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J Biol Chem, Vol. 274, Issue 6, 3315-3322, February 5, 1999

Architecture of a Complex between the ⁷⁰ Subunit of Escherichia coli RNA Polymerase and the Nontemplate Strand Oligonucleotide
LUMINESCENCE RESONANCE ENERGY TRANSFER STUDY

From the Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, St. Louis, Missouri 63104

We used luminescence energy transfer measurements to determine the localization of 5'- and 3'-ends of a 12-nucleotide nontemplate strand oligonucleotide bound to ⁷⁰ holoenzyme. Five single reactive cysteine mutants of ⁷⁰ (cysteine residues at positions 1, 59, 366, 442, and 596) were labeled with a europium chelate fluorochrome (donor). The oligonucleotide was modified at the 5'- or at the 3'-end with Cy5 fluorochrome (acceptor). The energy transfer was observed upon complex formation between the donor-labeled ⁷⁰ holoenzyme and the acceptor-labeled nontemplate strand oligonucleotide, whereas no interaction was observed with the template strand oligonucleotide. The oligonucleotide was bound in one preferred orientation. This observation together with the sequence specificity of single-stranded oligonucleotide interaction suggests that two mechanisms of discrimination between the template and nontemplate strand are used by ⁷⁰: sequence specificity and strand polarity specificity. The bound oligonucleotide was found to be close to residue 442, confirming that the single-stranded DNA binding site of ⁷⁰ is located in an -helix containing residue 442. The 5'-end of the oligonucleotide was oriented toward the COOH terminus of the helix.

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