RPB 2

RPB2 is the second largest subunit, 140kDa, and constitutes one of the core subunits, i.e is required for enzyme activity. This requirement for cell viability was assessed through disruption of the single chromosomal gene encoding for RPB2. The viability of the meiotic products was ascertained and the recessive lethal mutation produced the expected 50:50, live to dead cell ratio. Its structure and function are homologous to the ß subunit of E. coli RNA polymerase. There are nine major segments of homology that occur in relatively identical positions (Young 1987). In fact, close to one-third of the amino acids are identical, with comparable substitutions replacing the non-identical pairs (Young 1987). These comparisons were made using the National Biomedical Research Foundation protein sequence database. Although there are significant similarities between the protein sequences, the DNA sequences exhibit less homology. Sequence analysis identified motifs implicated in binding purine nucleotides and zinc ions (Young1987).

The enzyme appears to participate in nucleotide substrate binding and phosphodiester bond formation. In addition, the subunit is involved in binding DNA and the nascent RNA chain. It is relatively resistant to limited proteolysis and is not phosphorylated. Andre Sentenac and colleagues established that this subunit retained the nucleotide-joining active site of the enzyme by experimental labeling of the active site with nucleotide binding derivatives (Sentenac 1987). Essentially an ATP analogue, 4-formylphenly-gamma-ester of ATP, was covalently bound to polymerase I, II, and III. A phosphodiester bond was formed between the analogue and [a-32p]UTP. The purpose was to identify the subunit involved in catalyzing phosphodiester bond formation with a radioactive ribonucleoside triphosphate. The enzyme subunits were separated by gel electrophoresis and auto-radiographed the gel to detect the labeled subunit. The reaction was identified as occurring exclusively in the second largest subunit (Sentenac 1987). The nucleotide-binding site is located on the C-terminus between Asn946 and Met999 (Sentenac 1990).

Subunits
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