Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association.

Abstract

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.

RNA polymerase III interferes with Ty3 integration

“…Thus, one explanation for the difference in effectiveness of blocking between these two antisera is that antibody against the C53 subunit is directed distal to the region accessed by the integration complex, potentially presenting less of an obstacle to pol III binding at the target site. The C53 subunit is reported to be more loosely associated with the pol III complex [39] so it is also possible that it is dislodged from the polymerase by its interaction with the antibody, leaving the remainder of the polymerase available to bind to the target.…”

Functional impact of missense variants in BRCA1 predicted by supervised learning

“…Importantly, several missense variants were found to disrupt this interaction [64,65], suggesting that clustering of deleterious variants at solvent-exposed amino acid residue positions is indeed a useful indicator of binding site location. There is a large literature on the general topic of the relationship between deleterious mutations and binding sites [66][67][68][69][70][71][72][73][74].…”

Gene duplication and neofunctionalization: POLR3G and POLR3GL

“…The third subcomplex has partial structural similarity to TFIIE (Geiger et al 2010;Lefevre et al 2011). In Pol III, this subcomplex, which is detachable from the rest of the enzyme (Werner et al 1992(Werner et al , 1993Wang and Roeder 1997), contains the subunits POLR3C (RPC3/ RPC62) and POLR3F (RPC6/RPC39), with structural similarities to GTF2E1 (TFIIE-A) and GTF2E2 (TFIIE-B), respectively, as well as the subunit POLR3G (RPC7/RPC32-alpha), the only polymerase subunit without an identified counterpart in the other two transcription machineries.…”