Abstract
A method is described for the isolation of a yeast chromatin fraction highly enriched in ribosomal DNA sequences. In the presence of exogenous yeast RNA polymerase III, this purified chromatin actively synthesizes a set of 5 S ribosomal RNAs all of which have 5'-sequences identical with mature 5 S RNA but which end with a variable number (up to 10) of additional residues at the 3'-terminus. These extra nucleotides are precisely removed by a processing nuclease found in the chromatin supernatant fraction.
Highlights
A method is described for the isolation of a yeast S RNA
Sequences identical with mature 5 S RNA but which end with a variable number of additional Isolation of Yeast DNA-dependent RNA Polymerases-Yeast residuesat the 3*-terminus. These extra nucleotides arReNA polymerase I11 was isolated from the commercial strain, F1,as precisely removed by a processing nuclease found in described previously [19,20]
Eco RI Digestion of Nuclei and Chromatin Solubilization-Yeast nuclei were washed twice with PCK plus 0.1 mb~PMSF, Nuclei were digested in 5 m~ piperazine-N,N’-bis(2-ethanesulfoniaccid), obtain faithful transcription of 5 S rRNA genes with RNA 0.8 m~ CaCL, 6 rnM MgCL, 120m~ KQ, and 10%glycerol at a find polymerase 111 and yeast extracts have not proved routinely DNA concentration of 250 pg/ml
Summary
End with a variable number (up to 10) of additional Isolation of Yeast DNA-dependent RNA Polymerases-Yeast residuesat the 3*-terminus. Eco RI Digestion of Nuclei and Chromatin Solubilization-Yeast nuclei were washed twice with PCK plus 0.1 mb~PMSF, Nuclei were digested in 5 m~ piperazine-N,N’-bis(2-ethanesulfoniaccid) (pH 7), obtain faithful transcription of 5 S rRNA genes with RNA 0.8 m~ CaCL, 6 rnM MgCL, 120m~ KQ, and 10%glycerol at a find polymerase 111 and yeast extracts have not proved routinely DNA concentration of 250 pg/ml. We have alternatively attempted to isolate a 5 S chromatin transcription complex, a strategy which was used successfully for an analysis of transcription of chloroplast DNA [14] Study of such a chromatin complex couldprovide information about initiation and terminator factors. Digestion was carried out at0°C for 16 to 20 h
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