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. 1999 Sep 15;13(18):2439–2448. doi: 10.1101/gad.13.18.2439

Figure 3.

Figure 3

Figure 3

Figure 3

In vitro transcription by 3C3A RNAP. (A) RNAP3C3A is temperature resistant in vitro. RNAP3C3A was affinity purified from 397C cells harboring the pCYB2β′3C3A plasmid and used to transcribe the T7 A1 promoter-containing DNA fragment at the indicated temperatures. Reaction products were separated on a denaturing 20% polyacrylamide gel and revealed by autoradiography. (B) RNAP3C3A is assembly defective in vitro. RNAP3C3A and RNAPWT were subjected to a denaturation/renaturation cycle by the addition of 6 m guanidine-HCl, followed by dialysis into transcription buffer. The samples were then assayed in a steady-state transcription assay, using the T7 A1 promoter as a template. Reaction products were analyzed as in A. (C) Localized radical cleavage of iron-containing RNAP3C3A. RNAPs containing plasmid-borne, His6-tagged wild-type or 3C3A β′ were affinity purified from 397C E. coli cells grown in the presence of iron and in the absence of zinc. Affinity labeling, hydroxy-radical cleavage, and reaction-product analyses were performed as described in the legend to Fig. 2.