A helical twist-induced conformational switch activates cleavage in the hammerhead ribozyme

We have captured the structure of a pre-catalytic conformational intermediate of the hammerhead ribozyme using a phosphodiester tether formed between I and Stem II. This phosphodiester tether appears to mimic interactions in the wild-type hammerhead RNA that enable switching between nuclease and ligase activities, both of which are required in the replicative cycles of the satellite RNA viruses from which the hammerhead ribozyme is derived. The structure of this conformational intermediate reveals how the attacking nucleophile is positioned prior to cleavage, and demonstrates how restricting the ability of Stem I to rotate about its helical axis, via interactions with Stem II, can inhibit cleavage. Analogous covalent crosslinking experiments have demonstrated that imposing such restrictions on interhelical movement can change the hammerhead ribozyme from a nuclease to a ligase. Taken together, these results permit us to suggest that switching between ligase and nuclease activity is determined by the helical orientation of Stem I relative to Stem II.     

Folding of the hammerhead ribozyme: pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair.     

Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme

The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca²⁺, Mg²⁺, Mn²⁺, and Sr²⁺ have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed.     

An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method

We describe a chemical coupling procedure that allows joining of two RNAs, one of which contains a site-specific base analog substitution, in the absence of divalent ions. This method allows incorporation of nucleotide analogs at specific positions even into large, cis-cleaving ribozymes. Using this method we have studied the effects of substitution of G638 in the cleavage site loop of the VS ribozyme with a variety of purine analogs having different functional groups and pK(a) values. Cleavage rate versus pH profiles combined with kinetic solvent isotope experiments indicate an important role for G638 in proton transfer during the rate-limiting step of the cis-cleavage reaction.     

A modified approach to the measurement problem: objective reduction in the retinal molecule prior to conformational change

A new analysis of the measurement problem reveals the possibility that collapse of the wavefunction may now take place just before photoisomerization of the rhodopsin molecule in the retinal rods. It is known that when a photon is initially absorbed by the retinal molecule which, along with opsin comprises the rhodopsin molecule, an electron in the highest pi orbital is immediately excited to a pi* orbital. This means that a measurement or transfer of information takes place at the quantum level before the retinal molecule commences the conformational change from cis to trans. This could have profound implications for resolving some of the foundational issues confronting quantum mechanics.     

A Tris-induced change in the midpoint potential of Z,the donor to photosystem II,as determined by the kinetics of the back reaction

A new method of measuring the rate of the back reaction from the state Z⁺ P680 Q_A^? in Tris-washed chloroplasts is described. By using ratios of back reaction rates we demonstrate a Tris-induced change in the equilibrium between Z and P680 and attribute this change to an alteration of the midpoint potential of Z by Tris treatment. We also demonstrate that the previously observed inhibition of the back reaction by ADRY reagents can be localized at Z and understood in terms of electron donation to Z⁺ by ADRY reagents.