The complete catalytic cycle of EcoRV endonuclease has been observed by combining fluorescence anisotropy with fluorescence resonance energy transfer (FRET) measurements. Binding, bending, and cleavage of substrate oligonucleotides were monitored in real time by rhodamine-x anisotropy and by FRET between rhodamine and fluorescein dyes attached to opposite ends of a 14-mer DNA duplex. For the cognate GATATC site binding and bending are found to be nearly simultaneous, with association and bending rate constants of (1.45-1.6) x 10(8) M(-1) s(-1). On the basis of the measurement of k(off) by a substrate-trapping approach, the equilibrium dissociation constant of the enzyme-DNA complex in the presence of inhibitory calcium ions was calculated as 3.7 x 10(-12) M from the kinetic constants. Further, the entire DNA cleavage reaction can be observed in the presence of catalytic Mg(2+) ions. These measurements reveal that the binding and bending steps occur at equivalent rates in the presence of either Mg(2+) or Ca(2+), while a slow decrease in fluorescence intensity following bending corresponds to k(cat), which is limited by the cleavage and product dissociation steps. Measurement of k(on) and k(off) in the absence of divalent metals shows that the DNA binding affinity is decreased by 5000-fold to 1.4 x 10(-8) M, and no bending could be detected in this case. Together with crystallographic studies, these data suggest a model for the induced-fit conformational change in which the role of divalent metal ions is to stabilize the sharply bent DNA in an orientation suitable for accessing the catalytic transition state.

The chemical step of protein synthesis, peptide bond formation, is catalyzed by the large subunit of the ribosome. Crystal structures have demonstrated that the active site for peptide bond formation is composed entirely of RNA1. Recent work has focused on how an RNA active site is able to catalyze this fundamental biological reaction at a suitable rate for protein synthesis. Based on the absence of important ribosomal functional groups2, lack of a dependence on pH3, and the dominant contribution of entropy to catalysis4, it has been suggested that the role of the ribosome is limited to bringing the substrates into close proximity. Alternatively, the importance of the 2′-hydroxyl of the peptidyl-tRNA5 and a Bronsted coefficient near zero6 were taken as evidence that the ribosome coordinates a proton-transfer network. Here we report the transition state of peptide bond formation based upon kinetic isotope effect analysis at five positions at the reaction center of a peptidyl-tRNA mimic. Our results indicate that in contrast to the uncatalyzed reaction, formation of the tetrahedral intermediate and proton-transfer from the nucleophilic nitrogen both occur in the rate-limiting step. Unlike previous proposals, the reaction is not fully concerted, instead breakdown of the tetrahedral intermediate occurs in a separate fast step. This suggests that in addition to substrate positioning, the ribosome is contributing to chemical catalysis by changing the rate-limiting transition state.

Riboswitches contain structured aptamer domains that, upon ligand binding, facilitate helical switching in their downstream expression platforms to alter gene expression. To fully dissect how riboswitches function requires a better understanding of the energetic landscape for helical switching. Here, we report a sequencing-based high-throughput assay for monitoring in vitro transcription termination and use it to simultaneously characterize the functional effects of all 522 single point mutants of a glycine riboswitch type-1 singlet. Mutations throughout the riboswitch cause ligand-dependent defects, but only mutations within the terminator hairpin alter readthrough efficiencies in the absence of ligand. A comprehensive analysis of the expression platform reveals that ligand binding stabilizes the antiterminator by just 2–3 kcal/mol, indicating that the competing expression platform helices must be extremely close in energy to elicit a significant ligand-dependent response. These results demonstrate that gene regulation by this riboswitch is highly constrained by the energetics of ligand binding and conformational switching. These findings exemplify the energetic parameters of RNA conformational rearrangements driven by binding events.

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The ester bond of peptidyl-tRNA undergoes nucleophilic attack both in solution and catalyzed by the ribosome. To characterize the uncatalyzed hydrolysis reaction -a model of peptide release -the transition state structure for hydrolysis of a peptidyl-tRNA mimic was determined. Kinetic isotope effects were measured at several atoms that potentially undergo a change in bonding at the transition state. Large kinetic isotope effects of carbonyl oxygen-18 and α-deuterium substitutions on uncatalyzed hydrolysis indicate the transition state is nearly tetrahedral. Kinetic isotope effects were also measured for aminolysis by hydroxylamine to study a reaction similar to peptide bond formation. In contrast to hydrolysis, the large leaving group oxygen-18 isotope effect indicates the C-O3′ bond has undergone significant scission in the transition state. The smaller carbonyl oxygen-18 and α-deuterium effects are consistent with a later transition state. The assay developed here can also be used to measure isotope effects for the ribosome-catalyzed reactions. These uncatalyzed reactions serve as a basis to determine what aspects of the transition states are stabilized by the ribosome to achieve a rate enhancement.The ribosome catalyzes two chemical reactions during protein synthesis. During initiation and elongation, peptide bonds are formed by the nucleophilic attack of an aminoacyl-tRNA in the A-site on peptidyl-tRNA in the P-site. During termination, water acts as the nucleophile resulting in the release of the peptide chain from tRNA. The ribosome must be capable of selectively switching between these two catalytic activities, since hydrolysis during elongation would produce truncated proteins, and peptide bond formation in place of termination would result in read-through of stop codons.In the last decade, a wealth of structural information has been obtained to complement biochemical study of catalysis by the ribosome. Structures of the 50S ribosome demonstrated that the active site for peptide bond formation is composed entirely of RNA(1,2). Of the functional groups present in the active site, two have been identified as important for catalysis of peptide bond formation: the 2′-hydroxyl of A2451 in 50S RNA contributes about 10-fold (3), and the 2′-hydroxyl of the terminal adenosine of peptidyl-tRNA, which is adjacent to the leaving group, contributes at least 10 6 -fold(4). Brönsted(5) and kinetic isotope effect studies (6) indicate that the nucleophilic nitrogen is neutral in the transition state despite at least partial peptide bond formation. More recently, structures of 70S ribosomes bound to either release † This research was supported by NIH grant GM54839 (to SAS), NIH post-doctoral fellowship GM079980 (to DAH), and a Brown-Coxe fellowship (to VS).*Author to whom correspondence should be addressed: NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript factor 1 (7) or release factor 2 (8,9) have shown the location of the conserved GGQ motif in the active site for peptide release. Mut...