THE GENE COMPLEMENTING A TEMPERATURE-SENSITIVE CELL-CYCLE MUTANT OF BHK CELLS IS THE HUMAN HOMOLOG OF THE YEAST RPC53 GENE, WHICH ENCODES ASUBUNIT OF RNA POLYMERASE-C (III)

The temperature-sensitive BN51 cell cycle mutant of BHK cells arrests in G1 at the nonpermissive temperature (39.5-degrees-C). We have previ ously reported cloning the gene which complements this mutation. The c omplementing gene encodes a highly charged protein with a basic amino- terminal domain and an acidic carboxyl-terminal domain. We have recent ly found that the predicted BN51 protein shows significant homology to the 53 kilodalton subunit of RNA polymerase C (III) from Saccharomyce s cerevisiae. Consistent with this, antibodies raised to fusion protei ns containing BN51 coding sequences and antipeptide antibodies reveal that the BN51 gene encodes a 48 kilodalton protein which appears to be located primarily in the nucleus following subcellular fractionation and by immunohistochemistry. Analysis of RNA polymerase III activity i n temperature-sensitive BN51 cells by nuclear runoff transcription ass ay reveals a marked drop in RNA polymerase III transcription after 48 h at the nonpermissive temperature (39.5-degrees-C). This is correlate d with a significant decrease in low molecular weight RNAs after 48 h at 39.5-degrees-C. In addition, RNA polymerase Ill activity in S100 ex tracts of BN51 cells is more sensitive to heat inactivation at 39-degr ees-C than control extracts from BHK cells. When the yeast gene is int roduced into the BN51 cells in a mammalian expression vector, it weakl y complements the BN51 mutation in that it prevents cell death at 39.5 -degrees-C. The mechanism by which inhibition of RNA polymerase III ac tivity leads to arrest in G1 is unclear but is not due to a marked dec rease in total protein synthesis.