is 28-34% higher than direct biogas digestion that yielded 7.3-7.4 MJ/(kg dry oat straw). The rate of biogas formation from the fermentation residues was also higher than from the corresponding pretreated but unfermented oat straw, indicating that the biogas digestion could be terminated after only 24 days. This suggests that the ethanol process acts as an additional pretreatment for the biogas process.

Role of ribosomal protein L27 in peptidyl transfer.

The current view of ribosomal peptidyl transfer is that the ribosome is a ribozyme and that ribosomal proteins are not involved in catalysis of the chemical reaction. This view is largely based on the first crystal structures of bacterial large ribosomal subunits that did not show any protein components near the peptidyl transferase center (PTC). Recent crystallographic data on the full 70S ribosome from Thermus thermophilus, however, show that ribosomal protein L27 extends with its N-terminus into the PTC in accordance with independent biochemical data, thus raising the question of whether the ribozyme picture is strictly valid. We have carried out extensive computer simulations of the peptidyl transfer reaction in the T. thermophilus ribosome to address the role of L27. The results show a reaction rate similar to that obtained in earlier simulations of the Haloarcula marismortui reaction. Furthermore, deletion of L27 is predicted to only give a minor rate reduction, in agreement with biochemical data, suggesting that the ribozyme view is indeed valid. The N-terminus of L27 is predicted to interact with the A76 phosphate group of the A-site tRNA, thereby explaining the observed impairment of A-site substrate binding for ribosomes lacking L27. Simulations are also reported for the reaction with puromycin, an A-site tRNA analogue which lacks the A76 phosphate group. The calculated energetics shows that this substrate can cause a downward p K a shift of L27 and that the reaction proceeds faster with the L27 N-terminus deprotonated, in contrast to the situation with aminoacyl-tRNA substrates. These results could explain the observed differences in pH dependence between the puromycin and C-puromycin reactions, where the former reaction has been seen to depend on an additional ionizing group besides the attacking amine, and our model predicts this ionizing group to be the N-terminal amine of L27.

A model for how ribosomal release factors induce peptidyl-tRNA cleavage in termination of protein synthesis.

A major unresolved question in messenger RNA translation is how ribosomal release factors terminate protein synthesis. Class 1 release factors decode stop codons and trigger hydrolysis of the bond between the nascent polypeptide and tRNA some 75 A away from the decoding site. While the gross features of the release factor-ribosome interaction have been revealed by low-resolution crystal structures, there is no information on the atomic level at either the decoding or peptidyl transfer center. We used extensive computer simulations, constrained by experimental data, to predict how bacterial release factors induce peptide dissociation from the ribosome. A distinct structural solution is presented for how the methylated Gln residue of the universally conserved GGQ release factor motif inserts into the ribosomal A site and promotes rapid reaction with the peptidyl-tRNA substrate. This model explains key mutation experiments and shows that the ribosomal peptidyl transfer center catalyzes its two chemical reactions by a common mechanism.

Analysis of predictions for the catalytic mechanism of ribosomal peptidyl transfer.

The reaction mechanism of peptide bond formation on the ribosome is now becoming established by results from both experiments and computer simulations. Here, we analyze predictions from molecular dynamics simulations, as well as from new crystal structures, and examine their implications for the mechanisms of peptidyl transfer and peptidyl-tRNA hydrolysis. A number of computational predictions for the peptidyl transfer reaction, including quantitative energetics, stereochemistry, hydrogen bonding network, and role of solvent molecules, are found to be supported and confirmed by kinetic and structural data. The results show that this type of reaction calculations can provide important links between structure and function that cannot be obtained by experimental means.

Mechanism of peptide bond synthesis on the ribosome.

With the emergence of atomic-resolution crystal structures of bacterial ribosomal subunits, major advances in eliciting structure-function relationships of the translation process are underway. Nevertheless, the detailed mechanism of peptide bond synthesis that occurs on the large ribosomal subunit remains unknown. Separate x-ray structures of aminoacyl-tRNA and peptidyl-tRNA analogues bound to the ribosomal A- and P-sites, however, allow for structural modeling of the active complex in catalysis. Here, we combine available structural data to construct such a model of the peptidyl transfer reaction center with bound substrates. Molecular dynamics and free energy perturbation simulations then are used in combination with an empirical valence bond description of the reaction energy surface to examine possible catalytic mechanisms. Already, simulations of the reactant and tetrahedral intermediate states reveal a stable, preorganized H-bond network poised for catalysis. The most favorable mechanism is found not to involve any general acid-base catalysis by ribosomal groups but an intra-reactant proton shuttling via the P-site adenine O2' oxygen, which follows the attack of the A-site alpha-amino group on the P-site ester. The calculated rate enhancement for this mechanism is approximately 10(5), and the catalytic effect is found to be entirely of entropic origin, in accordance with recent experimental data, and is associated with the reduction of solvent reorganization energy rather than with substrate alignment or proximity. This mechanism also explains the inability of 2'-deoxyadenine P-site substrates to promote peptidyl transfer. The observed H-bond network suggests an important structural role of several universally conserved rRNA residues.