Recognition of tetrahedral 1,10-phenanthroline-cuprous chelates by transcriptionally active complexes does not depend on the sequence of the promoter.
BACKGROUND
The open complex formed at the initiation site of transcription within the active site of RNA polymerase is unique to actively transcribing genes and is thus an ideal target for the design of transcription inhibitors. Many redoxactive tetrahedral cuprous chelates of 1,10-phenanthroline (OP) or derivatives cleave the single-stranded template, principally at sequence positions -7 to -3, whereas the redox-inactive tetrahedral cuprous chelate of 2, 9-dimethyl-OP (neocuproine) blocks transcription, but does not cleave. The octahedral (OP)3-Fe2+ chelate has no effect. Different promoters can give different cleavage patterns. We therefore searched for structural determinants of the open complex that are important in the cleavage reaction.


RESULTS
Using site-directed mutagenesis, we systematically altered the nucleotides at the cleavage sites of the Escherichia coli lac UV-5-RNA polymerase open complex (positions -6 to -4), which are highly variable in E. coli promoters. Surprisingly, these changes had little effect on catalytic activity, on transcription inhibition by the cuprous complex of neocuproine and on the cleavage patterns generated by the cuprous chelates of OP derivatives. The scission pattern of a lac UV-5 promoter mutant in which the cleavage sites have the sequence of the trp EDCBA promoter is that of the lac UV-5 promoter, not the trp EDCBA promoter.


CONCLUSIONS
Nucleotide-specific interactions are not responsible for the observed cleavage patterns. The recognition of the tetrahedral OP chelate must be due to a specific structure of the single-stranded regions, determined by RNA polymerase-DNA interactions in the upstream regulatory region.
Gallagher
J
J
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095-1570, USA. Sigman@ewald.mbi.ucla.edu
Perrin
D M
DM
Chan
L
L
Kwong
E
E
Sigman
D
D
D002614:Chelating Agents / Q000378:metabolism; D003300:Copper / Q000378:metabolism* / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects; D018983:DNA Footprinting; D004277:DNA, Single-Stranded / Q000187:drug effects / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D007502:Iron Chelating Agents / Q000494:pharmacology; D007763:Lac Operon / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000378:metabolism* / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics*
D002614:Chelating Agents; D004277:DNA, Single-Stranded; D007502:Iron Chelating Agents; D010618:Phenanthrolines; D003300:Copper; C002701:neocuproine; D006861:Hydrogen Peroxide; D012321:DNA-Directed RNA Polymerases; C025205:1,10-phenanthroline