Ionic regulation in genetic translation systems.

Abstract

The polyelectrolyte theory can provide an interpretation of the interdependence of pH, ionic strength, and polyamines one observes in the activity of ribonuclease acting on RNA. According to this theory:(i) A nucleic acid-enzyme complex and the suspending medium may be considered as two phases in equilibrium, even though within limits, the complex is soluble in water.(ii) The enzymatic catalysis is under tight control of the electrostatic potential generated by the system. Consequently, modification in electrostatic potential will induce a concomitant change in activity.(iii) The electrostatic potential can be modified through action on the system of "modulators", either "external" (ionic strength, pH, temperature, etc.) or "internal" (specific ligands, substrates, protein factors, etc.).Similarities between the reaction of ribonuclease (ribonucleate 3'-pyrimidino-oligonucdeotidohydrolase; EC 3.1.4.22) and RNA and those observed with highly organized systems catalyzing DNA, RNA, and protein synthesis suggest that the electrostatic potential also provides an important regulatory mechanism in genetic translation. In this view, an essential function of nucleic acids is to provide their enzyme partners with polyanionic microenvironments within which their catalytic activities are controlled by variation in physicochemical parameters, including the proton concentration induced through "modulation" of the local electrostatic potential.The ionic environment plays an essential role in promoting and maintaining functional states of highly organized systems for genetic translation. Relatively mild changes following the addition of mono-, di-, and even polyvalent cations markedly affect activity. Activity response to ionic strength can be of two general types. As indicated in Fig. 1 We became familiar with the "catalytic implications of electrostatic potentials" in studying the characteristic behavior of several enzyme systems functioning within polyanionic environments on biomembranes and felt that they could be of interest for the investigation of the systems for genetic translation (1, 2). 1013The common features of such systems include the close interaction of their functional and/or structural proteins with polyanions such as DNA template, mRNA, and tRNA. According to the polyelectrolyte theory, these polyanions promote, in their immediate vicinity, strong electrostatic potentials that can be modulated and that are important at the level of protein and/or enzyme activity. The hydrolysis of RNA by ribonuclease A provides a simplified model that allows successful interpretation in qualitative terms of the characteristic responses depicted in Fig. 1. We could furthermore monitor the conversion of type I into type II using the polyamines, spermidine and putrescine that specifically bind nucleic acids as "internal modulators".This preliminary paper will be followed by a detailed report published elsewhere.MATERIALS AND METHODS Materials. Ribonuclease (type IA) (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase; E...

Regulation of Turnover GTPase Activity of Elongation Factor G: the 30‐S‐Coupled and 30‐S‐Uncoupled Reactions

“…Fig.8 shows that lowering the pH raised the K" optimum of the 30-S-coupled EF-G GTPase reaction. This suggests [24] that negative charges, probably from ribosomal RNA, are exposed in the vicinity of the catalytic center, upon interaction between the ribosomal subunits.…”

“…Moreover, Douzou and Maurel [24] have shown that they partake in ionic regulation at concentrations far below the amounts required to obtain similar effects with monovalent cations. This led to the distinction between polyamines as 'internal modulators', and monovalent cations or pH as 'external modulators', [24].…”

“…Moreover, Douzou and Maurel [24] have shown that they partake in ionic regulation at concentrations far below the amounts required to obtain similar effects with monovalent cations. This led to the distinction between polyamines as 'internal modulators', and monovalent cations or pH as 'external modulators', [24]. Fig.9 shows that also in our system, spermidine exerts a strong effect: it reduces the EF-G GTPase activity in the presence of the 50-S subunit alone (squares), and it changes the J curve obtained with both subunits from 'type I' (bell-shaped) to one closer to 'type 11' (steadily descending), probably by binding specifically to the phosphate groups of the ribosomal RNA [24].…”

“…This led to the distinction between polyamines as 'internal modulators', and monovalent cations or pH as 'external modulators', [24]. Fig.9 shows that also in our system, spermidine exerts a strong effect: it reduces the EF-G GTPase activity in the presence of the 50-S subunit alone (squares), and it changes the J curve obtained with both subunits from 'type I' (bell-shaped) to one closer to 'type 11' (steadily descending), probably by binding specifically to the phosphate groups of the ribosomal RNA [24]. An intriguing property of the assay system containing spermidine is its great stability against changes in salt concentration: thus, at 6 mM spermidine the Our results indicate that the EF-G-dependent EF-G GTPase activity dependent on both ribosomal GTPase activities expressed in the presence of both subunits was constant between 50 and 200 mM NH: , ribosomal subunits or of the 50-S subunit alone have while 30-S-uncoupled EF-G GTPase activity was the same basic mechanism; indeed, their V , K,, almost totally suppressed.…”

“…A possible approach to examine the interaction between EF-G and the ribosome was furnished by the theory of ionic regulation recently formulated by Douzou and Maurel [24] on the basis of the polyelectrolyte theory ; it envisages participation of locally concentrated electric charges in catalytic reactions. Experimentally, this effect manifests itself in an interdependence between pH and salt optima.…”