Interaction of Escherichia coli RNA polymerase with DNA in an elongation complex arrested at a specific psoralen crosslink site.
We have probed the interaction of Escherichia coli RNA polymerase with DNA in an elongation complex arrested by a site-specifically placed psoralen crosslink using DNase I footprinting techniques. The psoralen derivative 4'-hydroxymethyl-4,5',8-trimethylpsoralen was first placed at a specific site in the middle of a chemically synthesized double-stranded DNA fragment containing an E. coli RNA polymerase promoter at one end. The psoralen molecule was photochemically attached to two adjacent thymidine residues on opposite strands as a diadduct. Using this crosslinked DNA as the template for transcription, we found that the E. coli RNA polymerase was blocked at the psoralen diadduct, yielding a transcript 29 nucleotides long. The arrested elongation complex inhibited DNase I digestion of both the coding strand and the non-coding strand from about 22 nucleotides upstream to 15 nucleotides downstream from the diadduct. These results, which suggest that the unwindase and the catalytic sites of the polymerase are very close to each other, have been incorporated into a model of the transcription elongation complex.
Shi
Y B
YB
Department of Chemistry, University of California, Berkeley 94720.
Gamper
H
H
Van Houten
B
B
Hearst
J E
JE
D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D008957:Models, Genetic; D008969:Molecular Sequence Data; D013698:Templates, Genetic; D014307:Trioxsalen / Q000031:analogs & derivatives
C016507:hydroxymethyltrioxsalen; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D014307:Trioxsalen