Stimulation of Open Complex Formation by Nicks and Apurinic Sites Suggests a Role for Nucleation of DNA Melting in Escherichia coli Promoter Function

Xiao-Yong Li,William R Mcclure

doi:10.1074/jbc.273.36.23558

Xiao-Yong Li, William R Mcclure

Open Access

https://doi.org/10.1074/jbc.273.36.23558

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Abstract

We report the effects of depurination and prenicking at various positions of the phage lambda prmup-1Delta265 promoter DNA on the rate of open complex formation. We have found that depurination and prenicking at positions around the -10 region strongly stimulated the rate of open complex formation. Since nicking and depurination are known to destabilize DNA helical structure, our observations indicate that the instability of the -10 region is important for open complex formation. We further infer that (i) the nucleation of DNA melting, which occurs during the isomerization from the closed complex into the open complex, contributes to the rate of open complex formation; (ii) the nucleation of melting occurs around the -10 region; and (iii) the propagation of DNA melting from the nucleation region is not rate-limiting. In addition, we have found that depurination at several positions inhibited open complex formation. We used dimethyl sulfate modification protection studies to show that most of the guanine bases that are among these positions are in contact with RNA polymerase in the open complex.

Highlights

It is known that the Ϫ10 region of the promoters recognized by ␴70 holoenzyme is thermodynamically less stable than av
We found that the rate of open complex formation determined was similar on pretreated DNA as well as normal untreated DNA (␶obs between 17 and 21 min), and was comparable to the rate determined from a parallel experiment using the abortive initiation assay (␶obs ϭ 14 min, not shown)
This indicates that: (i) the structural instability of the Ϫ10 region is important for promoter function; (ii) the process of destabilizing a small region of promoter DNA around the Ϫ10 region, which we refer to as the nucleation of DNA melting, contributes to the rate of open complex formation; (iii) the nucleation of DNA melting may normally occur in the region where prenicking and depurination stimulated open complex formation; (iv) the propagation of DNA melting from the nucleation site(s) is not rate-limiting

Summary

Introduction

It is known that the Ϫ10 region of the promoters recognized by ␴70 holoenzyme is thermodynamically less stable than av-. We have tested whether the structural instability of the Ϫ10 region is important for promoter function (presumably in the nucleation of DNA melting) by carrying out depurination and prenicking studies. Both prenicking [14, 15] and depurination [16, 18] are known to destabilize DNA double-helical structure, and prenicking may increase DNA structural flexibility [19]. We found that both defects at positions around the Ϫ10 region had strong stimulatory effect on the rate of open complex formation on the prmup-1⌬265 promoter. DMS1 modification protection studies of the open complex to confirm that most of the guanine bases among these positions are likely to be involved in contacting RNA polymerase

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