FIG. 3.

(A) Synthesis of RNA in vitro. Preinitiation RNA replication complexes were isolated from 40-μl HeLa translation-replication reaction mixtures containing 2 mM guanidine HCl and the indicated RNAs after incubation at 30°C for 6 h. The complexes were resuspended in 40-μl reaction mixtures containing fresh HeLa S10 extract, translation initiation factors, and 25 μCi of [α-³²P]CTP. Reaction mixtures 2, 4, 6, and 8 also contained 100 μg of puromycin/ml; reaction mixture 9 contained 2 mM guanidine HCl. The reaction mixtures were incubated at 34°C for 2 h; the labeled RNA products were denatured with glyoxal and characterized by electrophoresis in a 1.1% agarose gel followed by autoradiography (38). The positions of migration of RNA markers run on the same gel and visualized by staining with ethidium bromide are indicated. (B) Stability of RNA templates. ³²P-labeled RNAs were synthesized and incubated for various times in HeLa translation-replication reactions, and samples were analyzed on agarose gels as described in Materials and Methods. Intact RNA migrating to the correct position on the gel was quantitated by phosphorimaging. The amounts of radioactivity at the start of the incubation were the same for both samples.