Structural Studies of RNA-dependent RNA polymerases

Salgado, Maria Paula Santos Cordeiro (2006). Structural Studies of RNA-dependent RNA polymerases. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000e94e

Abstract

Most RNA viruses possess an RNA-dependent RNA polymerase (vRdRP), responsible for viral genome replication and transcription. Furthermore, a number of eukaryotic organisms, including plants, fungi, protozoa and some metazoans, produce cellular RdRPs (cRdRPs) involved in RNA silencing mechanisms.

One of the best studied vRdRPs is that of the dsRNA bacteriophage Φ6. Structures of Φ6 RdRP (Φ6pol) in complex with RNA oligonucleotides revealed the basis for template specificity: the extra hydroxyl group leads to additional RNA-protein interactions, further stabilizing the template. Structures of "manganese-free" Φ6pol and a mutated form of the protein with lower affinity for the ion (E491Q mutant) provided some hints to the role of manganese. The structure of a complex of Φ6pol with RNA oligonucleotides and GTP with the catalytic magnesium ions substituted by calcium ions shows a distorted geometry of the initiation competent state, providing a molecular explanation of the calcium inhibitory effect. Finally, the structure of a mutated form of the protein (628QYKW632-SG mutant) prone to back-priming initiation revealed a set of contacts important for de novo initiation. Considering the high structural homology of Φ6pol with other vRdRPs, particularly from (+)ssRNA Hepatitis C Virus (HCV), insights into the mechanistic and structural details of Φ6pol are thought to be relevant to the general understanding of vRdRPs.

The dimeric structure of QDE-I, an RdRP from the fungus Neurospora crassa involved in RNA silencing, revealed a surprising similarity at the active site level to multi subunit DNA-dependent RNA polymerases (DdRPs). This implies a close evolutionary relationship between these erzymes and a possible connection between RNA silencing pathways and primordial RNA polymerisation mechanisms. Furthermore, an analysis based on the structures of several template dependent polymerases suggests that they have emerged more than once over the course of evolution.