Transcription of specific genes in isolated nuclei by exogenous RNA polymerases

Abstract

Mouse plasmacytoma (MOPC) 460 cells contain two chromatographic forms of RNA polymerase III (IIIA and IIIB) in addition to the major class I and II RNA polymerases. Nuclei isolated from these cells actively synthesize RNA. Among the discrete transcription products observed are the 5S and 4.5S RNAs and additional low molecular weight RNA species (approximately 5.8S, 6.3S, and 6.6S in size). The 4.5S RNAs appear to be tRNA precursors since they can be converted in vitro to 4S RNAs. Studies with alpha-amanitin have shown that the synthesis of these discrete RNA species, and other uncharacterized transcripts somewhat larger in size, is mediated by an endogenous RNA polymerase III activity(ies). Nuclear RNA synthesis is stimulated by exogenous purified RNA polymerases. Exogenous MOPC class III RNA polymerases stimulate the synthesis of each of the distinct low molecular weight species (including 5S and 4.5S RNAs) about 3-6 fold. The hybridization of nuclear transcripts to purified 5S genes (5S DNA) confirms that exogenous class III RNA polymerases stimulate (approximately 4 fold) the synthesis of ribosomal 5S RNA. The 5S RNA genes in nuclei are transcribed asymmetrically by both the endogenous and the exogenous class III enzymes. Exogenous RNA polymerase III from Xenopus laevis ovaries stimulates 4.5S and 5S RNA synthesis in MOPC nuclei as effectively as do the MOPC class III RNA polymerases. However, exogenous MOPC class I and II RNA polymerases do not stimulate 4.5S and 5S RNA synthesis, suggesting that this effect is specific for the structurally similar class III RNA polymerases.