FIG. 6.

Mutational analysis of globin cleavage/polyadenylation cassette. (A) Nonspecific transcription initiation at the globin poly(A) signal. Standard in vitro transcription reactions were carried out with 0.2 pmol of purified RNA polymerase and 0.2 pmol of the indicated templates at 30°C for 15 min. Lanes: 1, φX174/HinfI marker; 2, Polh/CFS-T; 3, SP6/CFS-T; 4, SP6/Polh-M3 as template in which AATAAA was changed to AGGAAA in the C-free cassette. Sizes are indicated in nucleotides. (B) Sequence requirements for termination. Lanes: 1, φX174/HinfI marker; 2, RNA transcripts synthesized from Polh/CFS-T plasmid; 3, RNA transcripts from Polh/CFS-M1 plasmid; 4, RNA transcripts from Polh/CFS-M2 plasmid; 5, RNA transcripts from Polh/CFS-T3 plasmid. (C) Sequences of the globin cleavage/polyadenylation cassette and three mutant versions. The relevant substituted regions are underlined.