RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (M). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophiic M,-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.The eukaryotic RNA polymerases are complex enzymes composed of multiple, distinct subunits. Saccharomyces cerevisiae RNA polymerase C activity is associated with a complex of at least 13 different polypeptides ranging from 10 to 160 kDa (4,11,13,49,56). A subset of the yeast RNA polymerase C subunits is homologous to the eubacterial RNA polymerase core enzyme. C160 and C128 are homologous to the Escherichia coli 1 and 13' subunits, respectively (1, 24). Two molecules of the a subunit are found in E. coli RNA polymerase. AC40 and AC19 each have a domain similar to a functionally important domain of the a subunit (8). It was thus proposed that one copy each of AC40 and AC19 in RNA polymerases A and C is functionally homologous to the ao homodimer of the E. coli RNA polymerase. A homodimer of the B44.5 subunit is likely to represent the a homolog of the B enzyme (29, 30). The two largest subunits along with the al homologs probably perform many of the same functions in polymerase assembly and the basic catalysis of transcription as do their homologs in the well-studied eubacterial enzyme. In contrast to these four core subunits, a function has not yet been assigned to the remaining nine small subunits. Five of these subunits (ABC27, ABC23, ABC14.5, ABC10a, and ABC10,B) are shared between all three nuclear polymerases and thus probably contribute to a common eukaryotic (and possibly archaebacterial) core enzyme (4, 56). The four small subunits specifically associated with RNA polymerase C (C82, C53, C34, and C31) seem destined to perform functions specific to transcription by this polymerase. The C82 (6), C34 (52), and C31 (37) proteins have all been shown to be necessary for yeast cell viability and for the synthesis of tRNA by RNA polymerase C. In this report, we show that C53 also performs an essential cellular function required for tRNA synthesis. Furthermore, we report a sequence similarity between C53 and the BN51 protein that may encode the human homolog of C53. MATERIALS AND METHODSStrains and media. The yeast strains used in this study are described in Table 1. CMY356 is a haploid meiotic segregant obtained after sporulation of CMY242 transformed with the plasmid pEMBLYc32...