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Nucleic Acids Research, 1981, Vol. 9, No. 16 3959-3978
© 1981

MOLECULAR BIOLOGY

Selective in vitro transcription by purified yeast RNA polymerase II on cloned 2 µm DNA

Paola Ballario1, Mario Buongiorno-Nardelli2, Francesca Carnevali1, Ernesto Di Mauro3 and Francesco Pedone2

1Centra di Studio per gli Acidi Nucleici, C.N.R., Istituto di Fisiologia Generale, Università di Roma 2Istituto di Istologia ed Embriologia della Facoltà di Scienze, Universita di Roma 3Cattedra di Biologia Molecolare della Facoltà di Scienze, Università di Roma 00185 Roma, Italy

Received May 12, 1981. The in vitro transcription properties of purified yeast RNA polymerase II have been analyzed on prokaryotic plasmids (pBR322 and pBR313) and chimaeric plasmids bearing yeast 2 µm sequences (BTYP1, BTYH2 and BTYH3).

Conditions for selective transcription of the 2µm DNA sequences in chimaeric plasmids have been determined. pBR322 and pBR313 are not transcribed by the purified RNA polymerase II when not bearing eukaryotic inserts. We show that the agarose gel electrophoretic analysis of ternary transcription complexes allows the localization of nascent RNA chains.

The RNA produced has been visualized by electron microscopy (nascent RNA hybridization loops) and by gel electrophoretic analysis.

All the observed properties are shared by RNA polymerase II purified by a conventional method (1) and by a rapid alternative procedure described herein. The peculiar properties of a partially purified form of RNA polymerase II are reported.


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