Aromatic amino acids in region 2.3 of Escherichia coli sigma 70 participate collectively in the formation of an RNA polymerase-promoter open complex

We recently proposed that a nontemplate strand base in the discriminator region of bacterial promoters, the region between the ؊10 element and the transcription start site, makes sequence-specific contacts to region 1.2 of the subunit of Escherichia coli RNA polymerase (RNAP). Because rRNA promoters contain sequences within the discriminator region that are suboptimal for interaction with 1.2, these promoters have the kinetic properties required for regulation by the RNAP-binding factors DksA and ppGpp. Here, we use zero-length cross-linking and mutational, kinetic, and footprinting studies to map RNAP interactions with the nontemplate strand bases at the junction of the ؊10 element and the discriminator region in an unregulated rRNA promoter variant and in the PR promoter. Our studies indicate that nontemplate strand bases adjacent to the ؊10 element bind within a 9-aa interval in 1.2 (residues 99 -107). We also demonstrate that the downstream-most base on the nontemplate strand of the ؊10 hexamer cross-links to region 2, and not to 1.2. Our results refine models of RNAP-DNA interactions in the promoter complex that are crucial for regulation of transcription initiation.promoter element ͉ RNA polymerase ͉ transcription initiation ͉ discriminator region ͉ Ϫ10 element I nteractions between bacterial RNA polymerase holoenzyme (RNAP; ␣ 2 ␤␤Ј ) and the promoter can determine not only its basal strength but also its regulation. (In this report, always refers to 70 , the major factor.) Four promoter elements are generally recognized as making sequence-specific contacts with RNAP (1, 2) ( Fig. 1): the UP element (bound by the C-terminal domains of the two ␣ subunits); the Ϫ35 hexamer (bound by region 4.2); the extended Ϫ10 element (bound by region 3.0); and the Ϫ10 hexamer (bound by region 2.3-2.4). Recently, an additional element immediately downstream of the Ϫ10 hexamer, within the discriminator region, was proposed to bind to region 1.2 (3, 4).The term ''discriminator'' was coined by Travers (5) more than 25 years ago to describe a GϩC-rich region downstream from the Ϫ10 hexamer in stable RNA (rRNA and tRNA) promoters, and it was proposed that the GϩC content of this region was important for maintaining proper regulation of stable RNA promoters (6, 7). High GϩC content was proposed to impede strand separation, leading to promoter regulation. However, it was found that a C to G substitution 2 nt downstream from the Ϫ10 hexamer in the rRNA promoter rrnB P1 (rrnB P1 C-7G) eliminated its regulation, suggesting that the actual sequence of the discriminator region, in addition to its high GϩC content, is crucial for control of transcription (3).Footprinting, photocross-linking, and genetic approaches led to the conclusion that the nontemplate strand base two positions downstream from the Ϫ10 element in the rrnB P1 C-7G promoter contacts 1.2. When the base at the analogous position in all other promoters investigated was a C (either naturally or by mutation), competitor-resistant complexes formed with RNAP were much shorte...

Section: Discussionmentioning

confidence: 99%

Fine structure of the promoter–σ region 1.2 interaction

Haugen

Ross

Manrique

et al. 2008

“…N-terminally his-tagged versions of wild-type 70 and the 70 mutants were purified as described after overproduction from the corresponding derivative of vector pLHN12-His (14). E. coli RNAP core enzyme was purchased from Epicentre, and holoenzymes were assembled by incubation of RNAP core (62.5 nM) with a 5-fold molar excess of the appropriate 70 at 37°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…E. coli RNAP core enzyme was purchased from Epicentre, and holoenzymes were assembled by incubation of RNAP core (62.5 nM) with a 5-fold molar excess of the appropriate 70 at 37°C for 10 min. C-terminally his-tagged AsiA was purified as described (15) and AsiA-containing RNAP holoenzymes were assembled by preincubating the appropriate 70 (in 5-fold molar excess over RNAP core) with different concentrations of AsiA in storage buffer (14) at 4°C for 10 min. RNAP core (62.5 nM) was then added, and the mixtures were incubated at 37°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…The value of this parameter is directly proportional to the amount of E 70 -cpmAsiA-FAM complex in solution. For these experiments, wildtype RNAP core enzyme was prepared by in vitro reconstitution as described (16), and unlabeled wild-type and mutant 70 subunits were purified as described (14). -cpm-AsiA-FAM complex by the unlabeled competitor protein, resulting in diminished intensity of the FRET signal.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme

Nickels

Garrity²,

Severinova³

et al. 2004

The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the ␤ subunit and conserved region 4 of the subunit, is a potential target for regulation. Here we provide genetic and biochemical evidence that the region 4͞␤-flap interaction is targeted by the transcription factor AsiA. Specifically, we show that AsiA competes directly with the ␤-flap for binding to region 4, thereby inhibiting transcription initiation by disrupting the region 4͞␤-flap interaction.T he bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytically proficient core enzyme (subunit structure ␣ 2 ␤␤Ј ) and a subunit that confers on the holoenzyme the ability to recognize specific promoter sequences (1). The primary factor in Escherichia coli is 70 , and the 70 -containing holoenzyme (E 70 ) typically recognizes promoters defined by two conserved sequence elements (the Ϫ10 and Ϫ35 hexamers) positioned roughly 10 and 35 bp upstream of the transcription start point (1). Most factors share four conserved regions (2), and of these, regions 2 and 4 interact with the Ϫ10 and Ϫ35 elements, respectively (1). Nevertheless, intact 70 recognizes specific promoter sequences only in the context of the holoenzyme because of critical conformational changes in 70 that take place when it associates with the core enzyme (3). These include an increase in the interdomain distance between regions 2 and 4 that is caused by an interaction between region 4 and the ␤-flap domain (4). The region 4͞␤-flap interaction is essential for the recognition of Ϫ10͞Ϫ35 promoters because it positions regions 2 and 4 of 70 for simultaneous interaction with the promoter Ϫ10 and Ϫ35 elements (4). This role of the region 4͞␤-flap interaction suggested that there may exist regulatory factors that inhibit transcription from Ϫ10͞Ϫ35 promoters by disrupting the region 4͞␤-flap interaction (4, 5).Here we examine the mechanism of action of the bacteriophage T4-encoded anti-factor AsiA. AsiA is known to bind tightly to region 4 of 70 and inhibit transcription from Ϫ10͞Ϫ35 promoters when complexed with the holoenzyme (6-10). The finding that AsiA binds to region 4 of 70 and inhibits transcription specifically from Ϫ10͞Ϫ35 promoters raises the possibility that AsiA works by disrupting the 70 region 4͞␤-flap interaction, a model that is consistent with recent NMR analysis (11). We provide genetic evidence that AsiA and the ␤-flap interact with overlapping determinants on 70 region 4. We then present complementary biochemical and biophysical evidence that AsiA works by a competitive binding mechanism, inhibiting transcription from Ϫ10͞Ϫ35 promoters by disrupting the region 4͞␤-flap interaction in the context of the holoenzyme. Materials and MethodsMutant Screen. Mutagenic PCR was u...

“…Both kinds require a 6-bp Ϫ10 sequence (consensus 5Ј-TATAAT-3Ј) located Ϸ7 bp 5Ј to the transcription start site. Functionally, the Ϫ10 element participates in RNA polymerase binding by interacting with the region 2.3-2.4 of 70 (2)(3)(4)(5)(6)(7)(8)(9)(10) and is part of an Ϸ15-nt putative single-stranded region in the open complex (4,8,(11)(12)(13)(14)(15). The first kind of promoters (Ϫ35 promoters) is more common and characterized by the presence of the Ϫ10 element as well as a 6-bp (consensus sequence 5Ј-TTGACA-3Ј) in the Ϫ35 position (16).…”

mentioning

confidence: 99%

A mutant spacer sequence between -35 and -10 elements makes the P _lac promoter hyperactive and cAMP receptor protein-independent

Liu

Tolstorukov

Zhurkin

et al. 2004

108

To determine whether the spacer region between the ؊35 and ؊10 elements plays any sequence-specific role, we randomized the GC-rich sequence ( ؊20 CCGGCTCG ؊13 ) within the spacer region of the cAMP-dependent lac promoter and selected an activatorindependent mutant, which showed extraordinarily high intrinsic activity. The hyperactive promoter is obtained by incorporation of a specific 10-bp-long AT-rich DNA sequence within the spacer, referred to as the ؊15 sequence, which must be juxtaposed to the upstream end of the ؊10 sequence for the hyperactivity. The transcription enhancement functions only in the presence of a ؊35 element. The spacer sequence enhanced both RNA polymerase binding and open complex formation. Isolated in the lac promoter, it also enhanced transcription when placed at two other unrelated promoters. Sequence analysis shows a low GC content and an abundance of stereochemically flexible TG:CA and TA:TA dimeric steps in the ؊18͞؊9 region and a strong correlation between the presence of flexible dimeric steps in this region and the intrinsic strength of the promoter.A promoter is a stretch of DNA sequence that encodes information for RNA polymerase binding and initiation of transcription. Genetic and statistical analysis of promoters in Escherichia coli defined two kinds of E 70 RNA polymerase holoenzyme-dependent promoters (1). Both kinds require a 6-bp Ϫ10 sequence (consensus 5Ј-TATAAT-3Ј) located Ϸ7 bp 5Ј to the transcription start site. Functionally, the Ϫ10 element participates in RNA polymerase binding by interacting with the region 2.3-2.4 of 70 (2-10) and is part of an Ϸ15-nt putative single-stranded region in the open complex (4,8,(11)(12)(13)(14)(15). The first kind of promoters (Ϫ35 promoters) is more common and characterized by the presence of the Ϫ10 element as well as a 6-bp (consensus sequence 5Ј-TTGACA-3Ј) in the Ϫ35 position (16). The Ϫ35 element also helps RNA polymerase binding through interaction with region 4.2 of 70 (2,5,17). It is believed that the spacer region between Ϫ35 and Ϫ10 (optimal length 17 bp) does not have any specific sequence requirement and simply facilitates the spatial alignment of the Ϫ10 and Ϫ35 elements in binding to the 2.4 and 4.2 regions of the factor (7, 17, 18). The second kind, called ''extended Ϫ10'' promoters, contains an extra 2-bp 5Ј-TG-3Ј sequence located 1 bp 5Ј to the Ϫ10 element, (consensus sequence Ϫ15 TGNTATAAT Ϫ7 ) (19). The DNA sequence immediately upstream of an extended Ϫ10 element may enhance the activity of the extended Ϫ10 promoter (20, 21). The TG element also binds to RNA polymerase by contacting the 3.0 (formerly 2.5) segment of 70 (22). The Ϫ35 promoters may be improved by the presence of the extended Ϫ10 sequence and vice versa.Many naturally occurring promoters are more or less inefficient because of the presence of non-consensus sequence elements or suboptimal spacer lengths and are resurrected by extra regulatory factors (23). The extra factor may be a DNA sequence, e.g., an UP element (24-26). The UP element, located imme...