In the 1950's, it was scientific belief that DNA was directly involved in protein synthesis, as the cellular function of RNA was not understood. However, based on the fact that reticulocytes and enucleated cells still produced proteins, some scientists deduced that DNA was not directly involved in protein synthesis. Polymerase phosphorylase was predicted to be responsible for cellular RNA synthesis (Losick et al 1976) until 1959 when Samuel B. Weiss and Leonard Gladstone first discovered DNA-dependent RNA polymerizing activity in the nuclear fraction of rat liver cells. Before then, active polymerase was always discovered in RNA extracts of bacterial RNA.
Weiss decided to run an experiment based on the assumption that RNA synthesis should be analogous to DNA synthesis (Losick et al 1976). He phosphorylated [32P]-labeled 5'CMP with ATP and crude brewer's yeast. The resultant [32P] was eluted on a Dowex-1-Cl column. A crude rat liver fraction isolated from the lower sedimenting fraction containing cell debris and nuclei showed incorporation of the [32P]-CTP into a TCA-precipitable fraction of RNA. Optimal incorporation of the labeled CTP occurred in the presence of other unlabeled ribonucleotide triphosphates as opposed to diphosphates. Addition of DNase slowed the incorporation, while addition of RNase significantly reduced the RNA chain length. However, Weiss and Gladstone did not indicate the presence of more than one class of RNA polymerase.
Later in 1969, Robert G. Roeder and William L Rutter separated and named the three mammalian RNA polymerases. They considered that there might be three different classes of RNA polymerases based on 3 assumptions:
1. Ribosomal RNA has a base composition different from other classes of RNA molecules.
2. Ribosomal genes are found in the nucleolus, while other classes of RNA molecules are dispersed in the nucleoplasm.
3. The regulation of ribosomal RNA synthesis and "DNA-like RNA," i.e. messenger RNA (mRNA) were independently regulated.
They isolated nuclei from rat liver cells. The RNA polymerase "activities" were dissolved and incompletely purified using sonication at a high ionic concentration, causing a high enzyme yield. They lowered the ionic concentration removed chromatin from the solution using centrifugation and ammonium sulfate fractionation. The enzymes were wholly purified using DEAE-Sephadex chromatography. Each polymerase was isolated and named according to its ammonium sulfate elution concentration. RNA polymerase I was isolated at the highest elution concentration. Polymerase II was isolated at medium salt concentration and polymerase III was isolated at the lowest concentration (Young 1991; Roeder and Rutter 1970).
The polymerases maintained functionally discrete properties after freezing, thawing, dilution, reconcentration, and second chromatographic purification. Polymerase II was characterized as easily soluble and found primarily in the nucleoplasm. Also, high ionic strength of Mn2+ increased mRNA synthesis and this activity was in the nucleoplasm. RNA polymerase I was localized in the nucleolus, while the functions of RNA polymerase III had not been characterized yet. In a subsequent experiment, the dynamic duo confirmed different activities for polymerases I and II at different stages of sea urchin embryo development. They found that during early cell division (blastula and postgastrula stages) synthesis of rRNA remains constant per cell attributed to the activity of RNA polymerase I, while mRNA synthesis increased in the cell. Thus, they confirmed that RNA polymerase II was responsible for the synthesis of "DNA-like RNA."