SNAP output for PDB entry 5msg [SNAP web server]

PDB-id

5msg; SNAP-derived features in text and JSON formats; DNAproDB

Class

viral protein

Method

X-ray (3.8 Å)

Summary

Influenza b polymerase bound to vrna promoter and capped RNA primer

Reference

Reich S, Guilligay D, Cusack S (2017): "An in vitro fluorescence based study of initiation of RNA synthesis by influenza B polymerase." Nucleic Acids Res., 45, 3353-3368. doi: 10.1093/nar/gkx043.

Abstract

Influenza polymerase replicates, via a complementary RNA intermediate (cRNA), and transcribes the eight viral RNA (vRNA) genome segments. To initiate RNA synthesis it is bound to the conserved 5' and 3' extremities of the vRNA or cRNA (the 'promoter'). 5'-3' base-pairing in the distal promoter region is essential to position the template RNA at the polymerase active site, as shown by a new crystal structure with the 3' end threading through the template entry tunnel. We develop fluorescence polarization assays to quantify initiation of cap-primed (transcription) or unprimed (replication) RNA synthesis by recombinant influenza B polymerase bound to the vRNA or cRNA promoter. The rate-limiting step is formation of a primed initiation complex with minimally ApG required to stabilize the 3' end of the template within the active-site. Polymerase bound to the vRNA promoter initiates RNA synthesis terminally, while the cRNA promoter directs internal initiation at a significantly lower rate. Progression to elongation requires breaking the promoter 5'-3' base-pairing region and favourable compensation by the emerging template-product base-pairs. The RNA synthesis assay is adaptable to high-throughput screening for polymerase inhibitors. In a pilot study, we find that initiation at the cRNA promoter is unusually susceptible to inhibition by 2'F-2'dNTPs.