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Structure of RNA-Dependent RNA Polymerase in Bacteriophage Φ6

Hildy Joseph '13 and Samuel Kaplan '14


I. Introduction

Bacteriophage Φ6 is a well-studied virus with several unique characteristics. It attacks Pseudomonas syringae, a plant pathogen. Φ6 has important homologies to the Hepatitis C virus (HCV); research suggests similar transcription initiation processes in the two viruses (Bressanelli et al. 2002). The Φ6 genome is comprised of three segments of double-stranded RNA totaling ~13.5 kbp (Mindich 1999).

RNA-dependent RNA polymerase (RdRp) is essential to all RNA viruses. In φ6, RdRp is the sole polymerase enzyme because the bacteriophage genome lacks a native DNA phase. Structural comparisons of the protein can demonstrate evolutionary relationships between viral strains and are useful for phylogeny construction (Bruenn 1991).

Φ6pol, an RdRp, is a viral polymerase that catalyzes both replication and transcription in Φ6. Φ6pol can use either ssRNA or dsRNA as a template, producing dsRNA in either case. A single-stranded region is transcribed from dsRNA during transcription in the 3' to 5' direction (Solgado 2004). Assembly of the initiation complex occurs de novo. Because synthesis can occur without the use of a primer, viral RNA can be synthesized more readily in host organisms, which do not constitutively express viral proteins (Solgado 2004).

II. General Structure

RNA-dependent RNA polymerase is a bacteriophage Φ6 enzyme that synthesizes de novo dsRNA from either single stranded or double stranded templates (Butcher et al. 2001). The 664-residue spherical protein resembles the canonical cupped right hand RNA polymerase structure, including the finger , palm , and thumb regions (Butcher et al. 2001; Koivunen et al. 2008). NTPs are recruited to binding sites (Butcher et al. 2001) within the substrate pore, which is adjacent to the RNA synthesis catalytic site. If necessary, a plough-like structure separates dsRNA without the need for an additional enzyme. The coding strand is directed into the template tunnel which is rich in basic amino acid residues (Bruenn, 2003). Further, Mn2+ is present in the binding site and acts, along with other ions, to stabilize protein-RNA interactions.

III. Structure of NTP pore and active site

NTPs are recruited into the enzyme through a substrate pore which stabilizes and properly orients them prior to polymerization. At the entrance of the NTP tunnel (site I), basic residues K223, R225, R268, and R270 interact with the phosphate groups of incoming NTPs, positioning them in the proper orientation to polymerize (Butcher et al. 2001; Poranen et al. 2008). The first incoming nucleotide, often CTP, interacts directly with both the template RNA and the RdRp itself. The NTP passes beyond site I into the subsequent binding site (site P) of the RdRp, where it stacks against Y630 . In addition, the NTP polyermerizes here, base pairing with the second residue of the template strand (T2). A second NTP then base pairs at the first template residue (T1) and at the P site (Poranen et al. 2008). This process continues until the nascent transcript has reached a critical length. Elongation then occurs once the daughter strand of dsRNA has displaced a C-terminal subdomain (Poranen et al. 2008).

IV. RNA Binding

RNA binds in short oligonucleotides to the RdRp in a channel known as the template tunnel. Within this tunnel, many amino acids act to stabilize the polymer and bind it tightly to the channel. Cytidine, the preferred 3’ nucleotide, is bound in a pocket known as site S, far past the main catalytic site. This NTP faces the critical loop region on Y630-K631-W632 and hydrogen bonds with the main chain carbonyl group of Q629 ; its base hydrogen bonds to the side chain of the neighboring K451 and the aromatic ring of Y295 and the ribose ring is stabilized by hydrogen bonds between the O2’ group and the side chain of T633 and the main chain of E634 . The second incoming NTP engages in hydrophobic interactions with R291 and A272 . The third incoming NTP base stacks with the previously bound NTP and hydrogen bonds with G275, M273, R204, and K543 (Salgado et al. 2004). Oligonucleotide binding is illustrated in this figure.

V. Metal-ion interactions

Mg2+ and Mn2+ are both necessary as bond-mediating metal ions to ensure φ6 RdRp function (Butcher et al. 2002). Mg2+ confers a greater degree of substrate specificity (Salgado et al. 2002). Mn2+ is inhibitory at high concentrations (Butcher et al. 2002), but at low concentrations coordinates the catalytic active site and facilitates NTP binding (Poranen et al. 2008). One of the primary binding sites for Mn2+ is D454 , which is located in the palm domain. Binding induces a conformational change in the side chain of the aspartic acid whereupon hydrogen bonds form between the residue and the daughter sequence. The sugar of the first nucleotide and the phosphate backbone of the second comprise the bonds. E491 and A495 are also important binding sites for Mn2+ (Poranen et al. 2008). In mutant forms lacking available Mn2+ ions, Q491 reorients to mediate a bond directly to A495 and D454, in a similar fashion to the Mn ion coordination in the wt (Poranen et al. 2008).

VI. References

Bressanelli, S., L. Tomei, F. Rey, and R. De Francesco. 2002. Structural analysis of the hepatitis C virus in complex with ribonucleotides. J. Virol. 76(7):3482-2492.

Bruenn, J. 1991. Relationships among the positive strand and double-stranded RNA viruses as views through their RNA-dependent RNA polymerases. Nucl. Acids Res. 19(2):217-226.

Butcher, S. J., J. M. Grimes, E. V. Makeyev, D. H. Bamford, and D. I. Stuart. 2000. A mechanism for initiating RNA-dependent RNA polymerization. Nature 410:235-240.

Koivunen, M. R. L., L. P. Sarin, D. H. Bamford. 2008. Structure-function insights into the RNA-dependent RNA polymerase of the dsRNA bacteriophage φ6. In: Segmented Double-stranded RNA Viruses: Structure and Molecular Biology, ed. J. T. Patton. Caister Academic Press, Norfolk, pp. 239-258.

Mindich, Leonard. 1999. Precise packing of the three genomic segments of the double-stranded-RNA bacteriophage.

Poranen, M. M, P. S. Salgado, M. R. L. Koivunen, S. Wright, D. H. Bamford, D. I. Stuart, and J. M. Grimes. 2008. Structural explanation for the role of Mn2+ in the activity of φ6 RNA-dependent RNA polymerase. Nucl. Acids Res. 36(20):6633-6644.

Salgado, P. S., E. V. Makeyev, S. J. Butcher, D. H. Bamford, D. K. Stuart, and J. M. Grimes. 2004. The structural basis for RNA specificity and Ca2+ inhibition of an RNA-dependent RNA polymerase. Structure. 12:307-316.

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