5MSG

Influenza B polymerase bound to vRNA promoter and capped RNA primer

Summary for 5MSG

• Entry DOI: 10.2210/pdb5msg/pdb
• Descriptor: Polymerase acidic protein, RNA-directed RNA polymerase catalytic subunit, Polymerase basic protein 2, ... (7 entities in total)
• Functional Keywords: influenza b virus rna-dependent rna polymerase, vrna promoter, capped rna primer, viral protein
• Biological source: Influenza B virus; More
• Cellular location: Virion : Q5V8X3
• Total number of polymer chains: 6
• Total formula weight: 277756.68

Authors

Cusack, S.,Guilligay, D. (deposition date: 2017-01-04, release date: 2017-02-08, Last modification date: 2024-03-06)

Primary citation

Reich, S.,Guilligay, D.,Cusack, S.
An in vitro fluorescence based study of initiation of RNA synthesis by influenza B polymerase.
Nucleic Acids Res., 45:3353-3368, 2017

PubMed 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.

PubMed: 28126917
DOI: 10.1093/nar/gkx043

Experimental method

X-RAY DIFFRACTION (3.8 Å)