Interaction of T7 RNA polymerase with DNA in an elongation complex arrested at a specific psoralen adduct site.
We have probed the interaction of T7 RNA polymerase with DNA in an elongation complex arrested by a site specifically placed psoralen diadduct or furanside monoadduct using DNase I footprinting techniques. The psoralen derivative, HMT (4'-hydroxy-methyl-4,5',8-trimethylpsoralen), was first placed at a specific site in the middle of a chemically synthesized double-stranded DNA fragment containing a T7 RNA polymerase promoter at one end. The psoralen molecule was photochemically attached either to 2 adjacent thymidine residues on opposite strands as a diadduct or to only 1 thymidine residue on the coding strand as a furan-side monoadduct. Using these psoralen-modified DNAs as templates for transcription, we found that T7 RNA polymerase was blocked at the psoralen adduct site and that the arrested elongation complex protected about 15 nucleotides upstream from the adduct on the coding strand and 20 nucleotides around the adduct on the noncoding strand from DNase I digestion. The two psoralen-modified DNA templates yielded identical RNA transcripts and DNase I footprints. In contrast, T7 polymerase protected only the coding strand from -20 to +8 in the initiation complex. These results suggest that the RNA polymerase undergoes a marked conformational change upon converting from an initiation complex to an elongation complex.
Shi
Y B
YB
Department of Chemistry, University of California, Berkeley 94720.
Gamper
H
H
Hearst
J E
JE
D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000201:enzymology*; D011564:Furocoumarins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D013604:T-Phages / Q000201:enzymology*; D014158:Transcription, Genetic; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D011564:Furocoumarins; C016507:hydroxymethyltrioxsalen; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D014307:Trioxsalen