Transcription of Superhelical DNA from Cell Nuclei

Abstract

Structures resembling nuclei may be released by gently lysing human or frog cells in solutions containing non‐ionic detergents and 1.95 M NaCl. These structures, or nucleoids, sediment in sucrose gradients containing intercalating agents in the manner characteristic of DNA that is intact, supercoiled and circular. They are depleted of nuclear protein and contain no endogenous RNA polymerase activity. We describe conditions for the transcription in vitro of these nucleoids by the RNA polymerase of Escherichia coli. We compared the kinetics of RNA synthesis directed by nucleoids and by DNA prepared using conventional procedures: nucleoids direct RNA synthesis at three to four times the rate of an equivalent weight of pure DNA and under appropriate conditions the DNA of each nucleoid can direct the synthesis of twice its own weight of RNA. Most of the newly‐synthesized RNA remains within the nucleoid. Experiments with the inhibitor, rifampicin, reveal that all RNA synthesis depends upon the initiation of new RNA chains and that nucleoids and pure DNA possess similar numbers of initiation sites. RNA synthesis directed by nucleoids has unusual kinetics: maximal rates are attained only after a lag of about ten minutes. Almost no lag is seen with pure DNA. The lag is due to the slow rate of formation of complexes of polymerase with nucleoid DNA that can initiate. Removal of supercoils by irradiation with γ‐rays or by the addition of the unwinding ligand, ethidium bromide, decreases the numbers of polymerase molecules able to initiate synthesis rapidly and increases the lag before maximal synthetic rates are achieved. Loss of super‐coiling does not alter the maximum synthetic rate. Supercoiling in eukaryotic DNA clearly influences the initiation of RNA synthesis. These results are discussed with reference to the effects of supercoiling on the transcription of prokaryotic templates.