Function and structure in phage Qbeta RNA replicase. Association of EF-Tu-Ts with the other enzyme subunits.

T Blumenthal,R A Young,S Brown

doi:10.1016/s0021-9258(17)33550-0

Abstract

Qbeta replicase is a complex of four nonidentical subunits readily dissociable into two subcomplexes: 30 S ribosomal protein S1 and the phage-coded polypeptide (Subunits I + II) and protein synthesis elongation factors EF-Tu and EF-Ts (Subunits III + IV). The affinity of the two subcomplexes for one another increases with increasing ionic strength. The enzyme is capable of initiation of RNA synthesis with synthetic templates only when in the low ionic strength conformation. Elongation of initiated polynucleotide chains is not affectedby ionic strength. Addition of Qbeta RNA to the enzyme also alters its quaternary structure: the EF-Tu-Ts cannot be covalently attached to the other enzyme subunits with bifunctional cross-linking reagents in the presence of RNA. This conformational change is not influenced by ionic strength. The addition of Qbeta RNA to the enzyme, does not result in the release of EF-Tu-Ts from the other enzyme subunits: whereas free EF-Tu-Ts binds GDP independently of salt concentration, this binding by Qbeta replicase is sensitive to high ionic strength and remains so in the presence of Qbeta RNA. Furthermore, RNA does not allow the release of EF-Ts from EF-Tu by GTP as measured by sensitivity of EF-Ts activity to N-ethylmaleimide.

Highlights

Qp replicase is a complex of four nonidentical subunits readily dissociable into two subcomplexes: 30 S ribosomal protein Sl and the phage-coded polypeptide (Subunits I + II) and protein synthesis elongation factors EF-Tu and EF-Ts (Subunits III + IV)
Samples were periodically removed to tubes containing aurintricarboxylic acid, a reagent that prevents QP replicase initiation but not elongation [19]. [3H]UTP was added and the reaction was incubated for 5 min at 25”. (This is approximately 3 times as long as required to complete the chains.)3 Fig. 1 demonstrates that the greatest initial rate of initiation was found at the lowest ionic strength tested
The association of Qfl replicase subunits has been shown to be sensitive to ionic strength: EF-Tu.Ts separates from the complex of Subunits I + II during glycerol gradient centrifugation in low ionic strength buffer [1]; significant amounts of elongation factors bound to the other replicase subunits after treatment with chemical cross-linkers are found only when treated at high ionic strengths [11]

Summary

Introduction

Qp replicase is a complex of four nonidentical subunits readily dissociable into two subcomplexes: 30 S ribosomal protein Sl and the phage-coded polypeptide (Subunits I + II) and protein synthesis elongation factors EF-Tu and EF-Ts (Subunits III + IV). The enzyme is capable of initiation of RNA synthesis with synthetic templates only when in the low ionic strength conformation. Addition of QP RNA to the enzyme alters its quaternary structure: the EF-Tu.Ts cannot be covalently attached to the other enzyme subunits with bifunctional cross-linking reagents in the presence of RNA This conformational change is not influenced by ionic strength. The addition of Qfl RNA to the enzyme, does not result in the release of EF-Tu.Ts from the other enzyme subunits: whereas free EF-Tu.Ts binds GDP independently of salt concentration, this binding by Q/3 replicase is sensitive to high ionic strength and remains so in the presence of Qp RNA. It can be further subdivided into two smaller complexes composed of the two larger subunits and EF-Tu.Ts [1, 2, 11]

Methods

Results

Conclusion