The nucleolus is the site of rRNA transcription, pre-rRNA processing and ribosome subunit assembly. The nucleolus assembles around clusters of ribosomal gene repeats during late telophase, persists throughout interphase and then disassembles as cells enter mitosis. The initial step in nucleolar formation is ribosomal gene transcription, which is mediated by Pol I (RNA polymerase I) and its associated transcription factors: UBF (upstream-binding factor), SL1 (selectivity factor) and TIF-IA (transcription initiation factor IA)/Rrn3. Ribosomal gene clusters, termed NORs (nucleolar organizer regions), are found on each of the five human acrocentric chromosomes. Though transcription is repressed during metaphase, NORs that were active in the previous interphase form prominent cytogenetic features, namely secondary constrictions. The main defining characteristic of these constrictions is under-condensation in comparison with the rest of the chromosome. Extensive binding of UBF over the ribosomal gene repeat is responsible for the formation of this chromosomal feature. During interphase, the majority of the Pol I transcription machinery, though present in nucleoli, is not actively engaged in transcription. Interaction with UBF bound across the gene repeat provides an explanation for how this non-engaged Pol I machinery is sequestered by nucleoli.

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