Studies with the <i>Escherichia coli</i> galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein

Ponnambalam, Sreenivasan; Spassky, Annick; Busby, Stephen J. W.

doi:10.1016/0014-5793(87)81214-0

Cited by 22 publications

(22 citation statements)

References 23 publications

(41 reference statements)

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“…at position Ϫ11. cAMP and CRP inhibited transcription from P3 not only with the wild-type DNA, as shown previously (33), but also in DNA containing 2-AP at position Ϫ11, Ϫ5, or ϩ1.…”

Section: Resultssupporting

confidence: 85%

“…2). In the latter examples, P3 was made generally in lower amounts relative to P1 RNA both from wild type and 2-AP-containing DNA templates, as was observed previously (33), except when the 2-AP was present at position ϩ3. With 2-AP at ϩ3, no P3 RNA was observed.…”

Section: Resultssupporting

confidence: 73%

“…CRP in conjunction with cAMP enhances the P1 promoter and represses P3 (21,31,33). Consistently, the presence of cAMP and CRP stimulated transcription 3-4-fold from P1 and inhibited the same from P3 with DNA templates without 2-AP (Fig.…”

Section: Resultssupporting

confidence: 54%

“…With 2-AP at ϩ3, no P3 RNA was observed. The ϩ3 position of P1 corresponds to the Ϫ11 position of P3 (33). Thus similar to P1, transcription initiation from P3 was abolished when 2-AP replaced adenine at the corresponding Ϫ11 position.…”

Section: Resultsmentioning

confidence: 91%

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

Lim

Lee

Roy

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…at position Ϫ11. cAMP and CRP inhibited transcription from P3 not only with the wild-type DNA, as shown previously (33), but also in DNA containing 2-AP at position Ϫ11, Ϫ5, or ϩ1.…”

Section: Resultssupporting

confidence: 85%

Section: Resultssupporting

confidence: 73%

Section: Resultssupporting

confidence: 54%

Section: Resultsmentioning

confidence: 91%

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

Lim

Lee

Roy

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…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).…”

Section: Resultsmentioning

confidence: 99%

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

Lee

Lim

2004

Journal of Biological Chemistry

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The conserved A:T base pair at the ؊11 position of the promoters in Escherichia coli is very sensitive to substitutions. In vitro transcription with the galP1 promoter having a natural or unnatural base in either strand at position ؊11 showed that only a purine base with no side group at C2 in the nontemplate strand is transcriptionally potent; neither a purine with an amino group at C2 nor a pyrimidine support transcription. The amino group at C6 in the omnipresent adenine at ؊11 does not play any role in promoting transcription. The nature of the base, complementary or noncomplementary, at ؊11 in the template strand also does not influence transcription. We propose that the adenine, by becoming extrahelical, interacts with an amino acid(s) of the 2.3-2.4 region of for which an unsubstituted C2 hydrogen is critical.Transcription initiation executed by a bacterial RNA polymerase holoenzyme (E) starts with its binding to a promoter DNA sequence (formation of a closed complex) and is followed by a multistep process leading to the formation of an open complex. The open complex is competent to initiate polymerization of ribonucleotides according to the DNA sequences of the template strand (1-4). Both DNA (Ϫ10 region of the promoter) and the multisubunit enzyme go through major conformational changes in becoming an open complex (4 -9). Although the exact structural changes that they undergo during open complex formation are unknown, recent crystallographic and fluorescence resonance energy transfer studies of various bacterial E and their parts provided some clue to the structure of the open complex (10 -15). It is believed that in a fully mature open complex, about 15 base pairs in DNA (from Ϫ12 to ϩ3 relative to the transcription start site) become single-stranded by the so-called "melting" process, which we will refer to as DNA deformation (16 -18). Several amino acid residues in the 2.3-2.4 region of the subunit were inferred to make consequential contacts with bases in the Ϫ10 region of the promoter: (4, 16, 18 -27). 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, 25-28). 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. EXPERIMENTAL PROCEDURESIn Vitro Transcription Assay-In vitro transcription reactions were performed in a 25-l volume following the procedure of Choy and Adhya (30). The 70 -saturated RNA polymerase from Escherichia coli was purchased from Epicenter Technologies (Madison, WI). The reaction mixture (...

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Correlation between the conformation of Escherichia coli −10 hexamer sequences and promoter strength: use of orthophenanthroline cuprous complex as a structural index.

et al. 1988

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The lac and gal control regions contain two functional overlapping promoters P1 and P2. Point mutations can shift transcription from P1 to P2 and vice versa. We show that the reactivity of DNA fragments towards nucleolytic attack with orthophenanthroline cuprous complex can be used to predict which promoter competes more efficiently for RNA polymerase binding. Furthermore, similar changes in reactivity are observed as closed complexes isomerize to form the final open complexes, provided that the functional start is taken as a reference. We found a correlation between the reactivity pattern of −10 regions in uncomplexed DNA and the rate of open complex formation.

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Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein

Cited by 22 publications

References 23 publications

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

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

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

Correlation between the conformation of Escherichia coli −10 hexamer sequences and promoter strength: use of orthophenanthroline cuprous complex as a structural index.

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