Architecture of the Maize Mitochondrial atpl Promoter asDetermined by Linker-Scanning and Point MutagenesisWILLIAM D. RAPP,t D. SHELLEY LUPOLD, SUSAN MACK, AND DAVID B. STERN*Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853
Received 8 June 1993/Returned for modification 3 August 1993/Accepted 23 August 1993
Plant mitochondrial promoters are poorly conserved but generally share a loose consensus sequencespanning approximately 17 nucleotides. Using a homologous in vitro transcription system, we have previouslyshown that an 11-nucleotide sequence within this region comprises at least part of the maize mitochondrial atplpromoter (W. Rapp and D. Stern, EMBO J. 11:1065-1073, 1992). We have extended this finding by using aseries of linker-scanning and point mutations to define the atlp promoter in detail. Our results show thatmutations at positions -12 to +5, relative to the major transcription start site, can decrease initiation rates tobetween < 10 and 401% ofwild-type levels. Some mutations, scattered throughout this region, have lesser effectsor no effect. Taken together, our data suggest a model in which the atpl promoter consists of a central domainextending from -7 to +5 and an upstream domain of 1 to 3 bp that is centered around -11 to -12. Becausemany mutations within this promoter region are tolerated in vitro, the maize atp) promoter is distinct from thehighly conserved yeast mitochondrial promoters.
Promoters of mitochondrial genes differ in vertebrates,yeasts, and plants. The 16- to 17-kb vertebrate mitochondrialgenome is symmetrically transcribed from one heavy-strandand one light-strand promoter. In vitro transcription systemshave been used to define the sequences of several of thevertebrate promoters, which vary in sequence content andrange in size from 8 bp in Xenopus laevis to approximately40 bp in mammals (reviewed in reference 6). Mammalianmitochondrial promoters contain a polymerase recognitionsite and an upstream binding site for a transcription speci-ficity factor. Initiation at the heavy-strand promoter alsoplays an essential role in DNA replication. Similar ap-proaches have shown that the Saccharomyces cerevisiaemitochondrial genome contains approximately 20 copies of ahighly conserved nonanucleotide promoter that has a singletranscription initiation site at the final adenine (3, 4, 8). TheRNA polymerase, which by sequence analysis resemblesbacteriophage T7 and T3 RNA polymerases (14), and atranscription factor with regions of similarity to bacterialsigma factors (13) form a complex that interacts with thepromoter region (21).
Mitochondrial promoter consensus sequences for variousplant species were first derived by inspection of regionssurrounding the 5' ends of primary transcripts, i.e., RNAsthat could be labeled in vitro with [a-32P]GTP and guanylyltransferase (reviewed in reference 10). Although a coreCRTA motif could be found in virtually all promoter regions,considerable variability was observed outside of, and occa-sionally within, the CRTA motif, even within a singlespecies. These observations raised questions concerning theselectivity of plant mitochondrial RNA polymerase. Indeed,maize mitochondrial genes have as many as nine transcrip-tion initiation sites (15) that contribute to the complexmRNA patterns observed by filter hybridizations.
t Present address: Department of Biology, University of Missou-ri-St. Louis, St. Louis, MO 63121.
To address plant mitochondrial promoter sequence re-quirements, we developed an in vitro transcription systemfrom maize mitochondria, based on a system first reportedfor wheat mitochondria (12). Using this system, we havepreviously shown that an 11-nucleotide consensus sequencefor maize mitochondrial promoters, which includes theCRTA motif, contains essential promoter elements of theatpl gene, which encodes the a subunit of the F1F0 ATPase(17). Deletion analysis showed that sequences upstream of-19 were not essential for full promoter activity in vitro. Inthe current study, we have used linker-scanning and pointmutagenesis to define the atpl promoter in more detail. Acore region comprises approximately five nucleotides, end-ing at the major initiation site. Both upstream and down-stream regions also contribute to promoter strength, indicat-ing that the atpl promoter spans a region of approximately17 bp.
MATERIALS AND METHODS
Preparation of transcription extracts. Pioneer hybrid 3377seeds were either germinated and grown in the dark for 3 to4 days or grown in field plots. Intact mitochondria wereisolated from etiolated maize seedlings or immature earsharvested from plants, and transcription extracts were pre-pared as previously described (17). Standard extracts wereprepared from etiolated shoots from approximately 6 kg ofseed, or from 150 to 200 immature ears. These extractsyielded sufficient protein for 50 to 200 assays, correspondingto 5 to 15 pg of protein per assay.
In vitro transcription reactions. Standard in vitro transcrip-tion reactions contained 50 ,ug of template DNA per ml andwere carried out as previously described (17). To correct fordifferential losses of in vitro transcripts during the phenol-chloroform extraction and ethanol precipitation steps and forgel loading differences, equal numbers of counts of a gel-purified 32P-labeled T7 transcript of known size were addedto the in vitro transcription reaction mixtures prior to phenolextraction. The amount of atpl transcript in each lane was
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MAIZE MITOCHONDRIAL atpl PROMOTER 7233
normalized following quantification of the T7 transcriptamount, using a PhosphorImager (Molecular Dynamics).For use as templates, plasmids were linearized with HindIII,and the reactions were phenol-chloroform extracted andethanol precipitated. DNA samples were first quantified bymeasuring their A260 in a microcuvette and then visualizedby agarose gel electrophoresis and staining with ethidiumbromide to verify their concentrations and also to confirmthat all the DNA was of a linear form. In vitro transcriptswere analyzed in 5% sequencing gels unless otherwiseindicated.Template DNA. The maize mitochondrial clone pBHO.7
(16) contains the atpl promoter and was used as a wild-typecontrol and to generate single-stranded DNA for site-di-rected mutagenesis, which was performed according tostandard methods (1). Linker-scanning mutants were gener-ated from matched 3' and 5' deletion clones. 3'--5' deletionconstructs were derived from plasmid pATP1(C2AT)19. Toconstruct this plasmid, an atpl promoter fragment wasgenerated by amplification of pBH0.7 with the M13 forwardprimer and BR1 (5'-GGQAAflCGGTCAGTCTCGTTGGCACGG-3', with an EcoRI site underlined; complementary topositions +26 to +46 of the atpl transcript) and cleavage ofthe amplification product with SpeI and EcoRI. This frag-ment was ligated into EcoRI- and SpeI-digested pBS(C2AT)19, which was obtained by recloning the EcoRI-HindIII insert from p(C2AT)19, a plasmid containing aguanosine-free (G-less) cassette (20), into pBluescript SK-(Stratagene). pATP1(C2AT)j9 was digested with KpnI andEcoRI and then incubated with exonuclease III, with ali-quots being removed at 20-s intervals. The ends wererepaired with nuclease S1 and the Klenow fragment of DNApolymerase and then ligated to PstI linkers. Followingdigestion with PstI, plasmids were size selected in andpurified from low-melting-temperature agarose, relegated,and transformed into Escherichia coli, and their endpointswere determined by sequencing of plasmid miniprep DNA.For 5'- 3' deletions, the -49 to +300 region of the atplpromoter was subcloned by amplifying a fragment of pBHO.7with primer BR12 (5'-GCGGATCCGGTGCCCAGGAATAATG-3', with a BamHI site underlined) and the T3 sequenc-ing primer. The product was cleaved with BamHI andHindIII and cloned into pBluescript SK-. This clone wasdigested with BamHI and SacI, and exonuclease III diges-tion was carried out as for the 3' deletions. 5' and 3' deletionclones with appropriately matched ends were combined bydigesting the deletion clones with PstI and XmnI and isolat-ing the atpl-containing fragment (PstI cleaves at the 5' endof the 5'--3' deletions and at the 3' end of the 3'-)5'deletions, and XmnI cleaves within the vector). The twopurified fragments were ligated to generate the final con-struct, which was verified by DNA sequencing.Primer extension and S1 nuclease protection. For primer
extension analysis, LiCl-precipitated total maize mitochon-drial RNA (23), prepared without aurintricarboxylic acid, orin vitro-synthesized atpl RNA was precipitated with ethanoland resuspended in 5 ,ul of H20. To the RNA were added 0.9,ud of 10 mM deoxynucleoside triphosphates, 0.9 ,ul of lOxpolymerase chain reaction buffer (100 mM Tris [pH 8.5], 60mM MgCl2, 500 mM KCl, 10 mM dithiothreitol), and 2 ,ul(approximately 2 ng) of the atpl-specific primer BR10 (5'-TAGGGCCAGCCTGGCTCAAC-3') which had been la-beled with [y-32P]ATP and polynucleotide kinase. The reac-tion was heated to 75°C for 5 min, annealed at 50°C for 5 min,and extended at 50°C for 15 min, using 0.5 pt (3.5 U) of avianmyeloblastosis virus reverse transcriptase (Promega). Five
microliters of 90% formamide-containing tracking dyes wasadded to the reaction mixture, which was denatured byheating and then analyzed in sequencing gels.A probe for S1 nuclease protection was generated by
annealing 32P-end labeled BR10 to alkali-denatured pBHO.7in 10 mM Tris (pH 8)-10 mM MgCl2 at 40'C for 15 min andextending the primer with the Klenow fragment of DNApolymerase and deoxyribonucleoside triphosphates. The re-action mixture was heated to 750C for 5 min, and the DNAwas digested with BamHI, ethanol precipitated, and electro-phoresed in a 1.2% alkaline low-melting-temperature agar-ose gel. The labeled single-stranded DNA fragment waslocated by autoradiography and purified. The labeled frag-ment (6 x 104 cpm) was annealed to the products of a10-fold-increased transcription reaction or to 35 ,ug of mito-chondrial RNA by ethanol precipitation and resuspension inS1 nuclease annealing buffer containing 80% formamide.After an overnight incubation at 370C, digestion was carriedout with 5 or 25 U of S1 nuclease according to standardprocedures (1).
RESULTS
Linker-scanning mutagenesis of the atl promoter. Ourprevious analysis of atpl 5' deletion mutants localizedessential promoter elements to a region downstream ofnucleotide -20, relative to the major transcription initiationsite (17). To define the upstream and downstream borders ofthe atpl promoter with more precision, an eight-base linkerwas substituted at various positions from -52 to +27 (Fig.1A). These plasmids were then used as templates for in vitrotranscription. Figures 1B and C show that linker substitu-tions extending from -52 to -15 had either no effect orminor effects on in vitro transcription activity (less than 30%deviation from wild-type activity), consistent with our pre-vious 5' deletion analysis. However, linker substitutions inthe -13 to +5 region significantly reduced (greater than 50%reduction from wild-type activity) or eliminated transcrip-tion (Fig. 1C and D), placing the 5' boundary of the atplpromoter between 11 and 14 bp upstream of the initiationsite. The 3' boundary of the promoter was localized to within5 bp downstream of the initiation site, as linker substitutionsfurther downstream had minor effects on transcription activ-ity (Fig. iD). The 16- to 19-bp promoter region delineated bylinker-scanning analysis includes the 11-nucleotide maizemitochondrial promoter consensus sequence (16) previouslyshown to contain an essential promoter element (17), plus anadditional 6 to 9 bp of upstream sequence, and is of a sizethat falls between those of the mammalian and yeast mito-chondrial promoters.
Site-directed mutagenesis of the alp) promoter. To explorethe roles of individual bases in atpl promoter activity,single- and double-point mutations were created in the -13to +6 region (Fig. 2), and these plasmids were used astemplates for in vitro transcription. Figure 3 shows thetranscription activities of representative templates, and theresults for these and other point mutants are summarized inFig. 4. The activities given in Fig. 4 represent the averages ofmultiple transcriptions for each template and thus mayappear to have minor differences from the gels shown in Fig.3.
Figure 3A shows the products of in vitro transcriptionreactions using templates with mutations at positions -12and -13, which are near the 5' boundary of the promoter.The double mutant pCG(-13/12) has less than 50% of
VOL. 13, 1993
A-5(.4 -( -3 -:ec -2X A20 1
wtL T T G~A ClteC G C; C ( C C AGGI A A TA A T C (..
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pLS(-452) CTCC g C~C A G-A A T A A T C TTlTTA T T A A C. C A'pLAI-ASITTGC~~~~~~~lTGGCCAGI.~~~~~~clIg~~~A~~eLTT '3 PTA~~~~ T \TT A TAAC T AAACA C A i,,1'iT..l U
pLSI-471 T T1 C C C~:MC U GC ICAIiGAAIAAg tg c'AIIAI1AAe AAA A 01 T A A T.,1 A T A A A A AC AAA T AA AAA T TpLS(-4)TGCCUG UICC G C;AAIT A A T C T T CIT T AT A~T T A CeAAe(TAe . AAAA
PLS(-45) T P Ci C C C C C ::A: U: A A T A A T G,TT::iT TAAR.TAA1 C C (I A A AG T A A .IA 7pLS(-l18) T T G C C ,1 Ci TTe' ' T A T T A T T A A .1eU A A A A A I C.P- A T A A A A A e' A AA C. IT'I A A\T
pl.S(-3l T T iC C C C C A C 'I A A AA: A Tel.e..*......TA.....A..P.A CA e, *A A A A CI T A A C1 ,T A 7 CAtAAgA AFL~~~ilTTCCIUUTUCI..l..AUUAA'I AAT'Ie~~~~~~~..........TA.U A A. e I . II T e l e~~ .A.l.ApLS(-I)TTCC~~~~~~~~~(,l~~~~le.CuCAGGAATAAP*Cle''l'P eATTA e.AA A~~~~~~~~~~~~~~~~~~~~A ce..AA AA A AAgCAA3PLS(-2) T TG C C GG GCC C A G GAAGAA T A A::: APTiAT T A e3eAA AAUp i A e
PLS(-29) T T G, C C C. G. T C C C A G G. A A T A A T C TTITTA1 A T T A A ' C A A 'A 'A C T A .A T, A 7 T A A A A A.C' AA A C; T. A A AAA T 3 ACCINpLS(-249) TT-rC CCIUC.ICC C C AUU,A APTAATCC 'T ITTAPT ATT: A e. eAA A AU0TA Ae T A T l AAAACA AAe A~ T ;Ap.. A T 1pLS(+A)TCCI.UUP CCCA GAA1 AAI'GT~eATA i APIA~e~Ge.A A A AUT A Ace PAT A A A, Ae AAA T I AA.' g.ACe A
pLSl-18OIT T G C C G. G. T C. C C C A G C A A T A A T G'C TiT T TTAAIAAiGTAAeA CeAAAA C AAAAAgIe~IT e Ag 4
B C D~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...pL wt52474TG53GG GC CA3AA 2AA- CT A I'TATTAA t:: TAACTA242 T8A16 A 1A+A+2+bA+9+Fi+T2O A
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FIG. 1. Transcription activities of linker-scanning mutants of the atpl promoter region. (A) Sequences of linker-scanning mutants. Thewild-type (wt) sequence from -52 to +27, relative to the major transcription initiation site, is shown on the top line, and the linker sequenceis shaded. Linker nucleotides that fortuitously match the wild-type sequence are capitalized. Transcription activities of templates weremeasured as described in Materials and Methods and are listed at the right. These values are relative to the wild-type level and represent theaverages of several experiments, and thus they may appear to differ slightly from the gels shown in panels B to D. (B to D) Gel analysis of32P-labeled in vitro transcripts from linker-scanning mutants, designated by the position at which the PstI linker begins relative to the majortranscription initiation site. The positions of the atpl runoff transcript and the T7 RNA loading standard (see Materials and Methods) areindicated on the left.
wild-type activity, as does the double mutant pTG(-13/12)(Fig. 4). As we have previously reported that mutating thesequence from -16 to -13 from GCCG to TATT does notreduce in vitro transcription activity (17), the reductionobserved for pTG(-13/12) is most likely due to the A-to-Gtransition at -12. Both pT(- 12) and pC(- 12) contain trans-versions at position -12, which decrease transcription toless than 50 and 40%, respectively, of the wild-type level.Together, these results suggest that an A in position -12 isnecessary for optimal transcription activity. Despite re-peated attempts, we were unable to generate mutants atpositions -11 and -10; thus, the sequence requirements atthese positions remain to be defined.A second region affected by mutagenesis extends from -7
to +6 (Fig. 4). We found at least one base change at each ofthese positions (except +3 and +4, for which we wereunable to generate mutants) that reduced transcription toless than 50% of the wild-type level (Fig. 3B and C and Fig.4). For example, a T-to-G transversion at -7 reduced
transcription to less than 40% of the wild-type level [pG(-7)in Fig. 3C], and an A-to-T transition at +5 reduced transcrip-tion to less than 30% of the wild-type level (Fig. 4). Theregion from -4 to +1, which we have termed the coreregion, is particularly sensitive to mutations, with at leastone base change at each of these positions causing more thana 70% reduction in transcription activity. The sequence ofthe core region is CGTAT, which conforms to a CRTA motifthat is highly conserved between plant mitochondrial pro-moters (5, 7, 16).As shown in Fig. 4, none of the mutations that we obtained
in positions -9 and -8 impaired transcription activity,suggesting that the atpl promoter can be divided into tworegions: a central domain extending from -7 to +5 thatsurrounds the transcription initiation site, and an upstreamdomain that includes position -12. The most stringentsequence requirements occur within the core region of thecentral domain, with positions -2 to +1 displaying the leasttolerance to mutations. Additional mutants will be needed to
pGG(.514) G GpT(-4) TpA(-3) ApC(-3) CpT(-3) TpA(-2) A:pG(-2) G:PC(-,)CpG(-1) GpT(-1) :TpA(+l) ApC(+) CpG(+2) GpG(+5) GpT(+5) T
pTG(+5/6) T G
FIG. 2. Point mutations in the atpl promoter region. The wild-type (WiT) sequence from -13 to + 10, relative to the major transcriptioninitiation site, is shown at the top. The mutants are designated by the base alterations and their positions. Mutations appearing in the samerow are present in the same construct. An empty column indicates that no mutants were obtained for that position.
determine the sequence requirements of the +2 to +4 upstream domain remains poorly defined because of ourpositions; however, the observation that a linker substitution inability to create single-base mutations at two positionsat positions +2 through +9 abolishes transcription (lane +2 within this region. However, as we have demonstrated ain Fig. iD) suggests sequence restrictions in this region. The requirement for an adenine at position -12, and alignments
:2A B C
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FIG. 3. In vitro transcription of atsp promoter region point mutants, determined by gel analysis of 32P-labeled in vitro transcripts frompoint mutants as designated in Fig. 2. Transcripts are indicated as for Fig. 1. An in vitro transcription reaction using a linker-scanning mutantwith 100% of the wild-type (wt) activity [lane pLS(-52)] was included in the gel shown in panel A as an additional positive control.
MmmT. W.
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7236 RAPP ET AL.
UPSTREAM CENTRAL DOMAINDOMAINF-Id CORE
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 +6 +7 +8 +9 +10G A A A A G T A A C G T A T T A A A A A C A Al
2100% TT T GC
90-99 __ IIII80-89 C G70-79 === = == T60-69 = === A50-59 = = = T GI40-49 T C G
C+G T +G___T+G
30-39 C __ T_ I_20-29 C T A -
10-19 . G+G G.10% A
FIG. 4. Transcription activities of point mutants and a workingmodel for the maize mitochondrial atpl promoter. The wild-typesequence is shown in boldface at the top. The far left columnindicates the transcription activity relative to the wild-type level foreach mutant, where the wild-type activity is 100%. Single mutationsare shown as single letters; for example, T and C have beenindividually substituted for A at position -9. Double mutants areindicated by a plus sign; for example, CG has been substituted forGA at positions -13 and -12. The extents of the two proposedfunctional domains of the atpl promoter are indicated at the top.The boundaries of the upstream domain are tentative (as describedin the text), so this region is indicated by a dotted line. The coresequence (indicated by a box) within the central domain is defined asdiscussed in the text.
of mitochondrial promoter sequences from maize and otherplant species suggest a preference for purines in this region(see Fig. 7), we have tentatively included positions -12 to-10 in this domain.
Initiation site selection in the atpl promoter. We and others(16) have previously observed multiple bands in primerextension analyses of atpl RNA isolated from plants or fromin vitro transcription reactions. To test independentlywhether such patterns result from experimental artifacts orindeed represent multiple transcription start sites, we ana-lyzed in vitro-transcribed atpl RNA by S1 nuclease protec-tion and compared the products with primer extensionproducts. Figure 5 shows that multiple 5' ends are detectedwith both methods. Using both primer extension and 25 U ofS1 nuclease, we detected 5' termini at positions +1, +2, and
SiT A PE 25u 5u A T
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* Mb~~~~~A
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FIG. 5. 5'-end analysis of atpl in vitro transcription products.RNA was transcribed in vitro from the wild-type atpl promoter andanalyzed by primer extension with fy-32P-labeled BR10 (PE) or bydigestion with 25 or 5 U of S1 nuclease (25u or 5u), followingprotection with a single-stranded DNA probe generated by exten-sion of y-32P-labeled BR10 on plasmid pBHO.7, as described inMaterials and Methods. A dideoxy sequence ladder was generatedby using primer BR10 and plasmid pBHO.7. Analysis was carried outin an 8% sequencing gel.
T A 3 X CL A T
T .A A
Wt (-5)ACGTA4TAAA(+5)
pG(+2) (-5)ACGrATGAAA(+5)
pC(+1) (-5)ACGTACTAAA(+5)
FIG. 6. 5'-end analysis of atpl promoter mutant in vitro tran-scription products. RNA was transcribed in vitro from the wild-typeatpl promoter and the indicated point mutants and analyzed byprimer extension with -y-3P-labeled BR10. Each transcription reac-tion was carried out with equal amounts of DNA and protein. Toequalize the intensities of bands in the final gel, the followingpercentage of each primer extension reaction was loaded: wild type(wt), 6%; pG(+2), 33%; pC(+1), 100%. The 5' termini observed foreach construct are underlined in the diagram. A sequence ladderwas generated as for Fig. 5.
+3. We have obtained identical results using RNA isolatedfrom immature maize ears (data not shown). We tentativelyconclude that there is a cluster of three transcription initia-tion sites within the atpl promoter.We observed reduced transcription activity for several
site-directed mutants with base changes within the transcrip-tion initiation region (Fig. 4). To test whether these muta-tions affected initiation site fidelity, in vitro-transcribedRNAs from two of these mutants were analyzed by primerextension. Figure 6 shows that while a transversion atposition +2 does not affect initiation site selection, a T--Ctransition at position +1 eliminates initiation at position +3and strongly reduces it at positions +1 and +2. Concomi-tantly, initiation sites appear to be activated at positions -1and -2. The new initiation sites are at A and T residues, thesame bases at which all initiation occurs in wild-type atpltemplates. This result may reflect a base preference fortranscription initiation at the atpl promoter.
DISCUSSIONOur detailed in vitro analysis of the maize mitochondrial
atpl promoter has revealed two distinctive features. First, ofall of the positions that we have examined, only -2, -1, and+ 1 appear to have a strict sequence requirement. This differsfrom the S. cerevisiae mitochondrial promoter, whose func-tion requires high sequence conservation at positions -7 to-1 (3). Second, we have found that base changes as farupstream as -12 and as far downstream as +5 can stronglyinfluence in vitro promoter activity. This finding indicatesthat the extent of the atpl promoter is at least 17 nucleotides,significantly longer than the yeast mitochondrial promoterbut shorter than mammalian mitochondrial promoters stud-ied to date. Determining whether the sequences downstreamof + 1 that influence transcription act at the level of initiationor elongation will require further experimentation.
FIG. 7. Comparison of plant mitochondrial promoter regions.(A) Maize mitochondrial promoter comparison. The maize atplpromoter is shown at the top, and below it is the IVT consensussequence derived from the in vitro transcription data presented inthis report. Below the IVT consensus sequence are sequences ofthree other promoters that are correctly transcribed in vitro, withtheir initiation sites underlined. A second consensus sequence canbe derived by inspection of these and other maize mitochondrialpromoter regions; this is presented for comparison with the IVTconsensus (see also reference 10). At the bottom, the two rRNApromoters are shown. Solid boxes around bases indicate those thatdeviate from the IVT consensus sequence, and a dotted boxindicates the core promoter sequence. (B) Comparison of mitochon-drial promoter sequences from maize, wheat, and soybean. Themaize (IVT) sequence is described above. The maize (16), wheat (7),and soybean (5) sequences were derived by comparing sequences towhich the 5' termini of primary transcripts map. A highly conservedCRTA motif [YRTA in the maize (IVT) sequence] is boxed. Arrowsindicate transcription initiation sites (the arrow and line over themaize sequence indicates a region within which transcription ini-tiates).
In general, our results suggest that two islands of baseswithin the atpl promoter region are critical to functionalityand that they are interspersed with positions whose precisesequences are unimportant. Interestingly, these two islandsare centered approximately 10 bp apart, corresponding toone helical turn. This might reflect the manner in whichproteins of the transcription apparatus interact with thepromoter region. Our preliminary model will have to betested by additional mutagenesis to delineate precise baserequirements and to determine whether the spacing of criti-cal sequence motifs is important.On the basis of the data presented in this report and on our
previously published results, we have derived a workingmodel for the atpl promoter consensus sequence (Fig. 7A).The consensus based on in vitro transcription data (IVT) isconservative because we have examined only a limitednumber of promoter variants. This consensus, A__NNYNRYRTAT, agrees well with a consensus sequence that canbe derived by inspection of other maize mitochondrialpromoters active in our in vitro system, although universalconservation at some positions, for example the CG found at-5/4 relative to the atpl start site, does not reflect anabsolute requirement for these bases for in vitro transcrip-tion. Figure 7B shows that the YRTA sequence within the
core region of the IVT consensus sequence aligns with thehighly conserved CRTA motif in consensus sequences formaize, wheat, and soybean mitochondrial promoters thatwere derived by comparing initiation sites of primary mito-chondrial transcripts (5, 7, 16).At the bottom of Fig. 7A are the sequences of the rrn18
and rn26 promoters, which represent two of the strongestpromoters in maize mitochondria, as determined fromrun-on transcription assays (16). However, in our in vitrotranscription system, the rn18 promoter functions veryweakly (unpublished data). In this regard, it is interesting tonote that each of these rRNA promoters differs from theconsensus sequence derived by either in vitro transcriptionor inspection. These differences are highlighted in Fig. 7Aand include a transversion at +1 (relative to the atpl startsite; the rnl8 and rn26 start sites are underlined) for rrnl8and transversions at positions -5 and -7 for rrn26. Theweak activity of the rnl8 promoter in vitro may reflect arequirement for additional or alternative transcription fac-tors and/or RNA polymerase. Biochemically distinct RNApolymerase activities that preferentially transcribe rRNAgenes have been found in chloroplasts (11, 18, 19), anddistinct activities are also found in the nuclei of eukaryoticcells. The ability to transcribe single-copy rRNA genes athigh rates may depend on the distinction of mRNA andrRNA transcription activities. In vertebrate mitochondria,enhanced transcription of rRNA may be accomplished bytranscription termination downstream of the rRNA genes(reviewed in reference 6).A conspicuous feature of maize mitochondrial transcrip-
tion is that in different promoters, transcription initiationoccurs at different positions relative to conserved promotersequences (15, 16). For example, Fig. 7A shows that al-though the atpl, atp6, coax3a, and cox3b promoters can bereadily aligned, transcription begins at positions +1, +3, +4,and +2, respectively, relative to the major atpl start site.We have recently confirmed these cox3 start sites by primerextension of in vitro-transcribed RNA (data not shown).Furthermore, transcripts originating from the same promoteroften have multiple 5' ends. For example, three major 5'termini, mapping to adjacent nucleotides, are observed foratpl transcripts (Fig. 5) (16). As these termini are detectedfrom both in vitro-generated and in vivo transcripts analyzedby primer extension and nuclease protection, they are mostlikely derived from clustered initiation events. In contrast,only a single initiation site is found for cox3b, and initiationoccurs at two adjacent bases for cox3a. Although we cannotrule out that 5'-end heterogeneity is created by nucleaseactivities present in our extract that are also active in vivo,these results raise intriguing questions about how the maizemitochondrial RNA polymerase interacts with the promoterto select the initiation nucleotide. We are currently alteringthe cox3 and atp6 promoters to determine whether mutationsthat strongly reduce atpl promoter function, e.g., at -2 and-3, also affect cox3 and atp6 transcription, even though theyare at different positions relative to the transcription startsite. Interestingly, start site choice appears to be less variantin both wheat (7) and soybean (5) mitochondria (Fig. 7B),although so far only the wheat cox2 promoter has beentested by in vitro transcription.
Altering the atpl + 1 position causes a change in start siteselection (Fig. 6). The data suggest that selection is alteredbecause of a preference forA or T as an initiating nucleotide,because the newly utilized start sites are T and A at positions-2 and -1, which are identical to the +2 and +3 start sitesin wild-type templates. However, many other plant mito-
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7238 RAPP ET AL.
chondrial transcripts initiate with G, which we have success-fully substituted at the atpl +2 position, and thus thepreference for A/T may not be stringent. In the yeast in vitrosystem, all base changes at + 1 allowed transcription, al-though purines gave higher activities than pyrimidines (3).When the -1/+1 dinucleotide TA was altered to AA, initi-ation was observed at both adenines, with a preference forthe first. There was no evidence for other instances ofmultiple initiation sites, as we have observed for maize atpl(Fig. 5).
Fluidity appears to be a general property of plant mito-chondrial promoters. Figure 7B shows an alignment ofnonmaize promoters whose RNAs have been directly se-quenced. Apart from the CRTA motif and the purine-richregion, little conservation is seen across species. The strik-ing amount of sequence variation observed to date, for thefewer than 20 promoters examined, raises the question ofwhether plant mitochondrial DNA is transcribed indiscrim-inately. Indeed, estimates of mitochondrial transcriptioncarried by solution hybridizations (2), filter hybridizations(22), and run-on transcriptions (9) all indicate that transcrip-tion of noncoding regions is extensive. How the specificity oftranscription initiation and degree of termination contributeto the extent and complexity of mitochondrial transcriptionis an issue to be addressed by a biochemical analysis of themitochondrial transcription apparatus.
ACKNOWLEDGMENTS
We thank Robin Tracy for stimulating discussions and MarcAlbertson of Pioneer Hi-Bred International, Inc., for generousprovision of Pioneer brand seed.
This work was supported by grant 91-37301-6419 from the U.S.Department of Agriculture. W.D.R. was the recipient of a postdoc-toral fellowship from the Department of Health and Human Ser-vices.
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