Mechanism of co-transcriptional cap snatching by influenza polymerase
Nature • • science
Key Points:
- The study engineered H7N9 influenza polymerase (FluPol) mutants, including PA(E119D) to impair endonuclease activity, and generated variants with altered interfaces to the Pol II elongation complex, confirmed by sequencing.
- Proteins including mutated human transcription factors DSIF and CAK kinase trimer were expressed and purified using modified protocols; in vitro transcription and capping of RNAs were performed with quality control by gel electrophoresis and fluorometric analysis.
- Endonuclease activity assays and in vitro FluPol transcription assays were conducted using labeled RNAs, Pol II, and elongation factors to measure cleavage and transcription efficiency, with statistical analysis performed on gel quantifications.
- Cryo-electron microscopy (cr
