Single ribosomal transcription units are linear, compacted Christmas trees in plant nucleoli.

Abstract

The rDNA transcription units are enormous macromolecular structures located in the nucleolus and containing 50-100 RNA polymerases together with the nascent pre-rRNA attached to the rDNA. It has not previously been possible to visualize nucleolar transcription units directly in intact nucleoli, although highly spread preparations in the electron microscope have been imaged as "Christmas trees" 2-3 microm long. Here we determine the relative conformation of individual transcription units in Pisum sativum plant nucleoli using a novel labelling technique. Nascent transcripts were detected by a highly sensitive silver-enhanced 1 nm gold procedure, followed by 3D electron microscopy of entire nucleoli. Individual transcription units are seen as conical, elongated clusters approximately 300 nm in length and 130 nm in width at the thickest end. We further show that there were approximately 300 active ribosomal genes in the nucleoli examined. The underlying chromatin structure of the transcribing rDNA was directly visualized by applying a novel limited extraction procedure to fixed specimens in order to wash out the proteins and RNA, thus specifically revealing DNA strands after uranyl acetate staining. Using this technique, followed by post-embedding in situ hybridization, we observed that the nucleolar rDNA fibres are not extended but show a coiled, thread-like appearance. Our results show for the first time that native rDNA transcription units are linear, compacted Christmas trees.