Conformation and DNA binding properties of a single-stranded DNA binding region of sigma 70 subunit from Escherichia coli RNA polymerase are modulated by an interaction with the core enzyme.
A derivative of the sigma 70 subunit from Escherichia coli RNA polymerase with specific fluorescence probes in conserved region 2.3 (DNA "melting motif") was prepared by replacing tryptophan residues at positions 314 and 326 of the wild-type sigma 70 with alanine. The remaining two tryptophan residues (Trp 433 and 434) of [Ala 314, 326]sigma 70 were biosynthetically replaced with 5-hydroxy-tryptophan (5OHTrp), a fluorescent tryptophan analogue with unique emission that can be selectively observed both in free 5OHTrp[Ala 314, 326]sigma 70 as well as in 5OHTrp[Ala 314, 326]sigma 70 bound to the core RNA polymerase. Fluorescence quenching experiments revealed that positions 433 and 434 were solvent exposed in free 5OHTrp[Ala314, 326]sigma 70. The binding of sigma 70 to core polymerase reduced the solvent exposure of these residues. In the presence of single-stranded oligonucleotides, fluorescence of 5OHTrp at position 433 and 434 was quenched approximately 65% and these residues became inaccessible to the solvent. Using fluorescence of 5OHTrp at positions 433 and 434 as a specific signal of DNA binding, we show that free sigma 70 bound single-stranded DNA weakly and did not discriminate between nontemplate and template strand of promoter DNA. Binding of sigma 70 to the core increased the affinity for binding nontemplate DNA, whereas the affinity to template or "nonspecific" DNA was reduced, resulting in a holoenzyme which could bind nontemplate strand approximately 200-fold better then the template strand. We concluded that Trp 433 and 434 of sigma 70 are located within a single-stranded DNA binding region of sigma 70 and that binding of sigma 70 to the core enzyme induced conformational changes in a single-stranded DNA binding region of the protein. As a consequence of these conformational changes, sigma 70 subunit gains the specificity for the nontemplate strand of the melted region in the "open" complex.
Callaci
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104, USA.
Heyduk
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D006916:5-Hydroxytryptophan; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005456:Fluorescent Dyes; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D012808:Sigma Factor; D006916:5-Hydroxytryptophan; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases