Sequences preceding the ninimal promoter of the Xenopus somatic SS RNA gene increase bindingefficiency for transcription factors
Wanda F.Reynolds
La Jolla Cancer Research Foundation, La Jolla, CA 92037, USA
Received June 1, 1989; Revised and Accepted October 6, 1989
ABSTRACTSequences preceding the minimal promoter play a role in the differential expression of the Xenopussomatic and oocyte-type 5S RNA genes. In this report, the somatic sequences between -32 and+37 are shown to increase transcriptional activity in microinjected embryos, yet have little to noeffect in microinjected oocyte nuclei. In vitro, these sequences increase activity in whole oocyteS150 extracts, but not in oocyte nuclear extracts. In S150 extracts, these somatic sequences facilitatebinding by a commonly required factor(s), other than TFHIA, which forms a stable complex withthe 5S gene. This transcriptional enhancement is also apparent in a reconstituted system using purifiedTFMA and partially purified TFIILB and TFHIC.
INTRODUCTIONThe two major classes of Xenopus 5S RNA genes are differentially regulated. The oocyte-type, present in 20,000 copies, is expressed during early oogenesis but is essentially inactivein adult cells; the somatic type, present in 400 copies, is expressed in both oocytes andadult tissues (1). The mechanism by which these related genes are differentially expressedin somatic cells is not completely understood. Transcription of either type by RNApolymerase HI requires at least three protein factors, referred to as TFIIIA, TFIILB andTFIIIC (2,3). Xenopus TFHIA has been identified as a 38 kdal protein (4). The activitiesin chromatographic fractions TFIIIB and TFHIC have only been partially characterizedand there is evidence that TFHIC represents more than one essential factor (5,6,7). Thesites of interaction by TFHIB or TFHIC with 5S DNA have not been established. Sinceinitial binding by TFIHA facilitates their binding (8), these factors are thought to interactat least in part through protein-protein contacts.TFIIIA binds the internal control region (ICR) or promoter of the 5S RNA gene (4).
This minimal promoter (+47 to +91) contains those sequences which are necessary andsufficient for transcription (9,10,11). Sequence differences within the ICR are partiallyresponsible for the differential expression of the somatic and oocyte 5S genes (11,12).However, as reported previously (13), somatic/oocyte sequence differences outside thiscore element have a significant effect on levels of expression in an oocyte S150 extract;the somatic sequences between -32 and +37 result in as much as ten fold higher activity.
In this study, the basis for the transcriptional enhancement by the somatic -32/+37region has been further investigated by competition transcription assays in oocyte extracts.The results show that the somatic -32/+37 region increases binding efficiency forcommonly required factors.
MATERIALS AND METHODSPlasmid DNAspXlsl 1-34 (13) contains the Xenopus laevis SS gene from pXlsl 1 (14) with 34 bp of native5' sequences. The major oocyte SS gene in pXlol76 (11) was derived from pXlo3l (15)by removal of the psuedogene. The hybrid o/somatic SS gene in pXlo/s40 (13) consistsof oocyte SS (pXlol76) sequences joined at +40 to the somatic 5S gene (pXlsl 1). Thehybrid s/oocyte gene in pXls/o30 (13) consists of somatic type (pXlsl 1) sequences joinedat +30 to the oocyte SS gene (pXlo176). pXlo/s47-56 (12) contains a hybrid oocyte 5Sgene (pXlol76) with somatic type base substitutions at positions +47, 53, 55 and 56.pXls5l0 contains sequences between -479 and + 30 of the somatic 5S gene in pXlsl 1.pXltmet contains a X. laevis tRNA gene (16).In Vitro transcription assays: oocyte S150 extractsWhole oocyte S150 extracts were prepared as previously described (17). Mature oocyteswere separated from immature stages by collagenase treatment of whole ovaries. The intactoocytes were centrifuged in two additional volumes of 20 mM TRIS-HCL, pH 7.6, 70mM KCI, 0.1 mM EDTA, 20% glycerol in an SW40 rotor for 30 minutes at 30K RPM.Transcription assays were performed in a 50 /J volume containing 40 ,d oocyte S150 extract,0.6mM ATP, CTP and UTP, 20%M GTP and 10tCi of a 32P GTP. Reaction mixtureswere incubated at room temperature for 2 hours. For template exclusion assays, the firstDNA was preincubated for one hour in the reaction mix, followed by the second DNAand &e32P GTP for a 2 hour incubation. Reactions were stopped by addition of 50mMEDTA, 0.1 % SDS, followed by phenol extraction and ethanol precipitation. RNA sampleswere resuspended in 90% formamide and electrophoresed in 6% sequencing gels or underpartially denaturing electrophoretic conditions (18) to allow resolution of the different 5SRNAs. Autoradiographic exposures were obtained with Kodak XAR-1 film. The amountof label incorporated was determined by excising the band and quantitating by liquidscintillation counting or by scanning laser densitometry.Oocyte nuclear extractsThe preparation of the oocyte nuclear extract was as described (19). Each reaction containedin a 15 tl volume the equivalent of 3-5 nuclei in J Buffer (10 mM Hepes, pH 7.4, 70mM NH4Cl, 7 mM MgCl2, 2.5 mM DTT, 10% glycerol, and 1 mM EDTA), 0.6 mMATP, CTP, and UTP, 20 yM GTP and 10 ,uCi of a32P GTP. The reaction was incubatedfor 2 hours at 22°C and processed as described above.HeLa cell extractsReactions contained in a 50 A1 volume, 20 ,d of the S100 extract of HeLa cells (20), 10mM Hepes, pH. 7.9, 60 mM KCI. 5mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.6mM ATP, CTP, UTP, 20 ,uM GTP and 10 yCi of a32P GTP. Incubation period andprocessing were as described above.Microinjection assaysThe germinal vesicles of mature oocytes were injected with 10 ng of DNA and 0.1 ACiof cX32P GTP in a 20 nl volume (21). For each DNA sample, 20-30 oocytes wereinjected. Following a 9-12 hour incubation, the eggs were homogenized in 0.5% SDS,50mM EDTA, followed by phenol extraction and ethanol precipitation.For embryo microinjection assays, 2-cell stage embryos were injected with 40ng of 5S
DNA and 0.1 tCi of a32PGTP in a 25nl volume and incubated for 9 hours. Cleavagewas largely disrupted under these conditions allowing dispersal of plasmid DNAs and labeledGTP.
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Fractionation of oocyte extractsTFIJIA was isolated from immature oocytes according to established procedures (22,23).Oocyte S150 and immature oocyte extracts were fractionated by phosphocellulose (WhatmanP11) chromatography essentially as previously described (2). The crude extract was appliedto a P11 column in 0. IM KCl, 10 mM HEPES, pH.7.6, 0.1 mM DTT, 0.1 mM EDTA,0.2 mM PMSF, 20% glycerol. TFIIIB was eluted with the same buffer containing 0.35M KCl, and TFUIC with 0.6 M KCl. These fractions were dialyzed against 100 mM KCl,10 mM HEPES, pH 7.6, 0.1 mM EDTA, 0.1 mM DTT, 10 ltM ZnCl2, 20% glycerol.Transcription reaction mixtures (100 IL volume, 4 hr incubation) contained these fractionsin addition to purified TFIIIA, 7 mM MgCl2, 0.6 mM ATP, CTP, UTP, 20 zM GTPand 10 ,uC of a32P GTP.
RESULTSSomatic sequences preceding the ICR increase transcriptional activity in microinjectedembryos, but not in microinjected oocyte nucleiTo investigate the significance of somatic/oocyte sequence differences preceding the ICR,these sequences were exchanged. The resultant chimeric genes are illustrated in figurelA; the o/somatic hybrid has oocyte 5S DNA sequences (pXlol73) preceding the geneand at positions +30 and +37, whereas the s/oocyte hybrid has somatic 5S DNA sequences(pXlsl 1) preceding the gene and at +30. In a previous report, the somatic-specific sequencesbetween positions -32 and +37 were shown to increase transcriptional activity by tenfold in vitro in oocyte S150 extracts (13). It was of interest to determine whether thesesequences play a role in the differential expression of these genes in vivo in embryoniccells. To address this question, a mixture of oocyte and somatic 5S plasmid DNAs (ata 5:1 oocyte/somatic concentration ratio) was microinjected into mature oocyte nuclei orembryos along with radiolabeled nucleotides (NTPs). Following a 9-12 hour incubationperiod, the RNAs were isolated and electrophoretically resolved in partially denaturinggels (Figure 1B). In microinjected oocytes, the somatic 5S gene was only ten fold moreactive than the oocyte 5S gene Oane 1), in agreement with previous studies (24,25). Theexchange of sequences preceding the ICR had little to no effect: the o/somatic hybrid wassimilarly ten fold more active than the s/oocyte hybrid (lane 2). Contrasting results wereobtained following microinjection of 5S DNA into two cell embryos. The somatic 5S genewas ca. 200 fold more active than the oocyte 5S gene (lane 4), in general agreement withprevious studies (24). Following the exchange of sequences preceding the ICR, thisadvantage decreased to 20 fold (lane 3). These results indicate that the somatic/oocytesequence differences preceding the ICR have a pronounced effect on the levels of geneexpression in embryonic cells but little to no effect in mature oocyte nuclei.
In these microinjection assays, the degree of radiolabel incorporation varies, and so asimilar amount of labeled RNA was loaded per each lane. Therefore it is possible to comparesignal intensities within a lane but not between lanes. For example, the fact that the somaticand o/somatic signals in lanes 3 and 4 are similar does not indicate that the activities ofthese genes are similar in microinjected embryos. When a mixture of these two DNAswas coinjected, the somatic 5S gene was 5-10 fold more active (data not shown).Somatic 5S sequences preceding the ICR increase transcriptional activity in whole oocyteextracts, but not in oocyte nuclear extractsIn vitro, as in vivo, the relative activities of the somatic and oocyte 5S genes are markedlydifferent in different types of cellular extracts. In S150 extracts of mature oocytes, the
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Figure 1. Microinjection of native and hybrid SS RNA genes into oocytes and embryos.A. 5S DNA sequences: The somatic 5S DNA sequence from position -32 through the 120 bp gene is shown(14). Positions which differ in the oocyte 5S gene in pXlol76 (15) are indicated below. The hybrid o/somatic5S gene has oocyte-type sequences at positions +30 and +37 and preceding the gene. The hybrid s/oocyte hassomatic-type sequences at position +30 and preceding the gene.
B. Microinjection assays: A 5:1 mixture of oocyte (0) to somatic (S) 5S plasmid DNA was microinjectedinto oocyte nuclei (lane 1) or two cell embryos (lane 4) along with labeled NTPs. A 5:1 mixture of s/oocyte(SO) to o/somatic (OS) plasmid DNA was microinjected into oocyte nuclei (lane 2) or embryos (lane 3). Followinga 9-12 hour incubation, RNA was purified and electrophoresed in partially denaturing conditions which resolvethe different RNAs on the basis of secondary stnrcture (18). An autoradiograph is shown.
somatic 5S gene is 50-100 fold more active than the oocyte 5S gene (25,26), whereasin oocyte nuclear extracts or in HeLa cell extracts, the somatic gene has only a four toten fold advantage (11, 27). It was of interest to determine whether the sequence differencespreceding the ICR were in part responsible for this differential expression. To answerthis question, the native and hybrid 5S genes were transcribed in oocyte S150 extracts,oocyte nuclear extracts and HeLa cell extracts. In oocyte S150 extracts (Figure 2, panelC), the somatic sequences preceding the ICR resulted in seven fold higher activity (comparelanes 5 and 6). Thus the 100 fold somatic transcriptional advantage in this extract (compare
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B C
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Figure 2. In vitro transcription of native and hybrid 5S genes.Reactions mixtures contained HeLa cell extract (A), oocyte nuclear extract (B), or oocyte S150 extract (C) and100 ng of somatic, o/somatic, o/s47-56, or oocyte 5S plasmid DNAs (lanes 1-4). Lanes 5-8 in panel C showa lighter exposure of lanes 1-4. Autoradiographs of denaturing gels are shown. The multiple bands observedin lanes 3 and 4 result from imperfect termination for the oocyte 5S gene.
lanes 1 and 4) is partly due to these sequences and partly to sequence differences withinthe ICR (compare lanes 1 and 3), as previously reported (12,13). In contrast, in oocytenuclear extracts (panel B), the somatic sequences preceding the ICR had little to no effecton transcriptional activity (lanes 1 and 2). In this extract, the somatic gene was only fourfold more active than the oocyte gene (lanes 1 and 4). Similar results were obtained withHeLa cell extracts (panel A); the somatic sequences preceding the ICR did not affect thelevel of gene activity. These findings demonstrate that the somatic -32/+37 region providesa transcriptional advantage in oocyte S150 extracts, but not in oocyte nuclear or HeLacell extracts.The somatic sequences preceding the ICR facilitate binding ofa factor(s) which is stablyassociated with transcription complexesIt is of interest to determine why the somatic -32/ + 37 region provides a transcriptionaladvantage in oocyte S150 extracts, but not in oocyte nuclear extracts. The transcriptionassays in figure 3 have provided some insight into the nature of this advantage. Inindependent reactions containing 200 ng of either DNA, the somatic 5S gene was severalfold more active than the o/somatic 5S gene (Figure 3, lanes 4 and 5). That concentrationof somatic 5S DNA was saturating in that the signal did not increase at higher DNAconcentrations (lanes 4, 9, and 14). This indicates a limiting component was depleted fromthe extract by 200 ng of somatic 5S DNA. In contrast, 200 ng of o/somatic DNA wasnot saturating; the signal continued to increase with 400 and 1000 ng of DNA (lanes 5,10 and 15) until equivalent to that of somatic 5S DNA (compare lanes 14 and 15). Thesedata indicate that a higher concentration of o/somatic 5S DNA is necessary to form themaximal number of transcription complexes in the oocyte S150 extract, suggesting a loweraffinity for transcription factors.
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Figure 3. Transcription assays in an oocyte S150 extract.Independent reactions: Reaction mixtures contained oocyte S150 extract and 200 ng of somatic (lane 4) or o/somatic(lane 5) 5S DNA. Transcripts are resolved on partially denaturing gels (18); the upper band corresponds to somatic5S RNA.Template exclusion assays: In lane 2, 200 ng of somatic 5S was preincubated for one hour in the reaction
mixture, and then 200 ng of o/somatic DNA was introduced along with labeled NTPs, and the reaction continuedfor an additional two hours. In lane 3, the DNAs were added in the opposite order.
Direct competition assays: In lane 1, the reactions contained 200 ng of both somatic and o/somatic DNAs.For all lanes 1-5, labeled NTPs were added to the reactions after one hour, and the reaction continued for twoadditional hours.
In lanes 6-1O, the protocol was identical except 400 ng of each DNA were present and in lanes 1 -l15, 1000ng of each DNA were present.
Template exclusion assays are one means to demonstrate stable binding of factors toDNA. In such assays, one template is preincubated in the extract to allow complexes toform and then a second template is introduced. If the first template depletes a commonlyrequired factor, transcription from the second template is excluded. Moreover, the
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concentration of DNA required to deplete a limiting factor provides a relative measureof factor affinity. Such assays have previously shown that TFIIIA, TFIIIB and TFIICform a stable complex with 5S DNA which is not disrupted by the passage of polymeraseand is resistant to challenge by a second template (32). There is, however, evidence thatsuch complexes are less stable on the oocyte 5S gene than on the somatic 5S gene in oocyteS150 extracts (28). To determine whether the somatic -32/+ 37 region increases the abilityto form stable complexes in oocyte S150 extracts, template exclusion assays were performed.In independent reactions containing 400 ng of somatic or o/somatic DNAs, the signalswere nearly equivalent in intensity (lanes 9 and 10). When 400 ng of somatic DNA waspreincubated in the extract, followed by 400 ng of o/somatic DNA, the o/somatic signalwas abolished (lane 2). However, when 400 ng of o/somatic DNA was preincubated, thesomatic signal was not completely abolished. Preincubation with 1000 ng of o/somaticDNA resulted in complete inhibition of the somatic signal. These data indicate that o/somaticDNA forms fewer stable transcription complexes than somatic 5S DNA, in that a higherDNA concentration was required to deplete a commonly required factor.
Direct competition assays provided further evidence that the somatic -32/+37 regionincreases binding efficiency for commonly required factors. In independent reactionscontaining 400 ng of either DNA, the somatic and o/somatic signals were nearly equivalent(lanes 9 and 10). However, in a mixed reaction containing 400 ng of both DNAs, thesomatic 5S signal was ten fold greater than that of the o/somatic gene (lane 6), indicatingpreferential binding of limiting factors to the somatic 5S gene.
In order to demonstrate that the observed competition was specific, control assays wereperformed with nonspecific DNA. Preincubation or coincubation with pUC18 did notdiminish the S or OS signals (data not shown). Similar control experiments can be seenin figure 4.The somatic -321+37 region does not independently bind factorsThe somatic 5S sequences preceding the ICR were subcloned and assayed as to abilityto deplete transcriptional components from the oocyte S150 extract in template exclusionassays. pXls5lO contains sequences extending from -479 to +30 of the somatic 5S genein pXlsl 1. Preincubation with up to 400 ng of either pXls5 10 (Figure 4, lanes 1-4) orpUC 18 (lanes 5-8) did not inhibit transcription of the somatic 5S gene. In contrast,preincubation with only 200 ng of a plasmid containing the intact somatic 5S gene, wassufficient to deplete a commonly required factor, thus inhibiting subsequent transcriptionof tDNAmet (lanes 10 and 11). tDNAmet, like the 5S gene, requires factors present inTFIIHB and TFIIIC. These results indicate that the somatic -479 to +30 region, whenseparated from the ICR, is unable to stably bind and deplete factors. This finding doesnot exclude the possibility that factors TFIIIB or TFIIC interact with these sequences withinthe intact 5S gene; the ICR is required for binding by TFIIIA, and binding by TFIIIAfacilitates or is required for binding by TFIIIC and TFIIIB (8).
Footprint competition studies were performed to demonstrate that the somatic/oocytesequence differences preceding the ICR do not affect binding by TFHIA. A labeled DNAfragment containing the oocyte 5S gene was incubated with saturating amounts of TFIIAand increasing concentrations of unlabeled somatic 5S or o/somatic 5S DNA. Thesetemplates had comparable ability to bind TFHIA as shown by equivalent loss of DNAseI protection on the labeled 5S DNA fragment (data not shown), indicating that the somaticsequences preceding the ICR do not influence TFIIIA binding efficiency, in agreementwith earlier studies showing that TFIIIA binding is influenced by sequences within the
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1 2 3 4 5 6 7 8 9 10 11 12
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Figure 4. Somatic SS sequences preceding the ICR do not independently bind transcription factors.Increasing concentrations (0, 100, 200, or 400 ng) of pXls51O containing sequences between -470 and +30of the somatic 5S gene (lanes 1-4) or the same concentrations of pUC 18 DNA (lanes 5-8) were preincubatedfor one hour in an oocyte S150 extract. Somatic 5S DNA (pXlsl 1)(200 ng) was then added along with labeledNTPs and the reactions continued for two hours. In lane 9, somatic 5S DNA (200 ng) was incubated for threehours; the increased signal, as compared to lane 1, is due to one hour of preincubation. In lane 10, somatic5S DNA (200 ng) was preincubated for one hour, and then tDNA"e' (800 ng) was added for two hours ofincubation. In lane 11, the reaction contained only tDNAmet (800 ng)(lower band). The control reaction in lane12 contained pUC 18 DNA (1 jg) and somatic 5S DNA (200 ng).
ICR (24,29), as well as a study showing that the somatic and oocyte 5S genes have equalaffinity for TFIIA (30).The somatic sequences between -32 and +37 increase transcriptional activity in areconstituted system with partially purified factorsThe experiments detailed above indicated that the somatic -32/ + 37 region increasesbinding efficiency for a factor, other than TFIIA, which forms a stable complex withthe 5S gene. The most likely candidates are factors present in fractions TFLIHB and TFHIC.To examine this possibility, these fractions were isolated and recombined with TFIIIAin a reconstituted transcription system. Fractions TFIIIB and TFIIIC were isolated fromoocyte extracts by phosphocellulose chromatography according to established procedures(2). The crude extracts were passed over phosphocellulose in 0.1M KCI and fractions TFIBand TFIIIC were obtained respectively as 0.35M and 0.6M eluates. RNA polymerase IHactivity cofractionates with TFIIIB. These fractions, when recombined with TFIIIA purifiedfrom immature oocytes, supported 5S transcription. Transcription was not observed witheither fraction alone (data not shown).The transcription reactions shown in figure 5A were performed with constant, optimal
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amounts of TFIIIA and TFIIIC, and variable concentrations of TFIIIB. At lower TFIIIBconcentrations, the somatic 5S gene was ten fold more active than the o/somatic 5S gene.
As the concentration of fraction TFIIIB was increased, that difference decreased to 3 fold.A TFIIIB concentration of 10 pt1!100 1I reaction produced a maximal signal for the somaticgene (lane 3), whereas 30 ,ul were required for a maximal signal for the o/somatic gene
(lane 11).Since RNA polymerase III copurifies in fraction TFHIB, these results could reflect
preferential interaction by this enzyme, rather than a factor, with the somatic 5S gene.
To investigate this possibility, the assays shown in figure 5A were repeated in the presence
of supplementary RNA polymerase Ill, obtained as described previously (31), with thesame results (data not shown). This finding is consistent with template exclusion assays(Figure 3) showing that the somatic -32/+37 region increases binding efficiency for a
factor(s) which forms a stable complex with the 5S gene, thus a component other thanRNA polymerase Ill.The reactions shown in figure 5B were performed with variable concentrations of TFEIIC
and constant amounts of TFIIIA and TFIIIB. The concentration of TFIIIB used in theseassays was optimal for the o/somatic 5S gene and slightly greater than optimal for thesomatic 5S gene. At all concentrations of TFIIC used in these experiments, the somatic5S gene was approximately twice as active as the o/somatic 5S gene. The level of activityfor both genes approached maximal when TFIIIC comprised 25-40 % of the reactionvolume. In some experiments, a slightly higher concentration of TFIIIC was required formaximal activity of the o/somatic gene than for the somatic gene (data not shown).
It is important to note that the same results were obtained with fractions TFIIIB andTFIIIC isolated from immature or mature oocyte S150 extracts (data not shown). Thisindicates that the differential expression is not due to stage specific forms of TFIIIB or
TFIIIC in the mature oocyte S150 extract.
DISCUSSIONThese findings demonstrate that the somatic 5S sequences preceding the minimal promoterincrease the binding efficiency for transcription factors. A previous study (13) showedthat somatic/oocyte sequence differences between -32 and +37 result in as much as tenfold higher activity for the somatic 5S gene in oocyte S150 extracts. In this study, templateexclusion assays, comparing the somatic and o/somatic 5S genes, showed that the somatic-32/+37 region increases the ability to stably bind a commonly required factor(s); a lowerconcentration of somatic 5S DNA was required to deplete a limiting component, thusattaining a maximal level of expression and precluding transcription of a second template.Similarly, in direct competition assays, the somatic 5S gene competed more effectively
Figure 5. Transcription reactions in a reconstituted system with chromatographic fractions TFIIIB and TFIIIC.(A). Reaction mixtures (100 t1) contained fraction TFIIIC (40 Al), purified TFIIIA, and 2, 5, 10, 20, 30, or
40 tl of fraction TFIIIB (lanes 1-6 and 7-12), along with 200 ng of somatic 5S (pXlsl 1-34) (lanes 1-6) or
o/somatic 5S DNA (lanes 7-12). pXlsl 1-34 contains the somatic 5S gene with 5' flanking sequences extendingonly to position -32.
(B). Reaction mixtures (100 1I) contained TFIIIB (30 Ml), purified TFIIIA, and 2.5, 5, 10, 15, 25, or 40 Alof fraction TFELIC (lanes 1-6 and 7-12). DNAs were the same as above. At top of each panel are autoradiographsof denaturing gels. The relative amounts of radiolabel incorporation was determined by scintillation countingof the corresponding regions of the gel.
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for commonly required factors. Finally, in independent reactions, a lower concentrationof somatic 5S DNA was required to attain a maximal signal; at higher DNA concentrations,the o/somatic gene attained the same maximal signal as the somatic 5S gene, suggestingthat higher DNA concentrations compensate for lower factor affinity.The template exclusion assays indicated that the somatic 5S gene has greater affinity
for one or more of the factors which form a stable complex with the 5S gene. Factorsknown to form stable complexes are TFIHA, and components of fractions TFIIIB andTFHIC (8,34). Since TFIIIA binds the somatic and o/somatic 5S genes with equal affinity,factors in TFIIIB or TFIIIC are most likely to be affected by the sequence differencespreceding the minimal promoter. When either TFIIIB or TFIIIC were made the limitingcomponent in a reconstituted system, the somatic 5S gene was more active than the o/somatic5S gene. Moreover, a lower concentration of TFIIIB was required for maximal activityfrom the somatic than the o/somatic 5S gene.The pol IH factors associate with 5S DNA in an interdependent manner. TFIIA binds
independently to the ICR, facilitating binding by TFIIIC, which in turn stabilizes TFIIIA(8, 34). Binding by TFIIIB requires the prior association of both TFIIIA and TFIIIC, andfurther stabilizes the TFIIIA/C complex (8,34). Due to this interdependence, the bindingcharacteristics of more than one factor may be affected by the somatic/oocyte sequencedifferences between -32 and +37. If TFIHC were to bind less efficiently to the o/somatic5S gene, this could reduce the number of potential binding sites for TFIIIB. Conversely,a less effective interaction with TFIIIB could decrease the stability of TFLIC binding.There is evidence that TFIIIC is less efficient at stabilizing the TFHIA/DNA complex onthe oocyte 5S gene in oocyte S150 extracts, apparently due to low TFIIIC concentrationsin these extracts (8,28). The results presented here suggest that the oocyte 5S sequencespreceding the minimal promoter may contribute to this reduced factor affinity. Thisinterpretation fits with current models for 5S gene regulation which suggest that decreasingconcentrations of factors during development, coupled with less efficient binding to theoocyte 5S sequences, contribute to the developmental inactivation of the oocyte 5S genes(28,33).There is recent evidence from DNAse I footprinting studies on yeast tRNA and 5S genes
that sequences upstream of the ICR interact with factors TFIIIB or TFIIIC (46,47,48).On the yeast 5S gene, TFIIIA protects a region within the ICR and the addition of TFIIICextends partial protection as far as -20 in the 5' direction as well as in the 3' direction(46). TFIIIB further extends the protected region to -45. Curiously, on the Xenopus 5Sgene, TFIIIC has not been found to extend the protection outside the region bound byTFIIIA (49). Moreover, in a crude nuclear extract capable of generating a high proportionof active complexes, only subtle differences are noted outside the TFIIIA footprint (50).In other class Im genes, such as tRNA genes and adenovirus VA I, TFIIC binds and protectsthe A and B block regions of the internal promoter in DNAse I protection assays (forreview, see 35). Although the 5S gene lacks a B block sequence, there is homology tothe A block between + 50 and +60 within the binding site for TFIIIA (36). A single pointmutation within this region at position +51 has been shown to be detrimental to TFIIICinteraction (37).
It is interesting that the somatic -32/ + 37 region increased gene activity in oocyte S150extracts but not in oocyte nuclear or HeLa cell extracts. This may reflect qualitativedifferences in the factor populations, perhaps postsynthetic modifications. Another possibilityis that the concentration of one or more factors are significantly different. The results
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obtained in the reconstituted system (Figure 5) suggest that the somatic -32/+37 regionincreases gene activity most significantly when factors are present in suboptimalconcentration. Thus, the differential expression of the somatic and o/somatic genes inmicroinjected embryos, as compared to microinjected oocyte nuclei, may reflect a decreasein the concentration of soluble factors following multiple rounds of DNA replication duringembyonic development.There are many examples of class Im genes in which sequences outside the core promoter
affect the level of gene expression. Sprague and coworkers initially reported that sequencesupstream of tRNA and 5S RNA genes of Bombyx mori were essential for transcriptionin homologous extracts (38,39). Upstream sequences have been shown to affect the levelof activity for tRNA, 5S and 7SL RNA genes from a variety of species (40-43). In thecase of mouse rRNA genes, a minimal promoter element was shown to be sufficient underoptimal transcriptional conditions, whereas under stringent conditions, upstream regionswere found to increase activity and augment stability of factor binding (44,45). Thesefindings bear similarity to those reported here; in oocyte nuclear extracts, the minimalpromoter was sufficient for maximal activity of the somatic and o/somatic 5S genes, whereasin oocyte S150 extracts, somatic sequences preceding the minimal promoter increased gene
activity and stability of factor binding. The minimal promoter may be sufficient underoptimal conditions to insure efficient binding by TFIIIC and TFIIIB, perhaps throughprotein-protein contacts with TFIA, whereas in suboptimal conditions, contacts with DNAsequences preceding the minimal promoter become increasingly important for maximalbinding efficiency.
ACKNOWLEDGEMENTSThe author thanks B. Mather, R. Maki, R. Oshima, and S. Evans for critical commentson the manuscript, N. Sheng for technical assistance, J. Gottesfeld for providing HeLacell extracts and N. Lyman and K. Sweeting for excellent secretarial assistance. Thisresearch was supported by a grant (GM34888) from the National Institutes of Health.
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