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  • Title: DNA-dependent RNA polymerases I and II from kidney. Effect of polyamines on the in vitro transcription of DNA and chromatin.
    Author: Jänne O, Bardin CW, Jacob ST.
    Journal: Biochemistry; 1975 Aug 12; 14(16):3589-97. PubMed ID: 1164498.
    Abstract:
    DNA-dependent RNA polymerases I and II were purified from pig kidney nuclei by chromatography on DEAE-Sephadex and phosphocellulose. When nonlimiting amounts of double-stranded DNA were used as the template, the in vitro transcription was markedly stimulated by spermidine and spermine. Maximal stimulation of RNA polymerase I occurred at 2-5 mM spermidine and 0.5-2 mM spermine, whereas optimal polyamine concentrations for RNA polymerase II were 5-10 and 1-5 mM for spermidine and spermine, respectively. DNA transcription by polymerase II was stimulated to a greater extent than that of polymerase I. Higher spermine (5-10 mM) concentrations were strong inhibitors of both polymerases under these conditions. The apparent Km of RNA polymerases I and II for UTP was unchanged at optimal polyamine concentration; under the same conditions the maximal reaction velocity was increased two- to three-fold and was essentially due to an increase in the rate of chain elongation. Thus, in a typical experiment the average chain length as determined by the UMP/uridine ratio increased from 570 to 1330 and the chain elongation rate increased from 0.64 to 1.44 nucleotides times sec-1 in the presence of spermine. When limiting quantities of native DNA were employed as the template, both RNA polymerases I and II were inhibited by 1-2 mM spermine. Kidney chromatin could be transcribed by homologous RNA polymerases with an efficiency ranging from 2 to 10% of that with native DNA. When chromatin was used in nonlimiting amounts instead of DNA, RNA polymerase II activity was again stimulated about two-fold at 2 mM spermine. Under these conditions, RNA polymerase I activity was inhibited by spermine. The inhibition of RNA synthesis in vitro at limiting quantities of templates (DNA or chromatin) could be overcome by preincubation of the enzyme with templates before polyamines were added. This inhibition thus appears to be due to a block in the initiation of RNA chains. Similar inhibition of transcription by RNA polymerase II was also observed with limiting quantities of chromatin as the template.
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