Active RNA polymerase I is fixed within the nucleus of HeLa cells

Abstract

We have investigated whether active RNA polymerase I, the enzyme responsible for transcribing ribosomal RNA, is immobilized by attachment to a large subnuclear structure in HeLa cells. As unphysiological salt concentrations induce artifacts, we have used isotonic conditions throughout the preparative and analytic procedures. Cells are encapsulated in agarose microbeads and lysed in Triton and a 'physiological' buffer; then soluble proteins and RNA diffuse out through the agarose pores to leave encapsulated chromatin. This can be manipulated without aggregation but is accessible to molecular probes; it retains the replicational and transcriptional activities of the living cell. After treatment with a restriction endonuclease, most chromatin can be removed from beads by electrophoresis: then active ribosomal genes and polymerase I remain behind. Active ribosomal genes are very accessible to nuclease digestion whilst the rest are even more inaccessible than inactive globin genes. Our observations confirm the complex organization of rDNA within nucleoli and are compatible with transcription occurring at fixed sites. A model for transcription involving an attached polymerase is presented.