Site-directed chemical modification for probing DNA-protein interactions. Osmium tetroxide modification of the -10 site of the lacUV5 promoter enhances open complex formation.
A new experimental approach, site-directed chemical modification, was used to explore relationships between RNA polymerase-promoter interactions and function. For this study, the lacUV5 promoter with an exposed -10 thymine on the non-template strand was constructed. Osmium tetroxide was selected as the thymine modifying reagent. Modification occurred predominantly at the exposed -10 T with 5-fold less reactivity at the -12 T residue. The isolated modified strand was used to reconstitute a lacUV5 promoter with -10 (-12) adducts. OsO4 modification at both the -10 and -12 positions of the lacUV5 promoter significantly enhances Escherichia coli RNA polymerase-promoter open complex formation relative to the unmodified promoter. DNase I cleavage sites at -7, -8, and -10 of the unmodified promoter were rendered insusceptible to scission in the modified promoter. However, no difference can be detected in the RNA polymerase footprints for unmodified versus modified open complexes. The latter are fully capable of productive transcription with comparable amounts of identical run-off transcripts to unmodified open complexes. A 16 degrees C reduction in Tm was found for a 14-base pair oligonucleotide duplex containing a single OsO4-bispyridine adduct. The latter result suggests that open complex formation appears to be enhanced due to promoter unpairing at the -10 (-12) adduct sites.
Chan
P T
PT
Department of Microbiology, University of Alabama, Birmingham 35294.
Sullivan
J K
JK
Lebowitz
J
J
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009992:Osmium / Q000494:pharmacology*; D009993:Osmium Tetroxide / Q000494:pharmacology*; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011485:Protein Binding; D015183:Restriction Mapping; D014158:Transcription, Genetic / Q000187:drug effects
D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D009992:Osmium; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D009993:Osmium Tetroxide