Purification of rat liver and mouse ascites DNA-dependent RNA polymerase I

Abstract

Three forms of RNA polymerase were assayed in nuclei and nucleoli isolated from rat liver and from Krebs II ascites cells. Assays of rat liver nuclei in the absence of exogenous DNA showed polymerase I accounted for 72% of the total activity, polymerase II for 17%, and polymerase III for 11%. The total activity in ascites nuclei was similar but the ratios of polymerase activities were different: polymerase I, 53%; polymerase II, 41%; and polymerase III, 6%. These values may reflect differences in the transcriptional activity of the nuclei. After isolation of nucleoli, both rat liver and ascites polymerase I accounted for 85% of enzyme activity. When exogenous calf-thymus DNA was added to nucleoli, there was a greater than 50% increase in activity suggesting that less than one-half of the polymerase I present was bound to endogenous template. Polymerase I was solubilized from either rat liver or ascites nucleoli by sonication at high ionic strength and subsequently purified by ion filtration, phosphocellulose, sucrose gradient centrifugation, and DNA-cellulose chromatography. The essentially homogenous ascites enzyme had a specific activity of 86 units/mg when assayed with native calf-thymus DNA and of 876 units/mg when assayed with poly(deoxycytidylic acid). Electrophoresis of the enzyme in sodium dodecyl sulfate indicated the presence of six subunits with molecular weights of 205 000, 125 000, 51 000, 44 000, 26 000 and 16 000. After the same purification procedure, the rat liver enzyme had a similar specific activity (98 units/mg) on native calf thymus and 362 units/mg on poly(deoxycytidylic acid).

Protein kinase activity of RNA polymerase I purified from a rat hepatoma: probable function of Mr 42,000 and 24,600 polypeptides.

“…The subunit composition and purity of hepatoma RNA polymerase I are comparable with those obtained in other laboratories (12,13,15).…”

Increased transcription and decreased degradation control and recovery of liver ribosomes after a period of protein starvation

“…The reaction stops very soon in all cases, suggesting that the elongation of RNA chains is the main activity that isolated nuclei retain. Normal liver nuclei incorporate 70pmol of UMP/mg of DNA in 10min, a value close to the 74pmol of UMP/mg of DNA in 10min reported for rat liver (Goldberg et al, 1977). The initial rate of transcription and final plateau value are increased in nuclei from re-fed livers as compared with normal livers, whereas no difference between the latter and nuclei from depleted livers can be detected.…”

Affinity Purification of Mammalian RNA Polymerase I

“…Immunopurification of RNA Polymerase I Using Affinitypurified Anti-A194 Antisera-Past purification protocols have resulted in preparations of vertebrate RPI consisting of two large polypeptides and as few as three or four smaller subunits (46,74,75). However, more recent studies indicate that purified murine RPI may consist of at least 11 subunits and two or three associated factors (24,53,76).…”