FIG. 4.

In vitro RNA synthesis mediated by the H510A polymerase. (A) Transcription initiation primed with ApG and globin mRNA primers. Transcription reactions were carried out in vitro with nuclear extracts from cells transfected either with pcDNA3 (C) (lanes 1 and 4) or with pcDNA-PB1, pcDNA-PB2, and wild-type (WT) (lanes 2 and 5) or mutant (H510A) (lanes 3 and 6) pcDNA-PA in the presence of ApG dinucleotide (lanes 1 to 3) or globin mRNA (lanes 4 to 6) primers. The positions of transcription products (TP) are indicated. The star indicates the position of a nonspecific band. (B) Transcription initiation primed with a capped RNA primer. Transcription reactions were carried out in vitro with nuclear extracts from cells transfected either with pcDNA3 (C) (lane 1) or with pcDNA-PB1 and pcDNA-PB2 either without pcDNA-PA (−PA) (lane 2) or with wild-type (WT) (lane3) or mutant (H510A) (lane4) pcDNA-PA in the presence of an 11-nt ³²P-labeled capped RNA. The positions of transcription products (TP) and the primer are indicated.