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

Abstract

We report the effects of depurination and prenicking at various positions of the phage prmup-1⌬265 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 ratelimiting. 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.Open complex formation is the major rate-determining step in the process of transcription initiation with E. coli RNA polymerase (E 70 ). Many of the previous studies have been based on a model containing two functional steps (1, 2) that include the initial binding of RNA polymerase to the promoter DNA to form the closed complex followed by the isomerization of the closed complex into the open complex. Some studies have also suggested that the isomerization step in the two-step model can be further dissected into two discrete steps: the rate-limiting isomerization step per se and a DNA melting step (3-10). In this extended model, the DNA melting step is argued to be very rapid under normal transcription conditions, and consequently it is normally not ratelimiting. It has been suggested that the isomerization step involves a major conformational change in RNA polymerase, which is thought to be the cause for the nucleation of DNA melting (10, 11).It is known that the Ϫ10 region of the promoters recognized by 70 holoenzyme is thermodynamically less stable than average DNA. The conserved Ϫ10 hexamer sequence has a melting free energy that is close to the maximum (least stable) possible based on a calculation using the nearest-neighbor thermodynamic data of Breslauer et al. (12). Therefore, as expected, Margalit et al. (13) have shown that 80% of the up and down mutations in the Ϫ10 region correlated qualitatively with the change in the melting free energy. A closer inspection showed that most of the mutations among the exceptions are located outside the Ϫ10 (hexamer) region, and consequently are not bona fide exceptions if only the melting free energy of the Ϫ10 region is important. There were several exceptions within the Ϫ10 region. However, in such cases it can be argued that the effect of the mutation on the specific contact between RNA polymerase and promoter DNA is larger than the effect of the melting free energy change, and con...

An Unsubstituted C2 Hydrogen of Adenine Is Critical and Sufficient at the –11 Position of a Promoter to Signal Base Pair Deformation

“…The Ϫ11 position did not accept a pyrimidine base (cytosine or thymine) either, suggesting a strict requirement of an adenine or a purine at Ϫ11 (Table I). Our observation of the essential nature of a purine base with an unmodified C2 hydrogen at Ϫ11 for promoter activity is consistent with a previous report that depurination of adenine, while stimulating open complex formation when at position Ϫ12, Ϫ10, or ϩ1, inhibited the process when at position Ϫ11 (28).…”

“…Although these amino acid residues were shown to strategically lie on one face of the subunit to be able to perform the two functions in open complex formation, RNA polymerase binding and DNA deformation (10,11), the details of the interactions from structural viewpoints remain unknown. Studies of transcription initiation with specifically designed DNA templates suggested that DNA deformation nucleates at around position Ϫ10/Ϫ11 (9,(25)(26)(27)(28). The omnipresent A:T base pair at the position Ϫ11 was specifically suggested to play a "master" role in signaling DNA deformation (29).…”

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

“…These results suggest that unpairing of the invariant A⅐T base pair Ϫ11 is a forerunner event and acts as the ''master'' of unpairing of bases. It has been suggested that DNA ''melting'' by RNA polymerase starts with a nucleation in the Ϫ12 to Ϫ4 region and extends up to ϩ2 position downstream (18). Our finding of Ϫ11 A⅐T being the master position further pinpoints the actual start of the process of the nucleation.…”

“…The consensus DNA sequence of the Ϫ10 region is 5Ј-TATAAT-3Ј in the nontemplate strand, with the first T being at the Ϫ12 position (1). To elucidate the molecular procedures of base unpairing during open complex formation, altered forms of DNA (depurinated, nicked, forked, or with single-stranded gap) in the Ϫ10 region were used as templates (10,(14)(15)(16)(17)(18)(19). These studies suggested that base unpairing involves an initial distortion or localized melting event around Ϫ11 (16,20).…”

Different Roles for Basic and Aromatic Amino Acids in Conserved Region 2 of Escherichia coli ς70 in the Nucleation and Maintenance of the Single-stranded DNA Bubble in Open RNA Polymerase-Promoter Complexes

“…Measurement of the size and location of the region of strand separation as a function of temperature shows that at low temperatures a small single-stranded region can be detected that, as the temperature is increased, expands toward the start site (3,(5)(6)(7). In addition, the introduction of nicks and mismatches in the Ϫ10 region is more effective in the acceleration of open complex formation than if such distortions are introduced at a more downstream position (8,9).…”