Ms. Puvvada et al., INHIBITION OF BACTERIOPHAGE-T7 RNA-POLYMERASE IN-VITRO TRANSCRIPTION BY DNA-BINDING PYRROLO[2,1-C][1,4]BENZODIAZEPINES, Biochemistry, 36(9), 1997, pp. 2478-2484
The interactions of several pyrrolo[2,1-c][1,4]benzodiazepine (PBD) an
titumor antibiotics with linearized plasmid pGEM-2-N-ras DNA have been
analyzed by quantitative in vitro transcription (QIVT) and in vitro t
ranscription footprinting (IVTF) methods. A concentration-dependent in
hibitory effect of the PBDs on transcription is observed using both te
chniques. The rank order for overall inhibition of transcription by th
e QIVT method is found to be: sibiromycin > tomaymycin > anthramycin >
DC-81 > neothramycin, whereas the IVTF experiments show a different r
anking: sibiromycin > anthramycin > neothramycin > tomaymycin. In addi
tion, stimulation of transcription was observed at low PBD concentrati
ons in both the QIVT and IVTF experiments. These results demonstrate u
nequivocally that the formation of PBD-DNA adducts at AGA-5' base sequ
ences on the transcribed strand results in transcription blockage for
all PBDs examined. Furthermore, the sequence of flanking base pairs ap
pears to influence the degree of blocking, with the sequences ACAGAAA-
5', AAAGATG-5', AGAGATA-5', and CAAGAAC-5' providing the most pronounc
ed blocks for all PBDs studied in this system. Neothramycin and tomaym
ycin cause additional blocks at some GGA-5' and TGA-5' sequences. Para
llel MPE-Fe(II) footprinting studies have revealed PBD binding sites o
n both the transcribing and nontranscribing strands, although all tran
scription blocks determined from the IVTF assays are due to drug bound
on the transcribing DNA template strand.