Escherichia coli RNA Polymerase Recognition of a {sigma}70-Dependent Promoter Requiring a -35 DNA Element and an Extended -10 TGn Motif

Escherichia coli ${sigma}$ ⁷⁰-dependent promoters have typically beencharacterized as either –10/–35 promoters, whichhave good matches to both the canonical –10 and the –35sequences or as extended –10 promoters (TGn/–10promoters), which have the TGn motif and an excellent matchto the –10 consensus sequence. We report here an investigationof a promoter, P_minor, that has a nearly perfect match to the–35 sequence and has the TGn motif. However, P_minor containsan extremely poor ${sigma}$ ⁷⁰ –10 element. We demonstrate thatP_minor is active both in vivo and in vitro and that mutationsin either the –35 or the TGn motif eliminate its activity.Mutation of the TGn motif can be compensated for by mutationsthat make the –10 element more canonical, thus convertingthe –35/TGn promoter to a –35/–10 promoter.Potassium permanganate footprinting on the nontemplate and templatestrands indicates that when polymerase is in a stable (open)complex with P_minor, the DNA is single stranded from positions–11 to +4. We also demonstrate that transcription fromP_minor incorporates nontemplated ribonucleoside triphosphatesat the 5' end of the P_minor transcript, which results in ananomalous assignment for the start site when primer extensionanalysis is used. P_minor represents one of the few –35/TGnpromoters that have been characterized and serves as a modelfor investigating functional differences between these promotersand the better-characterized –10/–35 and extended–10 promoters used by E. coli RNA polymerase.

INTRODUCTION

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Introduction

Recognition and binding of DNA promoter elements by RNA polymeraseset the start site for transcription initiation. In Escherichiacoli, these elements are recognized by a ${sigma}$ factor when ${sigma}$ is presentin RNA polymerase holoenzyme, ( ${sigma}$ plus core [ß, ß', ${alpha}$ , ${alpha}$ , and ${omega}$ ]) (15, 36). E. coli encodes several ${sigma}$ factors that canbe part of the holoenzyme, which are used under various conditionsof growth and stress (39). Each ${sigma}$ factor interacts with differentDNA sequences, and thus the recognition and usage of a givenpromoter is dependent upon the ${sigma}$ present in the holoenzyme. Thepresence of specific ${sigma}$ factors allows bacteria to coordinatethe expression of gene sets and is one of the major ways bacteriaregulate expression in response to changing growth conditions.

The primary ${sigma}$ factor of E. coli, ${sigma}$ ⁷⁰, is used during exponentialgrowth and belongs to a large family of prokaryotic primary ${sigma}$ factors related to each other by sequence, structure, and function(15, 39). Primary ${sigma}$ factors have four regions of similarity.It is known that residues in region 2 recognize a –10element (TATAAT) (37), residues in region 3 recognize an extendedTGn –10 motif (positions –15 to –13) (1),and residues in region 4 recognize a –35 element (TTGACA)(4). However, not all three of these promoter elements needto be present for promoter function. E. coli ${sigma}$ ⁷⁰-dependent promotershave typically been characterized as either –10/–35promoters, which have good matches to both the canonical –10and –35 sequences and do not require the TGn motif (32),or as extended –10 promoters (TGn/-10 promoters), whichhave the TGn motif and an excellent match to the –10 consensussequence and do not require a –35 element (2, 23, 26).

In addition to the sequence elements themselves, the distancebetween them is important for promoter recognition. Because ${sigma}$ ⁷⁰ regions 4 and 2 simultaneously contact the –35 andthe –10 elements, polymerase structure dictates the distancebetween the elements in a –10/–35 promoter (3, 10,38). The –35 and –10 elements are ideally separatedby a spacer length of 17 bp. Although this spacer length mayvary, transcription is affected by a change of even one basepair (35). Likewise, the distance between the –10 elementand the transcription start site is determined by the polymerasestructure. The transcriptional start site is typically locatedseven nucleotides downstream of the –10 element (–12TATAAT –7), with a preference for A as the incoming nucleotide(24, 28).

The consensus sequences for ${sigma}$ ⁷⁰-dependent promoters have beenstudied extensively and are well defined (16, 27, 34). Promoterelements for ${sigma}$ ⁷⁰-dependent promoters are initially assigned basedon sequences that match –10/–35 or extended –10consensus sequences at the appropriate distance from the +1transcription start. However, the bacteriophage T4 P_minor promoter(50), which was identified by its activity in vitro, is an exampleof a promoter that does not readily fit into either the –10/–35or the extended –10 promoter categories. Examination ofthe P_minor promoter region failed to locate good matches toany of the typical ${sigma}$ ⁷⁰ DNA elements at proper positions relativeto the transcriptional start site, which had been determinedby primer extension. Nonetheless, recognition of P_minor is specificfor polymerase containing ${sigma}$ ⁷⁰, since it is not recognized bypolymerase containing the closely related stationary phase ${sigma}$ factor, ${sigma}$ ³⁸ (50). In addition, P_minor is of interest becausethe formation of stable polymerase/P_minor complexes increaseswhen ${sigma}$ ⁷⁰ lacks the N-terminal 99 residues (region 1.1); othertested promoters have been either unaffected or negatively affectedby the lack of ${sigma}$ ⁷⁰ region 1.1 (50, 53).

To understand how P_minor is recognized and used by polymerasecontaining ${sigma}$ ⁷⁰, we have investigated how specific mutations withinthe P_minor promoter region affect transcription from this promoter.Here we define the minimal P_minor promoter and show that itfunctions in vivo as well as in vitro. We demonstrate that transcriptionfrom P_minor incorporates nontemplated ribonucleoside triphosphates(NTPs) at the 5' end of the P_minor transcript, which resultsin an anomalous assignment of the start site when primer extensionanalysis is used. The correct assignment of the start site suggeststhat P_minor has both a good –35 element and a TGn motifbut has an extremely poor –10 element. Our mutationalanalysis indicates that both the –35 element and the TGnmotif are required for efficient transcription and that theseelements compensate for the poor –10 element. P_minor representsone of only a few characterized –35/TGn promoters andis useful for comparing the properties of this class of promoterwith the well-characterized –10/–35 and TGn/-10classes.

MATERIALS AND METHODS

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Materials and Methods

Constructs and DNA templates. pFW11-P_null, pFW11-P₁, and pFW11-P₂ are pACYC184 derivativesthat have a EcoRI/SalI fragment without a promoter sequence(P_null), with a good promoter (P₁), or with a very good promoter(P₂) upstream of the 5' end of the lacZ gene (52). These plasmidsalso contain the 3' end of the lacI gene and the kanamycin resistancegene upstream of the promoter insert. Thus, homologous recombinationwill transfer the antibiotic resistance and the promoter sequenceinto an F' plasmid that contains the entire lac operon. To constructthe P_minor-lacZ transcriptional fusion plasmid pXBJ203, a PCRproduct was first obtained using pDKT90 (29) as a template,Pfu polymerase (Stratagene), and primers designed to createrestriction sites. The upstream primer annealed to pDKT90 startingat position –63 relative to the start of P_minor and includeda 5' EcoRI site, and the downstream primer annealed to pDKT90starting at position +4 and included a 5' SalI site. After isolationand digestion of this product with EcoRI and SalI, the fragmentwas ligated with pFW11-null that had been previously digestedwith EcoRI and SalI. The resulting plasmid is pXBJ203. ConstructspXBJ302, pXBJ402, and pXBJ503, which include the P_minor regionfrom positions –44 to +4, –35 to +4, or –29to +4, respectively, were created by inserting synthesized oligomersthat contained the desired region of P_minor with upstream EcoRIand downstream SalI sites into pFW11-P_null that had been previouslydigested with EcoRI and SalI. pIH4021(P_–33A), pIH4022(P_–31C), pIH4023 (P_{–14A–13A–12T}), pIH4024(P_{–14A–13A–12T–31C}), pIH4025 (P_–14A),pIH4026 (P_–16G), pIH4027 (P_{–12T–10T–7T}),pIH4028 (P_+1C), and pIH4029 (P_–11G), which include theP_minor region from the +4 to –35 positions with specificpromoter mutations, were constructed in the same manner usingthe appropriate oligomers to clone the promoter region. DNAsequence analyses (performed by the CBR-DNA Sequencing Facilityof the University of Maryland or the Facility for BiotechnologyResources of the Food and Drug Administration) confirmed thepromoter sequences in these various constructs.

Using the procedure of Whipple (52), pFW11-P_null, pFW11-P₁,pFW11-P₂, pXBJ203, pXBJ302, pXBJ402, pXBJ503, and pIH4028 (P_+1C)promoter constructs were transferred to single copy F' plasmidsby homologous recombination. The recombinant F' plasmids werethen transferred to the streptomycin-resistant E. coli strainFW102 by conjugation.

In some cases, templates for in vitro transcriptions were preparedby digesting plasmids, which had been previously isolated andpurified, with BglI. This digestion linearized the plasmidsat position +209, relative to the +1 position of P₂. Lineartemplates, which were used in transcriptions to identify theP_minor +1 (see Fig. 2A), were 120-bp PCR products containingonly the P_minor promoter. PCR was carried out with pIH4022,Pfu polymerase (Stratagene), and primers chosen to produce afragment from position –99 to position +21 relative tothe P_minor +1 start site.

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FIG. 2. The P_minor transcriptional start is identified. In vitro transcription reactions were assembled by adding 1.95 µl of a solution containing reconstituted polymerase (0.2 pmol core plus 0.5 pmol of ${sigma}$ ⁷⁰) in protein buffer I to 0.02 pmol of linear DNA in 2.05 µl of DNA buffer I. (A) Transcription was initiated by adding 1 µl of NTP mix to the protein-DNA mix containing P_minor DNA. As indicated, each NTP was added at the following concentrations: 1 mM UTP and 0.25 mM each ATP and GTP. The specific activity of [ ${gamma}$ -³²P]GTP or [ ${gamma}$ -³²P]ATP (where indicated by the asterisk) was 7 x 10⁵ dpm/pmol. Assignments of labeled RNA products are shown. The black arrow signifies 5-nt products, consistent with the migration of the pppACN₃ marker (not shown) kindly provided by N. Nossal. (B) Denaturing acrylamide gel showing the products of primer extension assays next to a P_minor DNA sequencing ladder. The unlabeled P_minor, P_+1C, and P₂ transcripts were generated by multiple-round transcription assays, which were initiated by the addition of 1 µl of NTP mix II. (C) Single-round transcription was initiated by adding 1 µl of NTP mix I with heparin. Transcripts arising from the P_minor, P_+1C, and P₂ promoters are indicated.

The labeled templates used in KMnO₄ footprinting were 156-bpPCR products containing only the P_minor promoter. PCR was carriedout with either pXBJ402 (P_minor) or pIH4023 (P_{–14A–13A–12A})template, Pfu polymerase (Stratagene), and primers chosen toproduce a fragment from position –99 to position +57,relative to the P_minor +1 start. Primers were 5' end labeledwith [ ${gamma}$ -^32P]ATP using T4 polynucleotide kinase (New England Biolabs)prior to PCR. Each reaction contained labeled primer that annealedto one strand, and unlabeled primer that annealed to the otherstrand. The ${gamma}$ -³²P-labeled PCR product was purified by gel electrophoresis.

Buffers and proteins. ${sigma}$ ⁷⁰ with an N-terminal His₆ tag was purified from E. coli BL21(DE3)/pLysE(46) cultures containing the rpoD plasmid (pET ${sigma}$ ^fl [20, 53]),as previously described (50), by denaturation of inclusion bodiescontaining the protein, Ni²⁺ resin affinity chromatography underdenaturing conditions, followed by a slow renaturation of theprotein. E. coli RNA polymerase core was purchased from EpicenterTechnologies. Protein buffer I contained 27 mM Tris-Cl (pH 7.9),54 mM Tris-acetate (pH 7.9), 52 mM NaCl, 40% (vol/vol) glycerol,0.9 mM EDTA, 0.007% Triton X-100, 0.24 mM dithiothreitol, 154mM potassium glutamate, 4.1 mM magnesium acetate, and 102.6µg of bovine serum albumin/ml. DNA buffer I contained21.9 mM Tris-Cl (pH 7.9), 43.4 mM Tris-acetate (pH 7.9), 71mM NaCl, 3.4% (vol/vol) glycerol, 0.5 mM EDTA, 0.15 mM dithiothreitol,219 mM potassium glutamate, 5.8 mM magnesium acetate, 146 µgof bovine serum albumin/ml, and 0.34 mM 2-mercaptoethanol. NTPmix I contained 1 mM each of ATP, GTP, and CTP and 25 µM[ ${alpha}$ -³²P]UTP (7 x 10⁵ dpm/pmol). NTP mix II contained 1 mM eachATP, GTP, CTP, and UTP. A set of NTP mixes was used for determiningthe P_minor +1. The NTPs present in each mix are indicated inFig. 2.

ß-Galactosidase assays. The level of ß-galactosidase activity in Miller unitsin FW102 lysates containing the single-copy F' plasmids withthe various promoter-lacZ constructs was determined as describedpreviously (41) except that cells were grown in the presenceof 30 µg of kanamycin and 100 µg of streptomycin/mland no IPTG (isopropyl-ß-D-thiogalactopyranoside)was added.

In vitro transcription assays. Transcription reactions were assembled as indicated in the figurelegends. Polymerase and DNA were incubated at 37°C for 10min, NTPs were added, and reactions were incubated at 37°Cfor an additional 8 min. When indicated, single-round reactionswere performed by including heparin (0.5 µl of 1 mg/ml)with the NTPs. Gel load solution (1x Tris-borate-EDTA, 7 M urea,0.1% bromophenol blue, 0.1% xylene cyanol FF) was added at avolume three times that of the reaction aliquots, and the reactionswere collected on ice. Each reaction solution was heated at95°C for 2 min before electrophoresis on 6% polyacrylamide-7M urea denaturing gels run in 1x Tris-borate-EDTA. After autoradiography,the films were scanned by using a Powerlook 2100XL densitometerand QuantityOne software from Bio-Rad, Inc.

Primer extension analyses. Primer extension reactions were carried out as described previously(50). Primer IGH107 is complementary to P_minor, P_+1C, and P₂transcripts from 155 to 171 nucleotides (nt) downstream of theSalI cut site, which is located from position +159 to position+175 relative to the P_minor +1A.

Potassium permanganate footprinting. Reactions were assembled as indicated in the legend of Fig.5. DNA-protein complexes were treated with potassium permanganateas described previously (29, 44). Potassium permanganate (0.5µl of a 50 mM solution) was added to each 4-µl reactionmixture, and the mixture was incubated for 2 min at 37°C.After the addition of 5 µl of stop solution (0.69 M sodiumacetate, 1 M 2-mercaptoethanol, 200 µg of salmon spermDNA/ml), the DNA was precipitated with ethanol and dried. Thepellets were resuspended in 100 µl of 1 M piperidine andincubated at 90°C for 30 min. The DNA was then precipitatedwith butanol, dried, and resuspended in gel loading solution.The DNA products were separated on denaturing gels as describedabove. As controls, G+A ladders were obtained using PCR productlabeled on either the top or the bottom strand (31).

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FIG. 5. Potassium permanganate footprints of P_minor (A) and P_{–14A–13A–12T} (B). Reactions were assembled by adding 1.95 µl of a solution containing reconstituted polymerase (E ${sigma}$ ⁷⁰; 0.4 pmol core plus 1.0 pmol of ${sigma}$ ⁷⁰) or core alone (0.4 pmol) in protein buffer I to 0.2 pmol of the indicated DNA in 2.05 µl of DNA buffer I. E ${sigma}$ ⁷⁰-dependent bands are marked by arrows and are numbered relative to the P_minor transcriptional +1 site. Nontemplate strand results for P_minor are consistent with the findings of Vuthoori et al. (50), but bands –6 and –4 are referred to as –2 and +1 in that study. Traces for the lanes are shown.

RESULTS

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Results

The 5' end of P_minor RNA has nontemplated nucleotides. Previously, P_minor was identified as a ${sigma}$ ⁷⁰-dependent promoteractive in vitro (50). Although this promoter is present withinT4 DNA, located about 70 bp downstream of the T4 middle promoterP_uvsX, P_minor RNA has not been observed after T4 infection (17),suggesting that it is not active for T4 under typical growthconditions. In a previous study (50), the +1 transcription startfor P_minor was identified as G (now position –3 in Fig.1A). This assignment was based on primer extension experimentsand the migration of the P_minor RNA on denaturing gels. However,typical ${sigma}$ ⁷⁰ elements are not observed at the correct positionsupstream of this assignment. Although an excellent –35element (TTGAAA) is seen from 27 to 32 bp upstream of this startsite, such a position would be highly unusual. Thus, we consideredthe possibility that the actual P_minor transcriptional startis 3 bp downstream of where the primer extension analyses indicated(+1A, Fig. 1). Primer extension acts as a measuring tape todetermine the distance from a fixed point to the 5' end of atranscript, which typically corresponds to the transcriptional+1 position. However, any extra nucleotides added to the transcriptwould result in a false measure of the transcriptional +1 position.This seemed plausible for the P_minor RNA since an A-rich sequencesurrounds the start of P_minor transcription (GGAAAAT, positions–3 to +4 in Fig. 1). Such an A-rich sequence could facilitateslippage of the polymerase during initiation, resulting in theincorporation of extra A's at the start of the P_minor transcript.To determine whether the P_minor RNA starting nucleotide is aG, as assigned by primer extension, or an A, consistent withpromoter element spacing, we carried out in vitro transcriptionwith either [ ${gamma}$ -³²P]GTP or [ ${gamma}$ -³²P]ATP (Fig. 2A). During transcriptiononly the initiating nucleotide retains the ${gamma}$ -phosphate; therefore,the labeled nucleotide must be incorporated at position +1 toresult in detectable product. Reactions in which [ ${gamma}$ -³²P]GTP wasadded yielded no observable product (Fig. 2A, lanes 1 and 2).In contrast, reactions that included [ ${gamma}$ -³²P]ATP produced bands(Fig. 2A, lanes 3 to 5). This indicates that in vitro P_minortranscription begins with an A rather than a G.

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FIG. 1. Promoter constructs. Sequences between EcoRI (GAATTC) and SalI (GTCGAC) cloning sites (enclosed in boxes) in pFW11 are shown. (A) P_null (pFW11-null), P₁ (pFW11-P1), and P₂ (pFW11-P2) are as described elsewhere (52). A 67-bp P_minor fragment is from pDKT90 (29). Promoter elements (–35, TGn, and –10) and the +1 start site are noted in red above the sequence. Note that the +1A starts of P₁ and P₂ are located within the SalI site, whereas the P_minor +1 is 8 nt upstream. Arrows indicate 5' end of the P_minor fragment before the EcoRI site in the P_-63, P_-44, P_-35, and P_-29 clones. (B) The P_-35 construct, used as wild-type P_minor promoter in the present study, is shown. Derivative promoters with the indicated changes are listed below P_minor. The dotted line indicates that the sequence is identical to P_minor.

The pattern of products seen in Fig. 2A suggests that stutteringoccurs at P_minor. The addition of [ ${gamma}$ -³²P]ATP alone (lane 3) resultedin a ladder of products, which is consistent with reiterativeincorporation of NTPs (42). The addition of [ ${gamma}$ -³²P]ATP and thenext templated base, UTP, produced three distinct bands (lane4). These bands migrate as four-, five-, and six-nucleotide(nt) products, which is consistent with a templated product(AAAU) plus products with one or two additional nucleotides.We assign these products as AAAAU and AAAAAU. The addition of[ ${gamma}$ -³²P]ATP, UTP, and GTP in the reaction (lane 5) resulted againin three major bands consistent with the 6-nt templated product(AAAUGU) plus products with one or two additional nucleotides.We conclude that the added transcript length of P_minor is dueto the incorporation of nontemplated A nucleotides. The sizesof the transcription products ( $≥$ 4) also suggest that the firstA (in the sequence 5'-GGAAAAUGU-3') is not the +1 nt. Rather,transcription appears to begin at one of the other A's.

To further examine the P_minor transcriptional start, we changedthe putative +1 nucleotide from an A to a C. C is the leastfavored nucleotide for beginning transcription, and changingthe +1 nt to a C will often result in selection of the +2 ntas the transcriptional start (24, 28). We reasoned that, ifthe second A is the transcriptional start, then the +1 C mutationwill disrupt the run of A's and prevent stuttering. To investigatethe effect of this mutation, we used a primer that annealed159 to 175 nt downstream of the assigned +1 start site of theP_minor transcript. As seen previously (50), after primer extensionthe product from P_minor RNA comigrated with the –3 positionof the P_minor DNA sequence. This is because of the additionalnontemplated nucleotides at the 5' end of the P_minor RNA. Incontrast, the primer extension product using the (P_+1C) RNAwas slightly shorter. Similar results were observed in a transcriptiongel (Fig. 2C), in which the P_+1C transcript migrated slightlyfaster than P_minor RNA. We conclude that interrupting the runof A's at the P_minor start site results in a loss of stuttering,and thus we assign the P_minor transcription start as the +1Adepicted in Fig. 1. As a control for these analyses, we usedthe P₂ transcript (Fig. 1 and see below). As expected, in theprimer extension assay (Fig. 2B) the P₂ product migrated correctly,with the +9 position of the P_minor DNA. (Note in Fig. 1 thatthe P_minor DNA construct has an extra 8 bp relative to the P₂DNA.) In addition, in the transcription gel (Fig. 2C) the P₂RNA migrated as a smaller product, which is consistent withits expected size of 209 nt.

Defining the minimal P_minor promoter. To precisely define the salient sequence features needed forthe recognition of P_minor, a set of lacZ transcriptional fusionswas designed based on the system of Whipple (52). A 67-bp fragmentknown to contain the P_minor promoter (–63 to +4) was firstcloned into pFW11 and designated P_–63 (Fig. 1). Similarconstructs were then made (P_–44, P_–35, and P_–29)with smaller promoter fragments, resulting in 5' nested deletionconstructs. pFW11-P₂ and pFW11-P₁, which contain the P_lacUV5-derived–10/–35 promoters P₂ and P₁, respectively, representedthe positive controls. In vivo, P₂ and P₁ produce high and moderatelevels of transcription, respectively (52) (see also Fig. 3A).pFW11-P_null, which lacks a promoter, was used as a negativecontrol. The lacZ transcriptional fusions were moved into F'plasmids, so promoter activity could be measured in vivo byß-galactosidase activity. The positive and negativecontrols behaved as expected (Fig. 3A), yielding significantor negligible activity, respectively. Strains carrying eitherP_–63 or P_–44 were less active than P₁, whereas P_–35and its derivative P_+1C yielded even more activity than P₁.However, the removal of the next six base pairs, containingthe sequence TTGAAA, significantly reduced P_minor activity (P_–29).We conclude that P_minor is active in vivo and that sequencesdownstream of position –36 are sufficient for this activity.In addition, these results suggest that the sequence from –35to –30 is required for significant P_minor activity invivo.

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FIG. 3. Minimal P_minor promoter defined. (A) Graph showing the ß-galactosidase assay activity (in Miller units) determined in vivo for each promoter. (B) A denaturing acrylamide gel showing the products of multiple-round in vitro transcription reactions is overlaid with a graph of the quantitation data. Transcription reactions were assembled as described in Fig. 2C. Multiple-round transcription was initiated by adding 1 µl of NTP mix I (without heparin). The amounts of RNA were determined by densitometry and are shown relative to P₂, which is set at 100. The values represent the average of three or more transcriptions.

In multiple-round in vitro transcription assays, we found thatthe relative activities of the promoters differed somewhat fromthat observed in vivo. However, deletion of the TTGAAA sequence(P_–29, Fig. 3B) was again deleterious, in this case eliminatingpromoter activity. Thus, because P_–35 contained the minimumfunctional P_minor promoter both in vivo and in vitro, this constructwas designated and used as wild-type P_minor for the rest ofthe present study.

Positions –35 to –30 of P_minor, TTGAAA, define the P_minor –35 element. To determine whether the TTGAAA sequence of P_minor (positions–35 to –30) functions as the –35 element,we investigated how specific mutations in this sequence affectedtranscription. Altering the –33G:C base pair within a ${sigma}$ ⁷⁰ –35 element (TTGACA) has been shown to significantlyreduce transcription from promoters because the C moiety isdirectly contacted by ${sigma}$ ⁷⁰ region 4 residues (22). The –33Amutation eliminated the production of P_minor RNA (Fig. 4), whereasthe –31C mutation, which makes the –35 element aperfect match to a consensus, dramatically increased transcription(Fig. 4). These results indicate that the TTGAAA sequence ofP_minor functions as the –35 promoter element and confirmthat this sequence is required for P_minor activity. In addition,other work has shown that the anti-sigma factor, AsiA (19),which blocks ${sigma}$ ⁷⁰ region 4 recognition of –35 elements,inhibits transcription from P_minor (20), further confirmingthe requirement for a –35 recognition element at P_minor.

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FIG. 4. TTGAAA functions as –35 element and is required for P_minor transcription. A denaturing acrylamide gel showing the products of single-round in vitro transcription reactions is overlaid with a graph of the quantitation data. Transcription reactions were assembled and carried out as described in Fig. 2C. The amounts of RNA were determined by densitometry and are shown relative to P_minor, which is set at 100. The values represent the average of three or more transcriptions.

The transcription bubble at P_minor ranges from position –11 to position +4. Identification of the P_minor –35 element and +1A suggestedthat GAAAAC should function as the –10 element based onlocation. However, such a sequence deviates substantially fromthe canonical ${sigma}$ ⁷⁰ –10 sequence, TATAAT (positions –12to –7) (26). Previous work has shown that the nontemplatestrand –11A is crucial for DNA melting and that transcriptionbubbles typically extend from this –11A to position +3(11, 25, 30). Thus, KMnO₄ footprinting, which reveals single-strandedthymines, is a reliable way to identify the –10 elementbecause it can identify the single-stranded T on the templatestrand opposite the nontemplate –11A.

We performed KMnO₄ experiments with DNA containing P_min_or andeither E ${sigma}$ ⁷⁰ or core alone (Fig. 5). When the nontemplate strandwas 5' end labeled (Fig. 5, top left), reactive bands occurredat positions +4, –4, and –6. A band was also visibleat position +6 but was ${sigma}$ ⁷⁰ independent. Footprinting using labeledtemplate strand produced eight bands representing every thyminefrom positions –11 to +3 (Fig. 5, top right). Therefore,in P_minor, the transcription bubble extends from –11Ato about position +4, as is typical (5, 7, 44), and the polymerase-DNAinteraction within the –10 element appears to be normalas judged by this assay. We conclude that GAAAAC (positions–12 to –7) is the functional –10 element,a finding consistent with data discussed above assigning the+1A as the transcriptional start site.

The TGn motif of P_minor compensates for its noncanonical –10 element. Although the P_minor –10 element (GAAAAC) deviates substantiallyfrom the consensus –10 sequence (TATAAT), P_minor has aTGn motif located just upstream at positions –15 to –13.To address whether the TGn was important for P_minor activity,we made a set of constructs to examine the roles of the TGnmotif and the –10 element (Fig. 1 and 6). When the –14Gwas changed to –14A, transcription was significantly reduced,a finding consistent with the idea that the TGn motif is requiredfor P_minor function. In contrast, the multiple mutation, –14A–13A–12T,had a positive effect on transcription. In this construct, theTGn motif was changed to TAn, but the –12G was also changedto T. Improving the –10 element allowed transcriptionin the absence of the TGn motif. These results suggest thatthe TGn motif of P_minor compensates for the poor –10 consensus.

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FIG. 6. P_minor requires TGn motif to compensate for a weak –10 element. A denaturing acrylamide gel showing the products of single-round in vitro transcription reactions is overlaid with a graph of the quantitation data. Transcription reactions were assembled and carried out as described in Fig. 2C. The amounts of RNA transcript were determined by densitometry and are shown relative to P_minor, which is set at 100. The values represent the average of three or more transcriptions.

Another consequence of the –14A–13A–12T mutationis that it creates a perfect –10 consensus sequence (TATAAT)at positions –17 to –13 relative to the P_minor start.The presence of this sequence could create a situation in whichthis perfect –10 sequence would be favored over the weaker–10 element of TAAAAC, present in this mutant. However,this is not the case since migration of the transcriptionalproduct is unchanged (Fig. 6), and KMnO₄ footprints with thismutant show a transcription bubble in the same location as thatseen with wild-type P_minor (Fig. 5, bottom). Thus, the functional–10 element is not changed in this mutant. Combining the–14A–13A–12T mutations with a –31C mutationcreated a promoter with a perfect –35 element and theimproved –10 element. As expected, transcription fromthis promoter was significantly higher than from wild-type P_minor(data not shown) and roughly the same as that seen with the–31C mutation alone. Finally, when the –12, –10,and –7 nucleotides were all changed to T, creating a –10element that perfectly matches consensus (P_{–12T–10T–7T}),transcription again was greatly improved (Fig. 6). As a control,we found that changing the –16A of P_minor, which is upstreamof the TGn motif, to a G had no effect.

As discussed above, the nontemplate A at position –11,which is also highly conserved among ${sigma}$ ⁷⁰-dependent promoters,is thought to lie at the transition between double-strandedDNA (upstream and including position –12) and the open(single-stranded) DNA within the transcription bubble (fromposition –11 to position +3) (11, 25, 30). To investigatethe importance of the –11A in P_minor, we changed the –11Ato a G. This change was particularly deleterious, resultingin a significant decrease in transcription (Fig. 6). We concludethat the –11A is a crucial determinant for P_minor activity.

DISCUSSION

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Discussion

Within promoter DNA, specific sequences provide the recognitionelements needed to define the start of transcription. E. coli ${sigma}$ ⁷⁰-dependent promoters have three well-characterized recognitionelements: –35 sequences, TGn, and –10 sequences.Previous work has shown that base determinants within theseelements are recognized by specific contact with ${sigma}$ ⁷⁰ residues.The ${sigma}$ ⁷⁰ region 4.2 residues R584 and E585 contact the –31Gand –33C, respectively, on the template (bottom) strandof the double-stranded –35 element (TTGACA) (4, 14, 45),whereas recognition of the TGn element is thought to arise bythe interaction of region 3 residues E458 and H455 (1, 43) withthe –14G:C base pair. For the –10 element, ${sigma}$ ⁷⁰ residuesrecognize and interact both with double-stranded DNA and withthe single-stranded base determinants formed at the transcriptionbubble. Region 2.4 residues T440 (45) and Q437 (51) are requiredfor recognition of the –12 T:A base pair in double-strandedDNA, facilitating closed complex formation (8, 9, 12, 37, 54).Aromatic residues Y425, Y430, W433, and W434 in region 2.3 promoteDNA strand separation beginning at –11 and interact withthe nontemplate strand of the –10 element in the opentranscription bubble (12, 21, 37, 40, 48, 49).

Despite the multiple contacts between ${sigma}$ ⁷⁰ and various base determinantsin the promoter DNA, recognition of all of these elements isnot biologically optimal because maximum promoter binding worksagainst the need to leave the promoter once transcription hasbeen initiated (reference 13 and references therein). In addition,deviation from consensus allows transcription to be conditionallyregulated by transacting factors, allowing the best level ofexpression for the environment rather than the highest levelof expression. Recognition of just the –10 and –35elements (–10/–35 promoters) or recognition of justthe extended –10 motif TGnTATAAT (TGn/-10 promoters) issufficient for excellent promoter activity (2, 26, 34). In fact,the absence of ${sigma}$ ⁷⁰ region 4 is nonessential for transcriptionfrom an extended promoter, indicating that the total loss ofthe ${sigma}$ ⁷⁰/–35 contacts can be tolerated if both the TGn andcanonical –10 sequences are present (23).

Although –35/-10 and TGn/-10 promoters have been establishedas ${sigma}$ ⁷⁰ promoter classes, previous work has suggested that perhapsany combination of the –35, TGn, and –10 modulesmight be acceptable for recognition (13, 33). A study of morethan 500 promoters (34) revealed that promoters with poorermatches to the canonical –10 sequences were more likelyto have the TGn motif than those with consensus –10 sequences.This finding led to the speculation that the TGn motif mightbe able to compensate for a less canonical –10 sequence.Recent work with the gapA P1 promoter of E. coli (47) has demonstratedthat the –35/TGn promoter is indeed a viable promoterarchitecture. Thus, the P_minor promoter, described here, andthe gapA P1 promoter represent the first well-characterizedmembers of a –35/TGn promoter class. Both of these promotershave excellent –35 sequences and the TGn motif. However,both of these promoters lack the conserved T at position –12within the –10 element. It is this contact in particularfor which –14G seems to compensate at P_minor, as seenby the activity of P_–14A versus P_{–14A–13A–12T}.In addition, P_minor also has a C rather than a T at the highlyconserved position –7. With both promoters, mutation ofeither the –35 or the TGn motif away from consensus essentiallyeliminates transcription, suggesting that at these promoters,the –35 and TGn elements are the primary elements forpromoter recognition. Thus, it seems that having a minimal numberof strong contacts is required, but these contacts can occurin any combination. Despite their deviations from the –10consensus sequence, both gapA P1 and P_minor retain the A nucleotideat position –11, and this base determinant is indeed crucialfor P_minor activity. Thus, the presence of the –11A, whichlies at the upstream edge of the single-stranded transcriptionbubble in the stable polymerase-promoter complex may be importantfor promoter melting despite whatever promoter modules are usedfor recognition.

Promoter elements are frequently assigned by an inspection ofsequences upstream of the identified +1 sequence. Because the–35/TGn class has not been previously appreciated, othermembers of this class may have escaped notice and instead havebeen assigned as –35/-10 promoters with less than optimalspacer distances. Indeed, gapA P1 was not originally identifiedas a –35/TGn promoter and was not included in the studyby Mitchell et al. (34).

Previous work in our lab failed to identify the correct promoterelements of P_minor in part because it is a member of the –35/TGnclass but also because P_minor RNA contains nontemplated NTPsat the 5' end. Consequently, primer extension analyses weremisleading, and the +1 of P_minor was incorrectly assigned (50).Although several promoters have been identified that generatetranscripts with nontemplated NTPs at the 5' end due to polymeraseslippage (6, 18, 42), in most of these cases, there is not awell-defined number of nontemplated bases incorporated. Rather,primer extension reveals a "stutter" stop as the primer is extendedalong RNAs of various lengths. However, with P_minor, primerextension gives a well-defined stop because of the additionof (mostly) three nontemplated bases. If this phenomenon occursat other promoters, then the incorrect assignment of the transcriptionalstart may be more common than is currently realized. Indeed,several promoters align with a distance between the –7and +1 that is less than the standard 6 bp (see the study byMitchell et al. [34]). However, studies examining start siteselection found that the 6-bp distance between the –10element and the start of transcription was strongly preferred,such that decreasing the distance by 1 bp moved the start sitedownstream (24, 28). This makes sense because start site selectionis dictated by polymerase structure, with the –10 nontemplatestrand DNA held by region 2.3, whereas the template +1 is inthe active site. Shorter distances between the transcriptionalstart site and –10 element might not be tolerated. However,polymerase slippage that specifically incorporates two to threenontemplated NTPs could be interpreted as a short distance betweenthe –7 and +1 positions. Our study suggests that a reconsiderationof the DNA elements of promoters with short distances betweenthe –10 element and the transcriptional start is warranted.

ACKNOWLEDGMENTS

We thank N. Nossal for the pppACn3 marker and F. Whipple forproviding the in vivo lacZ system. We are grateful to R. Bonocora,N. Nossal, and C. Turnbough for helpful discussions and to J.Lee for purification of the His₆-tagged ${sigma}$ ⁷⁰ protein.

This research was supported by the Intramural Research Programof the National Institutes of Health, National Institute ofDiabetes and Digestive and Kidney Diseases.

FOOTNOTES

* Corresponding author. Mailing address: Gene Expression and Regulation Section, Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 8, Room 2A-13, Bethesda, MD 20892-0830. Phone: (301) 496-9885. Fax: (301) 402-0053. E-mail: dhinton{at}helix.nih.gov.

Published ahead of print on 29 September 2006.

Present address: My Sister's Place, Domestic Violence Program,Washington, D.C.

REFERENCES

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