a t9g mutation in the prototype tata-box ......with rbcs, pcec, and pr-1a activators. the 9g...

A T9G Mutation in the Prototype TATA-BoxTCACTATATATAG Determines Nucleosome Formationand Synergy with Upstream Activator Sequences inPlant Promoters1[W]

Amol Ranjan2, Suraiya A. Ansari2, Rakesh Srivastava, Shrikant Mantri, Mehar H. Asif,Samir V. Sawant*, and Rakesh Tuli

National Botanical Research Institute, Council of Scientific and Industrial Research, Lucknow 226001, India

We had earlier reported that mutations to G and C at the seventh and eighth positions in the prototype TATA-boxTCACTATATATAG inhibited light-dependent activation of transcription from the promoter. In this study, we characterizedmutations at the ninth position of the prototype TATA-box. Substitution of T at the ninth position with G or C enhancedtranscription from the promoter in transgenic tobacco (Nicotiana tabacum) plants. The effect of T9G/C mutations was not lightdependent, although the 9G/C TATA-box showed synergy with the light-responsive element (lre). However, the 9G/C mutantsin the presence of lre failed to respond to phytochromes, sugar, and calcium signaling, in contrast to the prototype TATA-boxwith lre. The 9G/C mutation shifted the point of initiation of transcription, and transcription activation was dependent uponthe type of activating element present upstream. The synergy in activation was noticed with lre and legumin activators but notwith rbcS, Pcec, and PR-1a activators. The 9G mutation resulted in a micrococcal nuclease-sensitive region over the TATA-box,suggesting a nucleosome-free region, in contrast to the prototype promoter, which had a distinct nucleosome on the TATA-box.Thus, the transcriptional augmentation with mutation at the ninth position might be because of the loss of a repressivenucleosomal structure on the TATA-box. In agreement with our findings, the promoters containing TATAGATA as identified bygenome-wide analysis of Arabidopsis (Arabidopsis thaliana) are not tightly repressed.

The core promoter is essential for efficient andaccurate initiation of transcription in eukaryotic genes.The core promoter sequence is generally located be-tween –35 and +35 bp with respect to the transcriptionstart site (Smale, 2001) and mainly comprises twoimportant elements: the TATA-box and the initiatorelement. Various functional DNA motifs, known ascore promoter elements, assist in the recruitment,assembly, and initiation of the RNA polymerase IItranscription machinery. Transcription by RNA poly-merase II involves the recruitment of general tran-scription factors such as TFIIA, -B, -D, -E, -F, and -H,coactivators (such as the mediator complex), andpolymerase II to the promoter to form a preinitiationcomplex (PIC; Hahn, 2004). Several of the generaltranscription factors have been proposed as targets of

transcriptional activators, including TFIID, TFIIB, andTFIIF. TFIID is a multisubunit protein complex con-sisting of TBP and approximately 14 associated factorsthat plays a critical role not only in promoter bindingbut also as a transducer in conveying the upstreamregulating signals to the downstream general tran-scription machinery. The core promoter and its ele-ments are highly variable, so they can be a pointof regulation for gene-specific transcription (Zhuet al., 1995; Nakamura et al., 2002; Kiran et al., 2006;Bjornsdottir and Myers, 2008). Efficient transcriptiongenerally requires the synergistic action of multipleactivators bound at distinct sites upstream (or down-stream) of the promoter (Struhl, 1999). Activatorscontain a DNA-binding domain that specifically rec-ognizes enhancer elements and a physically separateactivation domain that stimulates transcription of thetarget gene (Brent and Ptashne, 1985; Hope and Struhl,1986; Hope et al., 1988). Activation domains have beenproposed to enhance transcription by a variety ofmechanisms. These include simple recruitment of thepolymerase II machinery to promoters (Klein andStruhl, 1994; Struhl, 1996; Ptashne and Gann, 1997),altering the conformation of components of the poly-merase II machinery (Roberts and Green, 1994; Chiand Carey, 1996), modifying chromatin structure(Workman et al., 1991; Croston et al., 1992; Kingstonet al., 1996), and affecting one or more steps after the

1 This work was supported by the Council of Scientific andIndustrial Research, Government of India, and a Sir J.C. Bosefellowship to R.T.

2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Samir V. Sawant ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.109.148064

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transcriptional initiation event (Rougvie and Lis, 1990;Yankulov et al., 1994; Krumm et al., 1995). Thesepossible mechanisms are not mutually exclusive, buttheir relative importance in vivo has yet to be estab-lished.Our analysis (Sawant et al., 1999) identified TCAC-

TATATATAG as the prototype TATA-box sequencepresent most commonly in highly expressed plantgenes. The TATA-box sequence determines the site oftranscription initiation and the efficiency of transcrip-tional complex formation. In recent years, the signif-icance of this element in regulating gene expressionin development-specific (Duan et al., 2002), tissue-specific (Hochheimer and Tjian, 2003; Grace et al.,2004), or organ-specific (Kloeckener-Gruissem et al.,1992) manner has also been reported. Light inducesthe expression of many genes in plants, such asribulose-1,5-bisphosphate carboxylase small subunit(rbcS) and chlorophyll a/b binding (cab) proteins.Response to light is mediated by at least four distinctfamilies of photoreceptors, which include phyto-chrome, cryptochromes, phototropins, and unidenti-fied UV B photoreceptors (Jiao et al., 2007). Thesephotoreceptors absorb the light signals and, in con-junction with molecular systems of the plant, regulatethe expression at the transcriptional and posttran-scriptional levels. Studies of light-mediated genome-wide expression during seedling development inArabidopsis (Arabidopsis thaliana; Ma et al., 2001; Jiaoet al., 2005) and rice (Oryza sativa; Jiao et al., 2005)revealed that light controls many growth and devel-opmental pathways. These pathways are believed totarget different transcription factors and/or light-responsive elements (lres) within the promoter of agene. These lres are conserved DNA modular arrays(CMAs) that are associated with light-responsive pro-moter regions and contain an I- and G-box cis-element.A 52-bp LRE, called CMA5, has been reported toconfer light-specific activation of transcription toeven heterologous promoters (Martinez-Hernandezet al., 2002). The core promoter region of severallight-regulated genes has a distinct TATA-box and/or initiator element. Nakamura et al. (2002) showedthat the majority of the photosynthesis nuclear geneslacked TATA-boxes. Instead, pyrimidine-rich initiatorelements that overlap the transcription initiation siteswere essential for light-responsive transcription. Inspite of many studies on light-mediated regulation,the role of the TATA-box sequence in light-regulatedcore promoters is poorly understood.We previously examined (Kiran et al., 2006) the

effect of point mutations in a 13-bp prototype TATA-box sequence (Sawant et al., 1999) in light-mediatedtranscription. The nucleotides at the seventh andeighth positions in the prototype TATA-box werereported as critical to light-dependent transcription.When T at the seventh or A at the eighth position wasmutated to G/C, the promoters continued to expressin the dark to a level comparable to that of theprototype TATA-box sequence in both the presence

and absence of the lre. However, the mutations led tofailure of the promoters to express in the light. Thesefindings established that the seventh and eighth posi-tions in the TATATATA-dependent core promoterwere critical to light-mediated transcription andwere not important for gene expression in thedark. In this study, the ninth position in the prototypeTATA-box was mutated to G/C, and the effect ontranscription was examined in the presence of differ-ent upstream activator sequences after transformationusing gusA as the reporter gene. This study shows thatthe ninth position plays an important role in theformation of nucleosome(s), assembly of the transcrip-tional complex, and interactions with the upstreamactivator sequences.

RESULTS

Substitution of G/C at the Ninth Position in theConsensus TATA-Box Leads to Enhancement

of Transcription

In an earlier work (Kiran et al., 2006), we haddemonstrated the critical role that nucleotides at theseventh and eighth positions play in the consensusTATA-box TCACTATATATAG for light-mediated tran-scription, both in the presence and absence of the52-bp lre (Martinez-Hernandez et al., 2002). This studyinvestigates the role of the ninth nucleotide in expres-sion from the same prototype minimal promoter(Pmec) containing TCACTATATATAG as a consensusTATA-box (Supplemental Fig. S1). The Pmec usedin this study is a 139-nucleotide-long typical TATA-dependent minimal promoter (Kiran et al., 2006), asshown by us bymutating TATATATA to GAGAGAGA.The lre was fused upstream of Pmec, Pmec 9G, andPmec 9C and cloned in binary vector pBI101contain-ing the gusA reporter gene. The resultant constructs,lre-Pmec, lre-Pmec 9G, and lre-Pmec 9C, were thentransformed in tobacco (Nicotiana tabacum) to obtainseveral transgenic lines. More than 12 independenttransgenic tobacco lines for each construct (lre-Pmec,lre-Pmec 9G, and lre-Pmec 9C) were analyzed for theexpression of the gusA reporter gene. Seedlings weregrown either continuously in the dark or with a 16-h-light/8-h-dark cycle for a period of 11 d beforefluorimetric GUS analysis.

In contrast to mutations at the seventh and eighthpositions (Kiran et al., 2006), mutation to G and C atthe ninth position exhibited enhancement of transcrip-tion in light. An increase by 8- to 9-fold was observedin lre-Pmec 9G and by 3- to 4-fold in lre-Pmec 9C incomparison with lre-Pmec (Fig. 1A). In the dark, theexpression of lre-Pmec 9G and lre-Pmec 9C increased byabout 5- and 3-fold, respectively (Fig. 1A). To studythis further, we generated several independent trans-genic lines of the prototype promoter and 9Gmutationwithout any activator region. The transgenic lines ofPmec 9G mutation in the TATA-box showed more than

Regulation of Nucleosome Formation by Consensus TATA-Box

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22-fold higher expression as compared with the trans-genic lines of Pmec with the prototype TATA-box (Fig.1B). Thus, the results indicate that the mutation in theTATA-box at the ninth position results in a morefavorable sequence architecture at the TATA-box,thus enhancing the level of transcription. Semiquanti-tative reverse transcription-PCR for the gusA tran-script showed that the GUS activity correlated wellwith the level of gusA mRNA in all cases (Fig. 1C).

The T9G/C Mutant Promoter Shows an Altered

Transcription Initiation Site

In order to study the mechanistic differences at theminimal promoters of lre-Pmec 9G and lre-Pmec 9C incomparison with lre-Pmec, we mapped the transcrip-tion start site of the three promoters. The result (Fig. 2)showed that lre-Pmec 9G and lre-Pmec 9C had multiplesites of transcription initiation. lre-Pmec 9G shared onecommon site with the lre-Pmec prototype construct,which showed only one transcription initiation site asreported earlier (Kiran et al., 2006). The mapping oftranscription sites is indicated on the sequence asarrows in Supplemental Figure S2. The multiple tran-scription start sites in 9G mutants suggest that thedifference in the TATA-box sequence may lead todifferences in the PIC formed on the core promoter.

Phytochrome-Mediated Transcription Enhancement IsLower in the T9G/C Mutant Promoter

The lre used in this study exhibits a 2- to 3-foldactivation of transcription in light in comparison withdark in the three constructs. We further examined thecontribution of light of different wavelengths in acti-vating transcription from these three constructs. Twotransgenic lines of each construct were grown incontinuous dark for a period of 9 d followed byexposure to continuous dark or to red, far-red, blue,or white light (16 h of light/8 h of dark) for 2 d beforethe seedlings were harvested to perform GUS assay.The prototype construct lre-Pmec showed 8.5-, 3.1-, and5.4-fold activation of transcription in red, far-red, andblue light, respectively, in comparison with dark,indicating the involvement of phytochrome in theregulation by lre (Fig. 3). The results are consistentwith our earlier results (Kiran et al., 2006). However, incomparison with the results obtained with the proto-type construct, both the mutants lre-Pmec 9G and lre-Pmec 9C showed significantly lower responses towardlight treatment. For lre-Pmec 9G, the activation oftranscription was 2.2-, 1.6-, and 1.1-fold, and for lre-Pmec 9C, it was 1.8-, 1.2-, and 1.8-fold in red, far-red,and blue light, respectively (in comparison with dark),which is significantly lower than the prototype (Fig. 3).The results indicate that the phytochrome-mediated

Figure 1. Role of ninth positionmutation in the TATA-box in light-responsive activator-dependent expres-sion of Pmec. A, GUS activity in 11-d-old light-grown(white bars) or dark-grown (black bars) transgenicseedlings containing lre-Pmec with prototype TATA-box and its variants with mutations lre-Pmec 9G andlre-Pmec 9C. B, GUS activity in 11-d-old transgenicseedlings containing minimal promoter Pmec (whitebar) or Pmec 9G (black bar) without any activatorregion. Error bars represent the SD of three indepen-dent experiments. The numbers of independent trans-genic lines (n) used in the analysis are indicated. MU,Methylumbelliferone. C, Semiquantitative reversetranscription-PCR carried out on light-grown (L) ordark-grown (D) transgenic seedlings of lre-Pmec, lre-Pmec 9G, and lre-Pmec 9C. The 26S rRNA levelsdetected by semiquantitative PCR are shown for com-parison.

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2176 Plant Physiol. Vol. 151, 2009



activation of transcription in the constructs lre-Pmec 9Gand lre-Pmec 9C was significantly less efficient ascompared with that in lre-Pmec.

Light Signaling in the T9G/C Mutant Promoter Operatesthrough a Calcium- and Sugar-Independent Pathway

Three different pathways have been identified inplants that act downstream of photoreceptors in light-mediated signal transduction: calcium, cGMP, andcalcium + cGMP. Phytochrome-dependent light acti-vation of cab and rbcS genes is mediated by calcium(Neuhaus et al., 1993; Wu et al., 1996). As the lre usedin this study was from the rbcS promoter of tobacco(Martinez-Hernandez et al., 2002), we were interestedto see the role of calcium in light signaling of lre-Pmec,

lre-Pmec 9G, and lre-Pmec 9C promoter constructs atdifferent concentrations. As expected, after 2 d incontinuous light, 10 mM calcium activated transcrip-tion from the prototype construct lre-Pmec by 2-fold,indicating a requirement for Ca2+ for lre-mediatedtranscription (Fig. 4A). The expression was inhibitedby EGTA, indicative of a requirement for Ca2+ ions foractivation. In contrast to this, lre-Pmec 9G and lre-Pmec9C promoters did not show any significant differencein the presence of calcium and EGTA. However, ageneral decline was observed in all three promoters inthe presence of 100 mM calcium, which could beindicative of a general cytotoxic effect of calcium atthis concentration (Fig. 4A).

In addition to the role of light in regulating theexpression of light-dependent promoters, metaboliz-able sugars also regulate the expression of promotersby a negative feedback regulation, leading to repres-sion of the light-dependent promoters (Krapp et al.,1991; Sheen, 1994). The lre used in this study contains asugar response element (S-box) that represses tran-scription in the presence of 25 to 50 mM Glc (Acevedo-Hernandez et al., 2005). In order to see the effect ofsugars at different concentrations on the expression oflre-Pmec, lre-Pmec 9G, and lre-Pmec 9C promoter con-structs, we developed several transgenic lines. Seed-lings harboring the lre-Pmec construct exhibited adecline in GUS activity at 50 mM Glc. In contrast tothis, seedlings with lre-Pmec 9G and lre-Pmec 9C didnot show a significant difference in GUS activity withrespect to control at any concentration of Glc (Fig. 4B).

The 9G-Dependent Transcriptional EnhancementSynergizes with Specific Upstream Activator Sequences

To evaluate if the synergy in augmentation of tran-scription from the 9G core promoter was general or

Figure 2. Mapping of point of transcription initiation from lre-Pmec,lre-Pmec 9G, and lre-Pmec 9C. Shown is primer extension analysisconducted with 32P-labeled primer (from +94 to +1 of the gusA gene)using RNA ligation-mediated RACE. The lanes C, G, T, and A representsequencing ladders. NC represents the nontemplate control, and thelanes lre-Pmec, lre-Pmec 9G, and lre-Pmec 9C represent primer-extended products of representative transgenic lines.

Figure 3. Effect of the quality of light on TATA-box-dependent geneexpression. GUS activities are shown in seedlings of transgenic lines oflre-Pmec (white bars), lre-Pmec 9G (checked bars), and lre-Pmec 9C(hatched bars) grown in dark for 9 d and then exposed to different lightconditions (i.e. 16 h of white light/8 h of dark [WL], continuous red[RL], far-red [FR], or blue [BL] light, and continuous dark [black bars])for 2 d. Intensities of the WL, RL, FR, and BL irradiations were 80, 10,0.86, and 15 mmol m22 s21, respectively. MU, Methylumbelliferone.





specific to certain upstream activator sequences, morecombinations were made. 9G was introduced in anumber of promoters, such as the constitutive syn-thetic promoter Pcec (Pcec 9G; Sawant et al., 2001), thechimeric seed-specific legumin promoter P1306 (leg-Pmec9G; Chaturvedi et al., 2007), the chimeric pathogen-inducible promoter PR-1a (PR-1a-Pmec 9G; Lodhi et al.,2008), and the native Arabidopsis rbcS promoter(AtrbcS-9G). Several transgenic tobacco lines wereobtained and were analyzed for their GUS activity. Incontrast to the results obtained with Pmec or lre-Pmec,the transgenic lines of Pcec 9G mutant showed at least4-fold reduction in GUS activity (Fig. 5A). AtrbcS-9Gshowed 30- and 11-fold reduction in GUS activityin transgenic lines when compared with the nativeAtrbcS with its prototype TATA box in light and dark,respectively (Fig. 5B). The seed-specific chimeric legu-min promoter leg-Pmec 9G showed around 8-foldhigher GUS activity in seeds of transgenic lines ascompared with leg-Pmec with the prototype TATA box(Fig. 5C). In the case of PR-1a-Pmec 9G, a mixedresponse was found. The mutant promoter showedslightly higher activity without salicylic acid inductionas compared with the prototype TATA box. However,when induced with salicylic acid, the expression ofGUS in the mutant lines decreased in contrast to theincrease in the prototype promoter (Fig. 5D). Thus, theresults indicate that the TATA-box and upstream acti-

vator sequences have a stringent specific requirementfor their synergy in function.

The 9G Mutation in the TATA-Box Results in aNucleosome-Free Region

We next examined the effect of the 9G mutation inthe TATA-box on nucleosome formation in this region.Mononucleosomal DNA template was prepared fromtransgenic lines of either Pmec or Pmec 9G showingcontrasting expression patterns and used as a templatein a PCR assay to determine nucleosome occupancy(Sekinger et al., 2005). Overlapping primers (Supple-mental Table S1) were designed for the TATA-boxregions (Supplemental Fig. S1). Semiquantitative PCRwith different groups of primers showed significantprotection from micrococcal nuclease (MNase) actionin the case of Pmec, indicative of a typical nucleosomeincorporating the TATA-box (Fig. 6). In contrast to theresults obtained with the Pmec transgenic line, thePmec 9G TATA region showed enhanced sensitivity toMNase and, hence, poor PCR amplification. The re-sults indicate the presence of a nucleosome-free regionaround the TATA-box with the T9G mutation (Fig. 6).The results are consistent in three independent trans-genic lines of Pmec (2b, 9b, and 4b) and Pmec 9G (12, 13,and 11) tested in this study. In order to establish thespecificity, the same mononucleosomal template was

Figure 4. Regulation of gene expression specific-ity by the TATA-boxmutations. Three independenttransgenic lines of lre-Pmec (white bars), lre-Pmec9G (black bars), and lre-Pmec 9C (gray bars)constructs were grown for 9 d in a 16-h-light/8-h-dark cycle onHoagland solution and exposedfor 2 d to the same solution supplemented with0.1, 1.0, 10, or 100mMcalciumor 10mMEGTA (A)and 25, 50, 100, or 200 mM Glc (B). All activitiesare expressed in relation to the activity of un-treated seedlings, taken as 1.0, under the given setof growth conditions.

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used to amplify the core promoter region of the pollen-specific promoter NTP303 (Weterings et al., 1995). Theposition of the nucleosome was not altered whenthe same groups of primers were used, indicatingthe specificity and the nucleosomal response (Fig. 6).Thus, a nucleosome-depleted region around theTATA-box in Pmec 9G is probably the reason for amore open structure and accessibility of the TATA-boxto other PIC components and the initiation of tran-scription with higher efficiency.

The TATAGATA Promoters in Arabidopsis Are NotTightly Repressed

We examined the 50-bp region upstream of thetranscription start site (Zhu et al., 1995; Gershenzonet al., 2006; Thomas and Chiang, 2006) in all the Arabi-dopsis genes in The Institute for Genome Researchdatabase for the presence of consensus TATA-boxsequence TATAGATA. We found 57 genes containingTATAGATA sequences upstream of the transcriptionstart site (Supplemental Table S2). However, 41 of thesehad more than one copy of the TATA-box within 50

bp upstream of the transcription start site. Only 16genes had only one TATAGATA and no otherTATA-like sequence in 50 bp upstream of the tran-scription start site. One of these, AT2G07716, isa pseudogene and was not considered for furtheranalysis. The 15 genes containing TATAGATA havediverse cellular functions (Table I). The expressionpattern of the genes containing TATAGATA wasexamined (except AT3G52345, which is a tRNAgene and hence not considered for the analysis) innine different tissues (germinated seed, seedling,young rosette, developed rosette, bolting, youngflower, developed flower, flowers and siliques, andmatured siliques) using the Genevestigator V3 tool(Hruz et al., 2008), which takes account of 4,070microarray experiments deposited in public data-bases for Arabidopsis (Supplemental Fig. S3). Thegenes containing TATAGATA showed consistentlyhigher expression, with intensities of hybridizationwell above 100 (Supplemental Fig. S3B, i–xiv) in allgenes except AT4G19720 (Supplemental Fig. S3Bv).In this case, although the expression is low, it isconsistently low in all the tissue conditions. Thus,

Figure 5. Expression of gusA in 11-d-old seedlings showing the synergisticeffect between TATA-box and differentupstream activator sequences. A, Con-stitutive synthetic promoter Pcec with-out (white bar) and with (black bar)T9G mutation. B, AtrbcS promoterwith (AtrbcS-9G) or without (AtrbcS)mutation in light-grown (white bars) ordark-grown (black bars) seedlings. C,Seed-specific legumin promoter P1306with (leg-Pmec 9G; black bar) or with-out (leg-Pmec; white bar) mutation. D,Pathogen-inducible promoter PR-1awith (PR-1a-Pmec 9G) or without(PR-1a-Pmec) mutation in uninduced(white bars) or salicylic acid-induced(black bars) condition. Error bars rep-resent the SD of three independentexperiments. The numbers of indepen-dent transgenic lines (n) used in theanalysis are indicated. F-test for signif-icance of variance (P value) is shown.MU, Methylumbelliferone.





the expression analysis of TATAGATA-containinggenes showed that the genes are always expressedat a higher level during plant development and thatnone of the genes is completely repressed. Theresults are in contrast to some of the TATATATA-containing genes we have examined; the results for14 TATATATA-containing genes are shown in Sup-plemental Figure S3D. These TATATATA-containinggenes show typical tissue-specific behavior: genes areexpressed during specific stages of expression andremain repressed in others. To further substantiateour results, we have checked the expression of sixdifferent tissue-specific genes (pollen specific[AT3G62230], seed specific [AT5G07190], stigma spe-cific [AT5G23960], gynoecium specific [AT4G32551],

microspore mother cell specific [AT2G45800], andsepal specific [AT1G05330]) as control genes usingGenevestigator software (Supplemental Fig. S3A,i–vi). These tissue-specific genes are significantly ex-pressed at the expected stages of plant developmentand remain completely repressed in other tissues(intensities of hybridization below 100), thus actingas good controls to substantiate our results on thealmost constitutive behavior of TATAGATA-containinggenes. Furthermore, we validated the microarray re-sults by quantifying nine TATAGATA-containinggenes by real-time quantitation of their transcripts indifferent tissues of Arabidopsis: leaf, root, seedling,and flower (Fig. 7). The quantity of transcripts of eachgene was normalized by the quantity of ubiquitingene in the respective tissues (see “Materials andMethods”). The normalized DCt (threshold cycle) forthe nine genes in different tissues showed that allnine genes were expressed ubiquitously at all stages(Fig. 7). Thus, the results indicate that TATAGATAmay be responsible for more favorable transcriptionalarchitecture at the minimal promoter and does notallow stringent repression.

We also identified 145 genes containing TATACATAout of which 35 genes contain TATACATA as theexclusive TATA-box in their minimal promoter region(Supplemental Table S4; i.e. within 50 bp upstream ofthe transcription start site). We further analyzed theexpression of TATACATA-containing genes usingGenevestigator; the expression profiles of the repre-sentative 14 genes are presented in SupplementalFigure S3C, i to xiv. The expression profiling of theTATACATA-containing genes showed that these genesare expressed consistently highly in all the tissues, asobserved in the case of TATAGATA-containing genes(Supplemental Fig. S3, compare B with C).

DISCUSSION

Our study shows that in spite of the prototypeTATA-box being only an eight-nucleotide sequence,individual position may determine specific aspects inpromoter function. Our earlier work (Kiran et al., 2006)showed that the seventh and eighth positions in theprototype core TATA-box TCACTATATATAG deter-mine light-induced promoter expression. The presenceof a G or C at these positions resulted in failure to forma light-specific transcription initiation complex. Thisstudy shows that the mutation of T at the ninthposition to G or C augments promoter function intransgenic tobacco lines both in the presence andabsence of lre. Thus, in contrast to the mutations atthe seventh and eighth positions (Kiran et al., 2006),the mutations at the ninth position do not alter light-specific promoter response (Fig. 1A). The expression inboth light and dark was enhanced equally as com-pared with the prototype promoter. The light-specificactivation in transcription is affected by the intracel-lular level of Glc (Acevedo-Hernandez et al., 2005),

Figure 6. Effect of the 9G mutation in the TATA-box on nucleosomeformation in the core promoter region. Shown here is the semiquan-titative PCR of Pmec and Pmec 9G core promoter regions to detectmononucleosome with four different sets of primers designed for theTATA-box region (Supplemental Table S1). The experiment was re-peated three times. The tobacco NTP303 promoter, used as an internalcontrol, is shown below. Identification of the transgenic line used toprepare each mononucleosome is given in parentheses.

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Ca2+ (Neuhaus et al., 1993; Wu et al., 1996), andphytochromes (Martinez-Garcia et al., 2000; Kimet al., 2002; Quail, 2002). However, in contrast to theprototype promoter with lre, the expression of theTATA-box with the 9G/C mutation was not altered byGlc, Ca2+ ions, or phytochrome. The results suggestthat the ninth position is not light specific. Instead, a Gor C at the ninth position provided sequence architec-ture more favorable to the assembly of PIC.

Transcript mapping (Fig. 2) showed that the 9G/CTATA-box led to a shift of the transcription initiationsite, suggestive of a different PIC formation. The PICcomposition depends upon the TATA sequence andinfluences transcription in yeast (Bjornsdottir andMyers, 2008). Additional factors were required forefficient in vivo transcription in certain TATA muta-tions. Similarly, in yeast and human, the PIC compo-sition or the site of PIC formation varies greatly,

Table I. The 15 genes with TATAGATA as the consensus TATA-box within 50 bp upstream of thetranscription start site in the genome of Arabidopsis

Serial

No.

Chromosome

No.

Arabidopsis

Genome

Initiative No.

OrientationPosition of

TATAGATAProtein Information

1 1 AT1G62935 Reverse 22 Unknown protein2 1 AT1G27490 Reverse 21 F-box family protein3 1 AT1G12970 Forward 32 Leu-rich repeat family protein4 1 AT1G15960 Reverse 26 Member of Nramp2 family5 1 AT1G32510 Forward 14 NAC domain-containing protein6 1 AT1G22800 Forward 43 Methyltransferase7 3 AT3G52345 Reverse 22 Pre-tRNA; tRNA-Cys (anticodon: GCA)8 3 AT3G46830 Reverse 9 Rab GTPase homolog9 3 AT3G20810 Forward 30 jumonji (jmjC) domain-containing

protein10 3 AT3G47000 Reverse 28 Glycosyl hydrolase family 3 protein11 3 AT3G03626 Reverse 11 Unknown protein12 4 AT4G27870 Forward 35 Integral membrane family protein13 4 AT4G18880 Reverse 33 Arabidopsis heat shock transcription

factor A4A14 4 AT4G19720 Reverse 42 Glycosyl hydrolase family15 5 AT5G04360 Forward 32 ATLDA/ATPU1; a-amylase/limit

dextrinase

Figure 7. Quantitation of the nineTATAGATA-containing genes in differ-ent tissues of Arabidopsis (leaf [stripedbars], root [crossed bars], seedling[checked bars], and flower [stippledbars]) by real-time PCR. Shown hereare the DCt values normalized againstubiquitin transcript in the respectivetissues.





depending upon the cellular or physiological state(Martinez, 2002; Muller and Tora, 2004; Thomas andChiang, 2006). These influence the rate of transcriptionand the start site selection. In both yeast and human,the TATA-containing promoters largely have singletranscription start sites (Hawkes and Roberts, 1999;David et al., 2006; Kuehner and Brow, 2006). However,a large proportion of the TATA-less genes show morethan one transcription start site (Yang et al., 2007;Juven-Gershon et al., 2008). The prototype TATA-boxused in this study has a plant consensus sequence(Sawant et al., 1999) that gives a single transcriptionstart site (Fig. 2).

The 9G mutation gave transcriptional activation ofthe prototype minimal promoter, without (Pmec) andwith lre (lre-Pmec) and legumin (leg-Pmec 9G) activatorsbut not with the constitutive (Pcec 9G), light-regulated(AtrbcS-9G), and salicylic acid-induced (PR-1a-Pmec9G) promoters. Thus, transcriptional enhancementdue to the 9Gmutation may not be general but specificto certain promoters, depending upon synergy withupstream activators. Functional specificity betweenthe upstream activator sequences and minimal pro-moters has been reported earlier (Mencia et al., 2002).Among plant promoters, we reported specificity be-tween the two components for stringent regulation ofthe PR-1a gene (Lodhi et al., 2008). Specificity infunctional compatibility has been reported betweenupstream and core promoter sequences for transcrip-tional activation in yeast, Drosophila, and mammaliancells (Butler and Kadonaga, 2001, 2002; Cheng et al.,2002; Li et al., 2002; Mencia et al., 2002; Gross andOelgeschlager, 2006; Muller et al., 2007).

Activation of transcription by the 9G mutation inthe TATA-boxmay be due to the formation of a PIC thatsynergizeswith specificupstreamsequence-dependentactivators. The 9G mutation in the TATA-box resultsin enhancing micrococcal nuclease sensitivity aroundthe minimal promoter (Fig. 6), suggesting that the 9Gmutation leads to either a nucleosome-free regionaround the TATA-box or a more open chromatinstructure. We have previously reported that theprototype TATA-box in Pmec used in this study isincorporated into a distinct nucleosome (Lodhi et al.,2008). The TATATATA sequence has a dyad symmetryand possesses a high capability for nucleosome for-mation (Boyle et al., 2008). Pyrosequencing of nucle-osomes (Owen-Hughes and Engeholm, 2007) andnucleosome mapping (Albert et al., 2007; Barskiet al., 2007; Lee et al., 2007; Mavrich et al., 2008;Valouev et al., 2008; Hall et al., 2009) have alsodemonstrated that stretches of AT or TA favor theformation of nucleosome over them. Thus, the 9Gmutation in TATAGATA might disturb dyad symme-try and TA stretch, affecting its ability to be packagedinto a nucleosome. The lack of stringent nucleosomalstructure over TATAGATA may result in higher ac-cessibility of the TATA-box region to PIC assembly.However, the synergy with certain upstream se-quences, and not others, suggests that the substitution

of 9T with G alters the composition or conformationof the PIC. The altered PIC may make better contactswith specific activator complexes.

Lomvardas and Thanos (2002) demonstrated thatexchanging the IFNb minimal promoter (which has adistinct nucleosome containing the TATA-box at rest-ing state that slides after viral induction) with the IL8minimal promoter (which does not have a nucleosomeover the TATA-box) resulted in a chimeric promoterthat showed higher background transcription. Thetranscript accumulation from the chimeric promoterfurther increased substantially following virus infec-tion as compared with that from the native IFNbpromoter. Thus, removal of the repressing nucleoso-mal architecture by replacing it with an alternativeminimal promoter (IL8) that does not form a nucleo-some over the TATA-box resulted in an overall in-crease in promoter activity. In the case of the tightlyregulated seed-specific b-phaseolin gene, the repres-sive state in other plant tissues is due to the presence ofa nucleosome over the TATA-box (Carranco et al.,2004). In developing seeds, the nucleosome gets re-modeled by the transcription factor PvALF.

Our computational analysis of the native Arabidop-sis promoters showed that in Arabidopsis, around 11%of protein-coding genes (Supplemental Table S5)are actually TATA containing, as per the consensusreported by Molina and Grotewold (2005). The TATA-containing genes are majorly dominated by TATA-TATA (990 genes) and TATAAATA (867 genes); thesetogether constitute around 57% of the total TATA-box-containing genes (1,857 out of 3,218; SupplementalTable S5). The TATA-consensus sequences TATAGATAand TATACATA are least preferred, being 0.18% and0.47%, respectively, of total protein-coding genes (Sup-plemental Table S5). The genes containing eitherTATAGATA or TATACATA did not show tight regu-lation during development, as analyzed by microarrayanalysis (Supplemental Fig. S3, B and C) or in real-timequantitation (Fig. 7), in contrast to the genes contain-ing TATATATA (Supplemental Fig. S3D). The resultsindicate that the TATA-consensus-like TATATATAmay favor nucleosome structure over the TATA-box,which is responsible for tight regulation of these genes.However, absence of stringent nucleosome structureover the TATA-box as a result of T9G or T9C consensusmay result in expression of these genes almost consti-tutively in different tissues during development.It will be interesting to map nucleosomes over theminimal promoter in the 15 Arabidopsis genes toevaluate our hypothesis further. Both TATAGATA andTATACATA show activation by the lre element (Fig.1A). However, the activation by TATAGATA is at least2-fold higher than that by TATACATA. Our resultssuggest that the TATAGATA sequence may be morepotent in removing chromatin-repressive structureover the TATA-box as compared with TATACATA.Thus, the promoters with TATAGATA sequencesare less likely to be stringently regulated and hencemay not be preferred in nature as compared with

Ranjan et al.




TATACATA. However, our hypothesis requires furtherevaluation.

MATERIALS AND METHODS

Construction of Mutations in Pmec and lre-Pmec

The constructs Pmec and lre-Pmec have been described by Kiran et al.

(2006). In this study, mutations in Arabidopsis (Arabidopsis thaliana) were

created at the ninth nucleotide position of the TATA sequence TCACTATA-

TATAG to G or C in Pmec by PCR using degenerate primers. A total of 10

random clones were picked for each primer and sequenced. A 52-bp light-

responsive element (lre), called CMA5 (Martinez-Hernandez et al., 2002), was

fused in single copy immediately upstream of Pmec 9G and Pmec 9C to obtain

the respective constructs under the control of lre. These were cloned into

binary vector pBI101 for transformation in tobacco (Nicotiana tabacum var Petit

Havana). A number of independent stable transgenic lines were generated to

take into account variations due to the site of insertion.

Site-Directed Mutagenesis

Mutations at the ninth position in the TATA-box of Pcec, AtrbcS, leg-Pmec,

and PR-1a-Pmec promoters were generated using P9GF (forward) and P9GR

(reverse) primers for Pcec, AtrbcS 9GF (forward) and AtrbcS 9GR (reverse)

primers for AtrbcS, LAP9GF (forward) and LAP9GR (reverse) primers for leg-

Pmec, and PRP9GF (forward) and PRP9GR (reverse) primers for PR-1a-Pmec

by site-directed PCR mutagenesis (QuikChange XL kit; Stratagene) according

to the manufacturer’s protocol. The clones were screened by DNA sequencing

for the desiredmutations using T3, T7, and P-10 primers. The sequences of the

primers used in the mutagenesis and sequencing are given in Supplemental

Table S1.

Plant Transformation

The gene constructs were cloned into the binary vector pBI101 (Clontech).

The recombinant vector plasmids were mobilized into Agrobacterium tumefa-

ciens strain LBA4404 by electroporation. Leaf disc explants were transformed

following the protocol of Horsch et al. (1985). Several independently trans-

formed plants were developed with each construct. Transgenic plants were

grown in a 16-h-light/8-h-dark cycle. The number of independent transgenic

lines tested in a given experiment are specified in the figures.

Growth Conditions and GUS Assay

After transformation, seeds of the T0 generation were collected and

homozygous lines were obtained through kanamycin (300 mg L21) selection.

Seeds of the homozygous lines from the T1 generation were used for detailed

studies. For light versus dark experiments, seeds were grown in a 16-h-light/

8-h-dark cycle or in complete darkness for 11 d before estimating the

expression of GUS using the fluorogenic substrate 4-methylumbelliferyl-b-D-

glucuronide (Jefferson and Wilson, 1991). For salicylic acid induction exper-

iments, seedlings were harvested and floated on water (uninduced) or 2 mM

salicylic acid (induced) in petri dishes and incubated in 16 h of light at 25�C62�C for 48 h before performing GUS assay. For assay in seeds, 50 mg of mature

seeds soaked in water for 4 h was used for GUS assay. After induction, the

seedlings were ground in liquid nitrogen, and GUS assay was performed as

described (Kiran et al., 2006). The product, 4-methylumbelliferone, was

quantified on a luminescence spectrophotometer (LS-55; Perkin-Elmer) with

excitation at 365 nm and emission at 455 nm. Total soluble protein in seedling

extracts was quantified using Bradford reagent (catalog no. 500-0006; Bio-Rad

Laboratories). Enzyme activity was expressed as pmol methylumbelliferone

released min21 mg21 soluble protein. Each experiment was performed at least

three times.

Light, Sugar, Calcium, and EGTA Treatment

For experiments on induction by light quality, 9-d-old dark-grown seed-

lings were exposed to white (80 mmol m22 s21), red (10 mmol m22 s21), blue (15

mmol m22 s21), and far-red (0.86 mmol m22 s21) light for 2 d before measure-

ment of GUS activity. For different sugar treatments, seedlings were grown for

9 d in a growth room at 25�C 6 2�C under a 16-h-light/8-h-dark photoperiod

with a light fluence of 80 mmol m22 s21 in Hoagland medium and transferred

to the same medium supplemented with 0, 25, 50, 100, or 200 mM Glc for 2 d

before estimating GUS activity. For calcium and EGTA treatments, seedlings

were grown as described above for 9 d and transferred to Hoagland medium

supplemented with different concentrations of calcium (0, 0.1, 1, 10, or 100 mM

CaCl2) as well as 10 mM EGTA for 2 d before estimating GUS activity.

RNA Extraction and Reverse Transcription

Total RNA was extracted from tobacco seedlings grown in Hoagland

medium in light/dark for 11 d. Tissue was frozen in liquidN2 and ground, and

RNA was extracted by Tri-Reagent (Sigma Pharmaceuticals). After DNase

treatment (Ambion), the integrity of RNA was tested by electrophoresis. The

first-strand cDNA was prepared from 2 mg of total RNA with SuperScript II

reverse transcriptase (Invitrogen) and an oligo(dT) primer at a temperature of

42�C according to the manufacturer’s instructions. The optimal number of

cycles for semiquantitative analysis was 28. The primers utilized for the GUS

gene were uidAF and uidAR (Supplemental Table S1), which gave an

amplified fragment of 1.87 kb.

RNA Ligation-Mediated RACE to Map gusA Transcriptsof lre-Pmec as Well as lre-Pmec 9G and lre-Pmec9C Constructs

Total RNA of lre-Pmec, lre-Pmec 9G, and lre-Pmec 9C light-grown transgenic

seedlings was used to perform 5# RNA ligation-mediated RACE using a kit

from Ambion. Primer extension was performed as described (Kiran et al.,

2006).

Nuclei Isolation and Digestion with MNase

Nuclear protein from tobacco leaves was prepared from plants grown in a

16-h-light/8-h-dark cycle. Tobacco leaf tissue was washed thoroughly with

distilled water and subjected to cross-linking in the presence of 1% formal-

dehyde for 30 min. The leaves were then rinsed with water, ground into

powder in liquid nitrogen, and suspended in homogenization buffer (1 M

hexylene glycol, 10 mM PIPES/KOH, pH 7.0, 10 mM MgCl2, 0.5% [v/v] Triton

X-100, 5 mM 2-mercaptoethanol, and 0.8 mM phenylmethylsulfonyl fluoride

[PMSF]). The homogenized extract was filtered sequentially through four

layers of muslin cloth, followed by 80-, 60-, 40-, and 20-mmnylonmesh. Nuclei

were pelleted at 3,000g for 10 min and resuspended gently with a soft paint

brush in wash buffer (0.5 M hexylene glycol, 10 mM PIPES/KOH, pH 7.0, 10

mM MgCl2, 5 mM 2-mercaptoethanol, 0.5% [v/v] Triton X-100, and 0.8 mM

PMSF). Nuclei were pelleted at 3,000g for 15 min, and the pellet was

resuspended in 5% Percoll (U.S. Biologicals) buffer (0.5 M Suc, 25 mM Tris,

pH 8.0, 10 mM MgCl2, 0.8 mM PMSF, 0.5% Triton X-100, 5 mM 2-mercaptoeth-

anol, and 5% Percoll) and loaded onto a 40% to 80% Percoll step gradient

containing a 2 M Suc pad at the bottom. The nuclei were collected from the 80%

Percoll-2 M Suc interface, washed in the wash buffer without Triton X-100, and

resuspended in lysis buffer (110 mM KCl, 15 mM HEPES/KOH, pH 7.5, 5 mM

MgCl2, 1 mM dithiothreitol, and 5 mg mL21 leupeptin). The nuclear prepara-

tion equivalent to A260 of 100 was incubated with micrococcal nuclease (300

units mL21; Fermentas Life Sciences) in a buffer containing 25 mM KCl, 4 mM

MgCl2, 1 mM CaCl2, 50 mM Tris-Cl, pH 7.4, and 12.5% glycerol at 37�C for 10

min. The reaction was stopped by adding an equal volume of 2% SDS, 0.2 M

NaCl, 10 mM EDTA, 10 mM EGTA, and 50 mM Tris-Cl, pH 8.0, and treated with

proteinase K (100 mg mL21; Ambion) for 1 h at 55�C. The cross-link was

reversed by heating at 65�C overnight. The DNA was extracted, and the

samples were incubated with RNaseA (100 mg mL21; Qiagen), extracted by

phenol-chloroform, and precipitated in ethanol. The DNAwas separated on a

1.5% agarose gel, and fragments of an average size of 144 to 160 bp were

purified.

Semiquantitative PCR Using Mononucleosome TemplateDNA to Detect Nucleosomes

Semiquantitative PCR was used to examine the nucleosomes on Pmec and

Pmec 9G promoters. The forward primers (TCPF1, TCPF2, and TCPF4) and the





reverse primers (TCPR1G, TCPR2G, TCPR3, and TCPR4) were used to

analyze protection of the core promoter region against micrococcal nuclease

digestion. The PCR efficiency of the primers was checked using tobacco

genomic DNA as template. A pollen-specific promoter (NTP303) of tobacco

was used as the internal control. The forward (NTP303F) and reverse

(NTP303R) primers were designed for semiquantitative PCR of the core

promoter region. The PCR products were visualized on a 1.5% agarose gel by

ethidium bromide staining. Sequences of the primers are given in Supple-

mental Table S1, and the positions are shown in Supplemental Figure S1.

Sequence Analysis of Upstream Promoter Regions

in Arabidopsis

The 500-bp upstream sequences of all the Arabidopsis genes were down-

loaded from the TAIR8 database (ftp://ftp.arabidopsis.org/home/tair/

Sequences/blast_datasets/TAIR8_blastsets/TAIR8_upstream_500_20080228).

The sequences in this data set are immediately upstream of the 5# untranslatedregion for those genes with annotated untranslated regions and upstream of

the translational start for the remainder. This data set comprised 33,281 genes.

From this data set, a new data set was made with 50 bp of sequence upstream

of the 5# end of each gene. This data set was used for the promoter analysis of

all genes from Arabidopsis.

For the protein-coding genes, all the loci for coding sequences were

extracted from the TAIR8 database (ftp://ftp.arabidopsis.org/home/tair/

Sequences/blast_datasets/TAIR8_blastsets/ TAIR8_cdna_20080412). From

this data set, all the redundant gene loci were removed (i.e. all alternative

spliced forms of the genes were removed), and this resulted in 27,379

nonredundant gene loci. These loci were used to extract the 50-bp upstream

sequences from the above-mentioned gene promoter database. We examined

the presence of consensus TATA-box and TATA consensus motifs (i.e.

TATAGATA, TATACATA, and TATAAATA) in the 50 bp upstream using the

Motif Scanner Program (Thijs et al., 2001). The nucleotide frequency matrix

(NFM) for the TATA-consensus was used from the NFM discussed by Molina

and Grotewold (2005). For all other motifs, pure NFM was used, giving 100%

frequency for each nucleotide at its respective position.

Microarray Analysis of Genes Having TATA-Box

Consensus Promoters in Arabidopsis

Using Genevestigator V3 (http://www.genevestigator.ethz.ch), we stud-

ied the expression patterns of the genes having different TATA-box consensus

across all 4,070 Affymetrix arrays. The profiles for various genes were

generated using the meta-profile analysis tool across different developmental

stages of Arabidopsis.

Real-Time PCR for the Quantitation of Genes with

TATAGATA Promoters in Arabidopsis

Total RNA was extracted from different tissues of the Arabidopsis plant

(leaf, root, seedling, and flower) by the Spectrum Plant Total RNA Kit (Sigma-

Aldrich) and treated with RNase free DNase (Ambion). The first-strand cDNA

was prepared from 2 mg of total RNAwith SuperScript II reverse transcriptase

and an oligo(dT) primer according to the manufacturer’s instructions. The

primers for the real-time analysis of nine target genes and one reference

ubiquitin gene (AT4G05320) was designed using Primer Express software

(Applied Biosystems); sequences of the primers are given in Supplemental

Table S1. Real-time PCR for nine target genes and the internal reference

ubiquitin gene was carried out using an ABI Prism 7000 sequence detection

system (Applied Biosystems) and SYBR Green chemistry. The Ct value for

each gene and reference gene was determined using ABI Prism 7000 SDS

software, and the Ct value was calculated as Ct = Ct (target gene) – Ct

(ubiquitin) for each particular tissue. The experiments were carried out with at

least three biological replicates and two technical replicates, and each exper-

iment was repeated twice.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers AT1G05330, AT2G45800, AT3G62230,

AT4G32551, AT5G07190, AT5G23960, AT4G05320, AT1G62935, AT1G27490,



AT4G19720, and AT5G04360.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Positions of the primers designed from the core

promoter regions of Pmec and Pmec 9G constructs are indicated by

arrowheads.

Supplemental Figure S2. The nucleotide sequence of the Pmec-gusA

cassette.

Supplemental Figure S3. Microarray expression profiles of genes contain-

ing TATAGATA, TATACATA, or TATATATA sequence along with tissue-

specific genes used as a control using Genevestigator V3.

Supplemental Table S1. Primer information for site-directed mutagenesis

(SDM), cloning and sequencing of constructs, detection of mononucleo-

somes, and semiquantitative and real-time PCR experiments.

Supplemental Table S2. List of 57 genes containing the consensus TATA-

box or its variant TATAGATA present within 50 bp upstream of the

transcription start site in Arabidopsis.

Supplemental Table S3. List of 145 genes containing the consensus TATA-

box or its variant TATACATA present within 50 bp upstream of the

transcription start site in Arabidopsis.

Supplemental Table S4. List of 35 genes containing the TATACATA

sequence present within 50 bp upstream of the transcription start site in

Arabidopsis.

Supplemental Table S5. Identification of TATAmotifs in250-bp upstream

promoter sequences of protein-coding and total genes in Arabidopsis.

ACKNOWLEDGMENT

We thank Prof. Randall H. Morse (Wadsworth Center) for critical reading

of the manuscript.

Received September 25, 2009; accepted September 30, 2009; published October

7, 2009.

LITERATURE CITED

Acevedo-Hernandez GJ, Leon P, Herrera-Estrella LR (2005) Sugar and

ABA responsiveness of a minimal RBCS light-responsive unit is medi-

ated by direct binding of ABI4. Plant J 43: 506–519

Albert I, Mavrich TN, Tomsho LP, Qi J, Zanton SJ, Schuster SC, Pugh BF

(2007) Translational and rotational settings of H2A.Z nucleosomes

across the Saccharomyces cerevisiae genome. Nature 446: 572–576

Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G,

Chepelev I, Zhao K (2007) High-resolution profiling of histone meth-

ylations in the human genome. Cell 129: 823–837

Bjornsdottir G, Myers LC (2008) Minimal components of the RNA poly-

merase II transcription apparatus determine the consensus TATA box.

Nucleic Acids Res 36: 2906–2916

Boyle AP, Davis S, Shulha HP, Meltzer P, Margulies EH, Weng Z, Furey

TS, Crawford GE (2008) High-resolution mapping and characterization

of open chromatin across the genome. Cell 132: 311–322

Brent R, Ptashne M (1985) A eukaryotic transcriptional activator bearing

the DNA specificity of a prokaryotic repressor. Cell 43: 729–736

Butler JE, Kadonaga JT (2001) Enhancer-promoter specificity mediated by

DPE or TATA core promoter motifs. Genes Dev 15: 2515–2519

Butler JE, Kadonaga JT (2002) The RNA polymerase II core promoter:

a key component in the regulation of gene expression. Genes Dev 16:

2583–2592

Carranco R, Chandrasekharan MB, Townsend JC, Hall TC (2004) Inter-

action of PvALF and VP1 B3 domains with the beta-phaseolin promoter.

Plant Mol Biol 55: 221–237

Chaturvedi CP, Lodhi N, Ansari SA, Tiwari S, Srivastava R, Sawant SV,

Tuli R (2007) Mutated TATA-box/TATA binding protein complementa-

tion system for regulated transgene expression in tobacco. Plant J 50:

917–925

Cheng JX, Floer M, Ononaji P, Bryant G, Ptashne M (2002) Responses of

Ranjan et al.




four yeast genes to changes in the transcriptional machinery are deter-

mined by their promoters. Curr Biol 12: 1828–1832

Chi T, Carey M (1996) Assembly of the isomerized TFIIA-TFIID-TATA

ternary complex is necessary and sufficient for gene activation. Genes

Dev 10: 2540–2550

Croston GE, Laybourn PJ, Paranjape SM, Kadonaga JT (1992) Mecha-

nism of transcriptional antirepression by GAL4-VP16. Genes Dev 6:

2270–2281

David L, Huber W, Granovskaia M, Toedling J, Palm CJ, Bofkin L, Jones

T, Davis RW, Steinmetz LM (2006) A high-resolution map of transcrip-

tion in the yeast genome. Proc Natl Acad Sci USA 103: 5320–5325

Duan ZJ, Fang X, Rohde A, Han H, Stamatoyannopoulos G, Li Q (2002)

Developmental specificity of recruitment of TBP to the TATA box of the

human gamma-globin gene. Proc Natl Acad Sci USA 99: 5509–5514

Gershenzon NI, Trifonov EN, Ioshikhes IP (2006) The features of Dro-

sophila core promoters revealed by statistical analysis. BMC Genomics

7: 161

Grace ML, Chandrasekharan MB, Hall TC, Crowe AJ (2004) Sequence and

spacing of TATA box elements are critical for accurate initiation from the

beta-phaseolin promoter. J Biol Chem 279: 8102–8110

Gross P, Oelgeschlager T (2006) Core promoter-selective RNA polymerase

II transcription. Biochem Soc Symp 73: 225–236

Hahn S (2004) Structure and mechanism of the RNA polymerase II

transcription machinery. Nat Struct Mol Biol 11: 394–403

Hall MA, Shundrovsky A, Bai L, Fulbright RM, Lis JT, Wang MD (2009)

High-resolution dynamic mapping of histone-DNA interactions in a

nucleosome. Nat Struct Mol Biol 16: 124–129

Hawkes NA, Roberts SG (1999) The role of human TFIIB in transcription

start site selection in vitro and in vivo. J Biol Chem 274: 14337–14343

Hochheimer A, Tjian R (2003) Diversified transcription initiation com-

plexes expand promoter selectivity and tissue-specific gene expression.

Genes Dev 17: 1309–1320

Hope IA, Mahadevan S, Struhl K (1988) Structural and functional char-

acterization of the short acidic transcriptional activation region of yeast

GCN4 protein. Nature 333: 635–640

Hope IA, Struhl K (1986) Functional dissection of a eukaryotic transcrip-

tional activator protein, GCN4 of yeast. Cell 46: 885–894

Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT

(1985) A simple and general method for transferring genes into plants.

Science 227: 1229–1231

Hruz T, Laule O, Szabo G, Wessendorp F, Bleuler S, Oertle L, Widmayer P,

Gruissem W, Zimmermann P (2008) Genevestigator V3: a reference

expression database for the meta-analysis of transcriptomes. Adv

Bioinform 2008: 420747

Jefferson RA, Wilson KJ (1991) The GUS gene fusion system. In S Gelvin, R

Schilperoot, eds, Plant Molecular Biology Manual, Vol B 14. Kluwer

Academic Publishers, Dordrecht, The Netherlands, pp 1–33

Jiao Y, Lau OS, Deng XW (2007) Light-regulated transcriptional networks

in higher plants. Nat Rev Genet 8: 217–230

Jiao Y, Ma L, Strickland E, Deng XW (2005) Conservation and divergence

of light-regulated genome expression patterns during seedling devel-

opment in rice and Arabidopsis. Plant Cell 17: 3239–3256

Juven-Gershon T, Hsu JY, Theisen JW, Kadonaga JT (2008) The RNA

polymerase II core promoter: the gateway to transcription. Curr Opin

Cell Biol 20: 253–259

Kim JI, Kozhukh GV, Song PS (2002) Phytochrome-mediated signal

transduction pathways in plants. Biochem Biophys Res Commun 298:

457–463

Kingston RE, Bunker CA, Imbalzano AN (1996) Repression and activation

by multiprotein complexes that alter chromatin structure. Genes Dev 10:

905–920

Kiran K, Ansari SA, Srivastava R, Lodhi N, Chaturvedi CP, Sawant SV,

Tuli R (2006) The TATA-box sequence in the basal promoter contributes

to determining light-dependent gene expression in plants. Plant Physiol

142: 364–376

Klein C, Struhl K (1994) Increased recruitment of TATA-binding protein to

the promoter by transcriptional activation domains in vivo. Science 266:

280–282

Kloeckener-Gruissem B, Vogel JM, Freeling M (1992) The TATA box

promoter region of maize Adh1 affects its organ-specific expression.

EMBO J 11: 157–166

Krapp A, QuickWP, Stitt M (1991) Ribulose-1,5-bisphosphate carboxylase-

oxygenase, other Calvin-cycle enzymes, and chlorophyll decrease when

glucose is supplied to mature leaves via the transpiration stream. Planta

186: 58–69

Krumm A, Hickey LB, Groudine M (1995) Promoter-proximal pausing of

RNA polymerase II defines a general rate-limiting step after transcrip-

tion initiation. Genes Dev 9: 559–572

Kuehner JN, Brow DA (2006) Quantitative analysis of in vivo initiator

selection by yeast RNA polymerase II supports a scanning model. J Biol

Chem 281: 14119–14128

Lee W, Tillo D, Bray N, Morse RH, Davis RW, Hughes TR, Nislow C

(2007) A high-resolution atlas of nucleosome occupancy in yeast. Nat

Genet 39: 1235–1244

Li XY, Bhaumik SR, Zhu X, Li L, Shen WC, Dixit BL, Green MR (2002)

Selective recruitment of TAFs by yeast upstream activating sequences:

implications for eukaryotic promoter structure. Curr Biol 12: 1240–1244

Lodhi N, Ranjan A, Singh M, Srivastava R, Singh SP, Chaturvedi CP,

Ansari SA, Sawant SV, Tuli R (2008) Interactions between upstream

and core promoter sequences determine gene expression and nucleo-

some positioning in tobacco PR-1a promoter. Biochim Biophys Acta

1779: 634–644

Lomvardas S, Thanos D (2002) Modifying gene expression programs by

altering core promoter chromatin architecture. Cell 110: 261–271

Ma L, Li J, Qu L, Hager J, Chen Z, Zhao H, Deng XW (2001) Light control of

Arabidopsis development entails coordinated regulation of genome

expression and cellular pathways. Plant Cell 13: 2589–2607

Martinez E (2002) Multi-protein complexes in eukaryotic gene transcrip-

tion. Plant Mol Biol 50: 925–947

Martinez-Garcia JF, Huq E, Quail PH (2000) Direct targeting of light

signals to a promoter element-bound transcription factor. Science 288:

859–863

Martinez-Hernandez A, Lopez-Ochoa L, Arguello-Astorga G, Herrera-

Estrella L (2002) Functional properties and regulatory complexity of a

minimal RBCS light-responsive unit activated by phytochrome, crypto-

chrome, and plastid signals. Plant Physiol 128: 1223–1233

Mavrich TN, Jiang C, Ioshikhes IP, Li X, Venters BJ, Zanton SJ, Tomsho

LP, Qi J, Glaser RL, Schuster SC, et al (2008) Nucleosome organization

in the Drosophila genome. Nature 453: 358–362

Mencia M, Moqtaderi Z, Geisberg JV, Kuras L, Struhl K (2002) Activator-

specific recruitment of TFIID and regulation of ribosomal protein genes

in yeast. Mol Cell 9: 823–833

Molina C, Grotewold E (2005) Genome wide analysis of Arabidopsis core

promoters. BMC Genomics 6: 25

Muller F, Demeny MA, Tora L (2007) New problems in RNA polymerase II

transcription initiation: matching the diversity of core promoters with a

variety of promoter recognition factors. J Biol Chem 282: 14685–14689

Muller F, Tora L (2004) The multicoloured world of promoter recognition

complexes. EMBO J 23: 2–8

Nakamura M, Tsunoda T, Obokata J (2002) Photosynthesis nuclear genes

generally lack TATA-boxes: a tobacco photosystem I gene responds to

light through an initiator. Plant J 29: 1–10

Neuhaus G, Bowler C, Kern R, Chua NH (1993) Calcium/calmodulin-

dependent and -independent phytochrome signal transduction path-

ways. Cell 73: 937–952

Owen-Hughes T, Engeholm M (2007) Pyrosequencing positions nucleo-

somes precisely. Genome Biol 8: 217

Ptashne M, Gann A (1997) Transcriptional activation by recruitment.

Nature 386: 569–577

Quail PH (2002) Phytochrome photosensory signalling networks. Nat Rev

Mol Cell Biol 3: 85–93

Roberts SG, GreenMR (1994) Activator-induced conformational change in

general transcription factor TFIIB. Nature 371: 717–720

Rougvie AE, Lis JT (1990) Postinitiation transcriptional control in Dro-

sophila melanogaster. Mol Cell Biol 10: 6041–6045

Sawant SV, Kiran K, Singh PK, Tuli R (2001) Sequence architecture

downstream of the initiator codon enhances gene expression and

protein stability in plants. Plant Physiol 126: 1630–1636

Sawant SV, Singh PK, Gupta SK, Madanala R, Tuli R (1999) Conserved

nucleotide sequence in highly expressed genes in plants. J Genet 78:

123–131

Sekinger EA, Moqtaderi Z, Struhl K (2005) Intrinsic histone-DNA inter-

actions and low nucleosome density are important for preferential

accessibility of promoter regions in yeast. Mol Cell 18: 735–748

Sheen J (1994) Feedback control of gene expression. Photosynth Res 39:

427–438





Smale ST (2001) Core promoters: active contributors to combinatorial gene

regulation. Genes Dev 15: 2503–2508

Struhl K (1996) Chromatin structure and RNA polymerase II connection:

implications for transcription. Cell 84: 179–182

Struhl K (1999) Fundamentally different logic of gene regulation in

eukaryotes and prokaryotes. Cell 98: 1–4

Thijs G, Lescot M, Marchal K, Rombauts S, De Moor B, Rouze P, Moreau

Y (2001) A higher-order background model improves the detection of

promoter regulatory elements by Gibbs sampling. Bioinformatics 17:

1113–1122

Thomas MC, Chiang CM (2006) The general transcription machinery and

general cofactors. Crit Rev Biochem Mol Biol 41: 105–178

Valouev A, Ichikawa J, Tonthat T, Stuart J, Ranade S, PeckhamH, Zeng K,

Malek JA, Costa G, McKernan K, et al (2008) A high-resolution,

nucleosome position map of C. elegans reveals a lack of universal

sequence-dictated positioning. Genome Res 18: 1051–1063

Weterings K, Schrauwen J, Wullems G, Twell D (1995) Functional dissec-

tion of the promoter of the pollen-specific gene NTP303 reveals a novel

pollen-specific, and conserved cis-regulatory element. Plant J 8: 55–63

Workman JL, Taylor IC, Kingston RE (1991) Activation domains of stably

bound GAL4 derivatives alleviate repression of promoters by nucleo-

somes. Cell 64: 533–544

Wu Y, Hiratsuka K, Neuhaus G, Chua NH (1996) Calcium and cGMP target

distinct phytochrome-responsive elements. Plant J 10: 1149–1154

Yang C, Bolotin E, Jiang T, Sladek FM, Martinez E (2007) Prevalence of the

initiator over the TATA box in human and yeast genes and identification

of DNA motifs enriched in human TATA-less core promoters. Gene 389:

52–65

Yankulov K, Blau J, Purton T, Roberts S, Bentley DL (1994) Transcrip-

tional elongation by RNA polymerase II is stimulated by transactiva-

tors. Cell 77: 749–759

Zhu Q, Dabi T, Lamb C (1995) TATA box and initiator functions in the

accurate transcription of a plant minimal promoter in vitro. Plant Cell 7:

1681–1689

Ranjan et al.




a t9g mutation in the prototype tata-box ......with rbcs, pcec, and pr-1a activators. the 9g...

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