FIG. 5.

Transcription initiation activity of PA mutants in vitro. 293T cells were transfected with plasmids expressing PB1 and PB2-TAP, either without PA (−PA), with a wild-type PA (WT), or with mutant PAs (K102A, D108A, and K134A). TAP-tagged polymerases were partially purified from cell extracts by immunoglobulin G-Sepharose and used in vitro. As a negative control (C), cell extracts from untransfected cells were prepared. (A) Silver staining of purified TAP-tagged polymerases separated by 7.5% SDS-PAGE. The positions of PB1, PB2-TAP, and PA are shown on the right. (B) Transcription activity on a model vRNA promoter of mutant polymerases primed with a globin mRNA primer. The positions of the 27- or 28-nt-long transcription products (TP) are indicated. (C) Transcription activity of mutant polymerases primed with an 11-nt ³²P-labeled capped RNA primer (see Materials and Methods). The positions of the 27- or 28-nt-long transcription products (TP) of capped RNA primer are indicated. (D) Endonuclease activity (see Materials and Methods) of mutant polymerases. TAP-purified polymerases were incubated with [³²P]poly(A)⁺-capped RNA. The positions of the poly(A)⁺-capped RNA and specific 11-nt cleavage product are indicated on the right. (D, E, and F) Quantification of results obtained in panels B, C, and D, respectively, by phosphorimaging analysis. Data are expressed as percentages relative to the wild type (mean ± standard deviation; n = 3).