Assembly of Q{beta} viral RNA polymerase with host translational elongation factors EF-Tu and -Ts

Abstract

Replication and transcription of viral RNA genomes rely on host-donated proteins. Qbeta virus infects Escherichia coli and replicates and transcribes its own genomic RNA by Qbeta replicase. Qbeta replicase requires the virus-encoded RNA-dependent RNA polymerase (beta-subunit), and the host-donated translational elongation factors EF-Tu and -Ts, as active core subunits for its RNA polymerization activity. Here, we present the crystal structure of the core Qbeta replicase, comprising the beta-subunit, EF-Tu and -Ts. The beta-subunit has a right-handed structure, and the EF-Tu:Ts binary complex maintains the structure of the catalytic core crevasse of the beta-subunit through hydrophobic interactions, between the finger and thumb domains of the beta-subunit and domain-2 of EF-Tu and the coiled-coil motif of EF-Ts, respectively. These hydrophobic interactions are required for the expression and assembly of the Qbeta replicase complex. Thus, EF-Tu and -Ts have chaperone-like functions in the maintenance of the structure of the active Qbeta replicase. Modeling of the template RNA and the growing RNA in the catalytic site of the Qbeta replicase structure also suggests that structural changes of the RNAs and EF-Tu:Ts should accompany processive RNA polymerization and that EF-Tu:Ts in the Qbeta replicase could function to modulate the RNA folding and structure.

Fig. 1.

Overall structure of the Qβ replicase. (A) Ribbon and (B) surface models of Qβ replicase. The Qβ replicase adopts a boat-like structure. The β-subunit, EF-Tu and EF-Ts are colored green, red, and blue, respectively.

Fig. 2.

Interactions between the subunits of the Qβ replicase. (A) Interactions between the β-subunit and the EF-Tu:Ts complex (interfaces I, II, and III). The thumb, palm, and finger domains and the helix-loop-helix (HLH) of the β-subunit are depicted by ribbon models and are colored orange, cyan, yellow, and pink, respectively. EF-Tu and -Ts are shown in surface models and are colored as in Fig. 1A. Interfaces I, II, and III are depicted in boxes. (B–D) Detailed view of the interactions in interfaces I, II, and III in A. The numbering of the amino acid residues of EF-Tu and -Ts is according to the literature (14, 44).

Fig. 3.

Structural differences between EF-Tu and -Ts. (A) A stereo-view comparison of the structures of EF-Tu:Ts in Qβ replicase (Tu and Ts are colored red and blue, respectively) and in the EF-Tu:Ts binary complex (14) (Tu and Ts are colored gray). The structural differences are observed in domain 2 of EF-Tu and a coiled-coil region in EF-Ts and are enclosed by boxes. (B and C) Detailed views of structural differences of EF-Tu and -Ts between the EF-Tu:Ts complex in Qβ replicase and the EF-Tu:Ts binary complex. The structures of EF-Tu and -Ts in the binary complex are colored gray. (D) The mutation or deletion of the amino acid residue(s) in the loop between f₂ and i₂ of EF-Tu reduced the complex formation and the expression of the Qβ replicase. (E) The deletion of the coiled-coil motif in EF-Ts reduced the complex formation and the expression of the Qβ replicase. The hexa-histidine tagged variants of EF-Tu (or EF-Ts) were coexpressed with wild-type EF-Ts (or EF-Tu) and the β-subunit and were purified by Ni-NTA column chromatography. The fractions eluted from the Ni-NTA column were separated by 10% (w/v) SDS/PAGE, and the gels were stained by Coomassie Brilliant Blue. “ Sup.” and “Ni” in the figures represent the total supernatant of the cell lysates and the eluted proteins from the Ni-NTA column, respectively.

Fig. 4.

Structure of the β-subunit. (A) A stereo view of the β-subunit structure and a schematic representation of the β-subunit. The thumb, palm, and finger domains and the HLH are colored as in Fig. 2A. (B) Superposition of the palm domain of the β-subunit (Ca-form, colored cyan) onto that of phi-6 RdRp (colored beige, PDB code: 1hhs, 21) (Left). A close-up view of the superposed catalytic core regions (Right). The carboxylates in the catalytic sites are depicted by stick models, and the corresponding amino acid residues are labeled in blue for the β-subunit and orange for phi-6 RdRp. The calcium ion in the structure of the β-subunit is colored brown. Only one metal ion was observed in the structure of the β-subunit of Qβ replicase. The manganese ion in the structure of phi-6 RdRp is colored blue.

Fig. 5.

RNA polymerization activities of core Qβ replicase variants. RNA replication activities of Qβ replicases containing β-subunit variants were assayed, using DN3 RNA as the template RNA (43). The activity (GMP incorporation activity) of the wild-type core Qβ replicase was defined as 1.0. The error bars in the graph indicate the standard deviations of more than two independent experiments.

Fig. 6.

RNA elongation model by Qβ replicase. (A) Substrate tunnels in Qβ replicase (tunnels I and II), shown in cross sections. Tunnel-I (t-I) and tunnel-II (t-II) are for the access of incoming nucleotides and a single-stranded template RNA, respectively. The template RNA, the growing RNA, and the incoming nucleotide are colored blue, red, and orange, respectively, and are shown in stick models. (B and C) Detailed view of the catalytic site of Qβ replicase, representing the elongation stage of RNA polymerization, in A. The incoming nucleotide and the 3′-nucleoside of the growing RNA are shown as stick models in B. Two magnesium ions are modeled in B using the structure of Norwalk virus RdRp (colored purple, PDB code: 3BSO), representing the RNA elongation stage (45). The nucleoside at the 3′-end of the growing RNA and the nucleoside of the template RNA that base-pairs with the nucleoside at the 3′-end of the growing RNA are shown in stick models in C.