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

Abstract

Isomerization of a closed to open complex of a promoter upon RNA polymerase binding involves base unpairing at the ؊10 region. After potassium permanganate sensitivity of unpaired thymine residues, we studied base unpairing at the ؊10 region during isomerization upon RNA polymerase binding at the P1 and P3 promoters of the gal operon. Substitution of adenine by 2-amino purine (2-AP) at the invariable A⅐T base pair at the ؊11 position of P1 and P3 prevented unpairing not only at that position but also at the other downstream positions, suggesting a ''master'' role of the adenine base at ؊11 of the template strand in overall base unpairing. 2-AP at ؊11 did not inhibit the formation of RNA polymerase⅐promoter complex and subsequent isomerization of the polymerase. Substitution of adenine by 2-AP at several other positions did not affect thymine unpairing. Changing the position of the amino group from C6 in adenine to C2 in 2-AP is mutational only at the master switch position, ؊11. U nder ordinary physiological conditions, transcription inEscherichia coli is executed by the RNA polymerase holoenzyme comprising ␣ 2 ␤␤Ј 70 subunits. Transcription initiation follows specific binding of RNA polymerase to the promotor. The initial binary complex (closed complex) undergoes structural changes (isomerization) in both proteins and DNA to make an open complex that is competent for subsequent templatedependent polymerization of ribonucleotides (1, 2). Whether isomerization of RNA polymerase and DNA occurs simultaneously or one after the other is unknown. The 70 subunit plays a key role in recognizing and making specific contacts with base pairs at the Ϫ35 and Ϫ10 regions of the promoter (3). In the open complex, DNA at the Ϫ10 region presumably becomes singlestranded for RNA polymerase to start reading the exposed template strand and polymerizing ribonucleotides (4-10). The bases in the nontemplate strand make specific interactions with amino acid residues of 70 (11). The consequence of these interactions is believed to stabilize the unstable nature of the unpaired DNA region (11-13). 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). In this paper, we provide results by using intact duplex DNA to demonstrate that the adenine residue itself at the position Ϫ11 of the nontemplate strand plays a master role in the initiation of base unpairing. Materials and MethodsMaterials. E. coli RNA polymerase holoenzyme was purchased from Epicentre Technologies (Madison, WI). cAMP receptor protein (CRP), purified to 98% homogeneity by FPLC (Amersham Pharmacia) from an E. coli strain carrying the crp gen...

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

“…3, the wild type template (with adenine at Ϫ11) produced two transcripts, a 135-nt 1 -long P1 RNA and a 121-nt-long P3 RNA (lanes 1 and 2). The P3 RNA was not made when a supercoiled plasmid DNA was the template (29,33). The DNA template having 2-aminopurine at Ϫ11 of the P1 promoter did not yield transcripts from P1 (lane 3).…”

“…Primer 1, whose sequence is shown, was 105 nt long (from Ϫ75 to ϩ30 positions) and contained a different base at the Ϫ11 position in each PCR reaction. The reverse primer was from ϩ283 to ϩ259 of the plasmid pSA852 DNA sequence (29). The PCR products were 358 bp long.…”

“…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). We investigated the properties of the Ϫ11 base pair, which are critical in interacting with and in signaling base pair deformation.…”

“…358-bp-long DNA templates for transcription containing adenine, 2-aminopurine, 2,6-diaminopurine, or purine (experiments 1, 8, 12, and 16 in Table I) were prepared by PCR using the pSA852 plasmid DNA as template as described previously (29). The position and the size of the primers are indicated in Fig.…”

The promoter spacer influences transcription initiation via σ 70 region 1.1 of Escherichia coli RNA polymerase

“…1 A) (12)(13)(14). The result of these changes generates a complex in which the promoter is unwound from Ϫ11 to around ϩ3, and the protection footprint extends to around ϩ25 (9,11,(15)(16)(17)(18)(19)(20)(21). In addition, RPo is normally competitor resistant, although RPo at the very strong ribosomal promoters does not follow this rule (22,23).…”