Transcription of mouse ribosomal RNA gene with inactive extracts is activated by NAD+ in vitro

S100 extract prepared from rapidly growing mouse FM3A cells (approx. 5 x 10(5) cells/ml) transcribed ribosomal RNA gene (rDNA) much more actively in vitro than that from stationary phase cells (1-2 x 10(6) cells/ml). When the inactive S100 extract was preincubated with NAD+, rDNA transcriptional activity was restored almost to the level of the active extract. The extract activated with NAD+ exhibited a gel-shift band in the gel mobility shift assay and enhancement of protection of the sequence between -44 and -8 nt from the initiation site from exonuclease III digestion. Such an extract labeled with [32P]NAD+ was analyzed by immunoprecipitation with anti-RNA polymerase I (pol I) antibody; a protein with M(r) 130 kDa was detected. In contrast, the polypeptide was hardly labeled in the active extract. 3-Aminobenzamide, a specific inhibitor of poly ADP-ribosylation, did not inhibit the activation by NAD+. These results suggest that the activation by NAD+ is due to enhancement of the formation of initiation complex by mono ADP-ribosylation of the second-largest subunit (130 kDa) of pol I.