Characterization of the RNA Synthesizing Activity of Isolated Kidney Nuclei

Abstract

The limiting factor in RNA synthesis by isolated kidney nuclei is RNA nucleotidyltransferase at high salt concentrations but at low salt concentrations template availability becomes limiting.oc-Amanitin inhibits 85 % of the activity at high salt concentrations but only 20-50 % of the activity at low salt concentrations. Exogenous DNA is utilized at low salt concentrations [up to O·lM (NH4hS041 but not at high salt concentrations. The effect of increasing salt concentration is mainly to cause an increase in the length of chains synthesized. Initiation rates are not increased by high salt concentrations. The apparent Km for UTP is 8-10 11M at high salt concentrations, indicating that assays performed at low UTP concentrations are likely to give inaccurate results. The activation energy for the reaction at low salt concentration is less than that for the reaction at high salt concentration. The RNA synthesizing capacity of kidney nuclei is dependent on the method of isolation, and preparation by a modification of the Chauveau method (Chauveau et al. 1956) yields the most active nuclei.

Characterization of double-stranded ribonucleic acid sequences present in the initial transcription products of rat liver chromatin

“…(1) The synthesis of RNA in vitro was performed under conditions limiting elongation of RNA chains while optimizing initiation specificity (low RNA polymerase/DNA ratio; low ionic strength; unsheared template) (Cox, 1973;Story & Wheldrake, 1975;Tsai et al, 1975). The short RNA sequences obtained under these conditions could be preferentially transcribed on palindromic DNA sequences, since they are particularly rich in double-stranded 'hairpins'.…”