Fig. 1.

RNAPII-ser2 localizes to the mitotic kinetochore and is associated with active kinetochore activity. (A) Antibody 4H8_{total RNAPII} immunostained the kinetochores of HeLa cells, as shown by the colocalization with CREST anti-centromere signals (arrows). (B) Antibody 8WG16_{unphosphorylated}, specific for unphosphorylated RNAPII did not immunostain HeLa mitotic kinetochores. (C) Antibody H14_phosphoSer5, which recognizes RNAPII phosphorylated at serine 5 (RNAPII-ser5) associated with transcription initiation, showed no staining of the kinetochores. (D) H5_phosphoSer2, specific for elongating RNAPII, stained the kinetochores of mitotic HeLa cells (arrows). (E) Combined immunofluorescence/DNA-FISH (antibody H5_phosphoSer2 and 10q25 band-specific 153G5 BAC probe) of mouse ES mardel (10) cells showed the presence of RNAPII-ser2 at the mardel (10) neocentromere (arrow) as well as at endogenous mouse kinetochores. (F) RNAPII-ser2 was present only at the active neocentromere of the PD-NC4 chromosomes (arrow) but not at the inactive native centromere (arrowhead, as indicated by the weaker CREST signals attributed to residual CENP-B). (G) Schematic depicting the change in RNAPII phosphorylation status across the transcription cycle. At the promoter, RNAPII is unphosphorylated. At transcription initiation, RNAPII is phosphorylated at serine 5 (S5), but before the RNAPII complex is competent for transcription elongation it must be phosphorylated at serine 2 (S2). (Scale bars: 5 μm.)