A Study of the Structure of Trypsin-Like Serine Proteinases. 2. A Study of Tryptophan Fluorescence in Variants of Miniplasminogen with an Altered Primary Structure

Biophysics - Abstract—Variants of miniplasminogen with an altered primary structure have been designed to study previously described changes in tryptophan fluorescence during plasminogen...     

A Study of the Structure of Trypsin-Like Serine Proteinases: 1. Study of Mini-Plasminogen Activation Using Tryptophan Fluorescence

Biophysics - Abstract—It has been shown that the curve of the time dependence of tryptophan fluorescence during plasminogen activation by urokinase is well correlated with kinetic curves of...     

Properties of Antigenomic HDV Ribozyme Consisting of Two RNA Chains

B:LS ribozyme, a trans-variant of naturally occurring HDV ribozyme, has been constructed. The ribozyme consists of a substrate-containing LS chain and a catalytic B chain and differs from previously constructed trans-ribozymes in the length and nucleotide sequence of its oligonucleotide chains (33 and 34 bp, respectively). The chains readily associate with each other at room temperature, at which the LS cleavage reaction is negligible, which makes it possible to investigate association of the intact chains. At the same time, the self-cleavage rate constant for the trans-ribozyme B:LS at 50°C is close to those for the previously studied permuted cis-ribozymes, especially the LSB variant. In addition, the dependence of trans-ribozyme on reaction conditions (Mg²⁺ concentration, pH, and temperature) resembled that of cis-ribozyme. Similar to other trans-ribozymes, B:LS ribozyme demonstrated the ability for multiple turnover of the B strand with an excess of the substrate LS chain. The kinetic model of the self-cleavage reaction for B:LS is presented at http://www.cardio.ru/labgen/RZ_r.html. Taken together, our results show that the novel trans-variant of HDV ribozyme can be used as a model for analyzing the process of HDV ribozyme self-cleavage.     

Properties of antigenomic hepatitis delta virus ribozyme cis- and trans- analogs

A series of permuted variants of antigenomic HDV ribozyme and trans-acting variants were constructed. The catalytic activity study of the ribozymes has shown that all the variants were capable of self-cleaving with equally biphasic kinetics. Ribonuclease and Fe(II)-EDTA cleavage have provided evidence that all designed ribozymes fold according to the pseudoknot model and the conformations of the initial and cleaved ribozyme are different. A scheme of HDV ribozyme self-cleavage reaction was suggested. The role of hydrogen bonds in the reaction was evaluated by substitution of ribose in the ribozyme for deoxyribose. It was found that the 2'-OH group of U23 and C27 is critical for the reaction to occur; the 2'-OH group of U32 and U39 is important, while 2'-OH groups of other nucleotides of loop 3, stem 4 and stem 1 are unimportant for the cleavage activity.     

Interaction between RNA molecules of a two-component trans analog of antigenomic HDV ribozyme

A study was made of the association of the RNA components forming a B:LS two-component rans analog of the antigenomic HDV ribozyme. The B:LS ribozyme differed from known trans ribozymes in the sizes and nucleotide sequences of its components (33 and 34 nt, respectively), the topology of its functional parts, and the lack of a very short cleavage product. Compared to the cis ribozyme, B:LS showed similar dependences on the reaction conditions (Mg²⁺ concentration, pH, temperature) and a similar biphasic kinetic curve of self-cleavage. The kinetic model of B:LS self-cleavage (available at www.cardio.ru/labgen/RZ_e.html) describes a possible cause of the biphasic kinetic curve as a change in the rate-limiting step of consecutive conformational transitions accompanying self-cleavage. Another possible cause is an interaction between the molecules involved in cleavage, i.e., multimerization of whole ribozyme molecules with their components or the reaction products. B:LS provides a convenient model for studying such interactions, since the mode of component binding allows generation of 1B:2LS and 2B:1LS complexes as well as complexes with the cleavage products. Nondenaturing PAGE was used to study the factors affecting association and dissociation of the ribozyme components. The possibility of interactions between the RNA components of the cis and trans ribozymes was demonstrated experimentally. It was shown that the ribozyme is capable of multimerization when LS is in excess over B and that the cleavage products are not significantly involved in this process. The results suggest intermolecular interactions for the cleavage of the natural cis ribozyme.     

Kinetic Self-Cleavage Models for Permuted Variants of the Antigenomic HDV Ribozyme

The kinetic characteristics have been studied for noncircularly permuted variants of the human hepatitis delta virus antigenomic ribozyme to find out the cause of the two-phase kinetics of the self-cleavage reaction. Different ways of reaction initiation, suboptimal conditions, and jumpwise changes of reaction conditions have been used, and the temperature dependences have been studied. A correlation has been shown between the apparent kinetic constant of the first reaction phase and the portion of the ribozyme molecules that self-cleaved during the first phase. Partial restoration of the initial reaction characteristics has been shown by the reinitiation of reaction being stopped after completing the first phase. On the basis of all the data obtained, a scheme of the self-cleavage reaction has been proposed including: (i) activation of the ribozyme with energy of 40–50 kcal/mol and a characteristic time of several deciminutes under optimal reaction conditions; (ii) fast and reversible reaction of the phosphodiester bond cleavage; (iii) reaction leading to isomerization of the 3",5"-phosphodiester bond to the 2",5" bond in the self-cleavage site with a characteristic activation time of tens of minutes; and (iv) practically irreversible conformational change leading to fixation of the cleavage by immobilization of the 5"-terminal nucleotide of the product in the center of the formed structure and displacement of the 3"-terminal nucleotide to the periphery. The latter process has a characteristic time of tens of minutes and a low activation energy.