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).