Structure of the Qbeta replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins

Abstract

The RNA-dependent RNA polymerase core complex formed upon infection of Escherichia coli by the bacteriophage Qbeta is composed of the viral catalytic beta-subunit as well as the host translation elongation factors EF-Tu and EF-Ts, which are required for initiation of RNA replication. We have determined the crystal structure of the complex between the beta-subunit and the two host proteins to 2.5-A resolution. Whereas the basic catalytic machinery in the viral subunit appears similar to other RNA-dependent RNA polymerases, a unique C-terminal region of the beta-subunit engages in extensive interactions with EF-Tu and may contribute to the separation of the transient duplex formed between the template and the nascent product to allow exponential amplification of the phage genome. The evolution of resistance by the host appears to be impaired because of the interactions of the beta-subunit with parts of EF-Tu essential in recognition of aminoacyl-tRNA.

Fig. 1.

Dimer and monomer of the Qβ replicase core. (A) Resolution of the TEV-digested fusion protein into dimer and monomer by gel filtration in a buffer containing 500 mM NaCl. (B) Silver stained gel and (C) its radioactivity pattern after nondenaturing PAGE of RQ135 RNA (lane 1), the monomer (lane 2), the dimer (lane 6), and complexes formed by the monomer and dimer in the presence of the RNA and GTP (lanes 3 and 7), plus CTP and [α-³²P]UTP (lanes 4 and 8) or CTP, [α-³²P]UTP, and ATP (lanes 5 and 9), and in the presence of GTP and no RNA (lane 10). The dots on the gel are radioactive markers identifying the positions of nonradioactive bands in C. Dot “ds” marks the position of the double-stranded RNA product.

Fig. 2.

Structure of the Qβ replicase core complex. (A) Cartoon representation of the monomeric core replicase containing β-subunit (Green, labeled “βS”) in complex with EF-Tu (Yellow) and EF-Ts (Dark Red). (B) The domains of the β-subunit. The thumb domain is colored blue, the palm domain green, the fingers domain magenta, and the bridge region gray. (C) The PEP (Green Sticks) binding site on the β-subunit; a hydrogen bond with Arg262 of EF-Tu (Yellow) is shown as a dashed line. Binding of PEP possibly stabilizes the interaction of EF-Tu Phe261 with the hydrophobic cluster in the β-subunit.

Fig. 3.

Intermolecular contacts within the Qβ replicase. Hydrogen bonds and electrostatic interactions are shown as dashed lines. (A) Details of the β-subunit (βS): β-subunit interface. (B) Contacts between the EF-Ts (Dark Red) coiled-coil motif and the β-subunit (Green) thumb domain. (C) The interface between the β-subunit and EF-Tu (Yellow) involving the T helix and bridge helix from the β-subunit and domains 1 and 3 from EF-Tu. (D). The complex of EF-Tu and aa-tRNA in the same orientation as in C. (E) Close-up of the β-subunit T-helix interaction with EF-Tu domain 2. (F) Insertion of the β-subunit thumb domain between EF-Tu domain 3 and EF-Ts.

Fig. 4.

RNA binding and surface properties of the β-subunit. The double-stranded RNA in the internal cavity of the β-subunit (colored as in Fig. 2B) was docked by comparison with the structure of the Norwalk virus RdRp, PDB ID code 3BSN. Template and product strand are labeled “T” and “P,” respectively, and 3′ and 5′ ends are labeled as well. (A) Cross-section through the β-subunit displaying the suggested template and NTP entrance channels together with the template exit channel. (B) Cartoon representation of the core replicase indicating the four putative channels. (C) Surface representation displaying the electrostatic potential of the β-subunit colored according to charge (Blue 10 kt/e, White 0, Red -10 kt/e) with EF-Tu and EF-Ts shown in cartoon mode. The positively charged blue surface patch containing elements of the bridge and thumb domains of the β-subunit is a possible binding site for the S1 protein, template, and product. The thumb, bridge, and fingers domains of the β-subunit are indicated on the surface representation with a black outline.