The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.RNA polymerases belong to two different classes. One is represented by the RNA polymerases of SP6, T-uneven phages, and mitochondria that have polymerizing activity associated with a single polypeptide. The second class is made up of multisubunit enzymes found in eubacterial, archaebacterial, and eukaryotic cells (see references 41 and 47 for reviews). The two largest subunits of these enzymes are well conserved in all cellular enzymes, as indicated initially by immunological studies (reviewed in reference 40) and by subsequent comparison of the amino acid sequences deduced from the DNA sequences of the cloned genes (see references 41 and 47 for reviews). Comparison of the largest subunits of eukaryotic RNA polymerases A, B, and C and of the archaebacterial enzymes disclosed eight regions of homology, which have been termed domain a to domain h (20,41,47). Similarly, the second-largest subunit can be divided into a succession of conserved domains (41, 46). Most but not all of these domains are also present in the cognate eubacterial large subunits P' and ,B. Since the two largest subunits account for around 70% of the molecular mass of RNA polymerases, it is reasonable to propose that conserved domains participate in the catalytic functions of these enzymes. Some biochemical and genetic evidence supports this view. For instance, mutations affecting the nucleotidebinding site (34) and transcription termination (18,22) are located within conserved domains of the P-like subunit.Little is known of the structural or catalytic roles played by the largest subunit of RNA polymerases. Several genetic analyses have shown that RNA polymerase conditional mutations in the largest subunit tend to map in the conserved domains (14,38,53). Moreover, the fact that all a-amanitinresistant mutations affect the largest subunit of the B enzyme (see reference 37 for a review) suggests that the largest subunit is implicated in chain elongation. In the mouse, a-amanitin resistance results from an amino acid change in the strongly conserved domain f (3).The two large subunits are also involved in zinc binding.

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...