THE NUCLEOLAR ARCHITECTURE OF POLYMERASE-I TRANSCRIPTION AND PROCESSING

The nucleolus, the site of transcription and processing of the major ribosomal genes, generally reveals three distinct ultrastructural components in conventional thin-section electron micrographs (fibrillar centres, dense fibrillar component and granular component), We show here that different parts of the transcription and transcript processing pathway can be mapped to the different nucleolar components in pea root cells, This study shows the full three-dimensional arrangement of the different domains by in situ hybridization and confocal microscopy, and their correspondence with the major ultrastructural components of the nucleolus is revealed by parallel serial section electron microscopy, The active rDNA is widely dispersed in discrete foci, the larger of which, at least, correspond to well-defined fibrillar centres. A probe to the external transcribed spacer (ETS) sequence of the pre-rRNA transcripts labels clearly demarcated regions surrounding the foci of rDNA, and which we show correspond to the dense fibrillar component, Finally, a probe to the entire 45S transcript shows a higher concentration in regions corresponding to the granular component, surrounding the dense fibrillar component labelled by the ETS probe, The changes in structure that occur with heat shock show that nucleolar organization is dynamic and dependent upon transcriptional activity, These results show that the various RNA processing events are spatially highly organized and suggest a vectorial or radial model of transcription and transcript processing, where nascent and newly completed transcripts occupy zones surrounding the genes, which are in turn surrounded by regions containing the older more mature transcripts.