Function and Structure in Phage Q@ RNA Replicase ASSOCIATION OF EF-Tu.Ts WITH THE OTHER ENZYME SUBUNITS*

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.

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 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 affected by ionic strength. Addition of QP 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 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. 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.
Qp phage RNA replicase (nucleoside triphosphate:RNA nucleotidyltransferase (RNA dependent)) is composed of four nonidentical subunits, three of which are coded for by the host, Escherichia coli (1, 2). The host polypeptides are part of the protein synthetic machinery in uninfected cells: the largest (M, = 70,000) has recently been found to be 30 S ribosomal protein Sl (3,4). The smaller two are protein synthesis elongation factors EF-Tu' and EF-Ts (M, = 45,000 and 35,000, respectively) (5). The phage-coded subunit (M, = 65,000) must be responsible for the high specificity of the enzyme for its natural template (6), since the other subunits are host-coded and are found in RNA bacteriophage RNA replicases with different template specificities (7).2 The phage-coded subunit is capable of performing the elongation reaction on synthetic templates in the absence of the host-coded subunits (8). Subunit I is required only for initiation of transcription of Q/3 RNA (9), while EF-Tu and EF-Ts are necessary for initiation of transcription of synthetic templates (8)  The enzyme is composed of one each of the four subunits (11). It can be further subdivided into two smaller complexes composed of the two larger subunits and EF-Tu.Ts (1, 2, 11). Using the technique of intramolecular protein cross-linking we have recently found that the two smaller complexes are associated in a different, "tighter," complex at high ionic strength, than at low ionic strength (11). In addition, we have shown that both GDP binding by EF-Tu and [3H]GDP exchange catalyzed by EF-Ts are inhibited by increasing ionic strength when these polypeptides form part of the replicase enzyme, although with the individual factors these activities are not influenced by salt concentration (8). We report here that increasing ionic strength results in a concomitant decrease in the ability of the enzyme to initiate transcription of synthetic templates, but not elongation of initiated RNA molecules.
There is considerable evidence that addition of RNA to QP replicase alters the quaternary structure of the enzyme: when either synthetic templates (1, 8) or Qp RNA (2) are mixed with the enzyme (with or without GTP) and the mixture is subjected to zone sedimentation on sucrose gradients, the EF-Tu and EF-Ts are separated from the other enzyme subunits which remain bound to the RNA. Furthermore we have shown (11) that when QP RNA is added to the enzyme the EF-Tu.Ts can no longer be cross-linked to the other enzyme subunits by dimethyl suberimidate.
These results suggest the possibility that the presence of RNA results in the release of EF-Tu and EF-Ts from the enzyme. In this paper we present evidence that the conformational change induced by Qp RNA EF-Tu. triphosphate (18)) at a variety of salt concentrations. Samples were periodically removed to tubes containing aurintricarboxylic acid, a reagent that prevents QP replicase initiation but not elongation (19). [3H]UTP was then 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 (no added NaCl). There is an inverse relationship between ionic strength and rate of initiation of Qp replicase on this template. The effect of ionic strength on the rate of elongation was also measured: ample time (5 min at 25") for complete initiation on poly(A,C) was allowed at low ionic strength before the addition of NaCl. (Formation of aurintricarboxylic acid-resistant initiation complexes is complete within 1 min at 25".)3The [3H]UTP was then added to permit elongation and sequential samples were removed and precipitated.
Again reinitiation was prevented by the addition of aurintricarboxylic acid with the UTP. The results (Fig. 2)