.::) 1990 Oxford University Press
Sequence
Nucleic Acids Research, Vol. 18, No. 8 2117
variation in transcription factor IIIA
Chris J.Gaskins and Jay S.Hanas Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA Received November 28, 1989; Revised and Accepted March 13, 1990
ABSTRACT Previous studies characterized macromolecular differences between Xenopus and Rana transcription factor IIIA (TFIIIA) (Gaskins et al.,1989, Nucl. Acids Res. 17, 781 - 794). In the present study, cDNAs for TFIIIA from Xenopus borealis and Rana catesbeiana (American bullfrog) were cloned and sequenced in order to gain molecular insight into the structure, function, and species variation of TFIIIA and the TFIIIAtype zinc finger. X. borealis and R. catesbeiana TFIIIAs have 339 and 335 amino acids respectively, 5 and 9 fewer than X. laevis TFIIIA. X. borealis TFIIIA exhibited 84% sequence homology (55 amino acid differences) with X. laevis TFIIIA and R. catesbeiana TFIIIA exhibited 63% homology (128 amino acid changes) with X. Iaevis TFIIIA. This sequence variation is not random; the Cterminal halves of these TFIIIAs contain substantially more non-conservative changes than the N-terminal halves. In particular, the N-terminal region of TFIIIA (that region forming strong DNA contacts) is the most conserved and the C-terminal tail (that region involved in transcription promotion) the most divergent. Hydropathy analyses of these sequences revealed zinc finger periodicity in the N-terminal halves, extreme hydrophilicity in the C-terminal halves, and a different C-terminal tail hydropathy for R. catesbeiana TFIIIA. Although considerable sequence variation exists in these TFIIIA zinc fingers, the Cys/His, Tyr/Phe and Leu residues are strictly conserved between X. laevis and X. borealis. Strict conservation of only the Cys/His motif is observed between X. laevis and R. catesbeiana TFIIIA. Overall, Cys/His zinc fingers in TFIIIA are much less conserved than Cys/Cys fingers in erythroid transcription factor (Eryf 1) and also less conserved than homeo box domains in segmentation genes. The collective evidence indicates that TFIIIA evolved from a common precursor containing up to 12 finger domains which subsequently evolved at different rates.
INTRODUCTION Transcription of 5S ribosomal RNA genes in Xenopus oocytes requires RNA polymerase HI and at least three additional factors (1,2). One of these proteins, transcription factor IHA (TFIIHA), has a mass of about 40,000 Daltons and promotes transcription by binding to the 50 bp internal control region (ICR) of the 5S
RNA gene (1,3,4). TFIHA contains zinc and requires the metal for function (5). The amino acid sequence of Xenopus laevis TFIIIA (6) contains at least nine repetitive domains (fingers), each with the potential to coordinate a zinc ion between two cysteine and two histidine residues (7,8). In vitro mutagenesis studies suggest a region comprising the N-terminal fingers has a major role in forming DNA contacts with the 3' portion of the 5S RNA gene ICR (9,10,1 1) whereas the C-terminal tail has a major role in promoting transcription (9). Although TFIIIA was the first DNA binding protein shown to require zinc for function and contain zinc fingers, many other gene regulatory proteins containing amino acid repeats similar to those found in TFIIIA have since been described in a wide variety of organisms (12). Types of zinc fingers found in eukaryotic gene regulatory proteins include the Cys/His motif exemplified by TFIIIA and the Cys/Cys motif exemplified by steroid hormone receptors (13) and the erythroid transcription factor (14). Zinc fingers are common evolutionary threads connecting major classes of eukaryotic DNA binding proteins. Recently, the examination of species variation in the erythroid transcription factor revealed that the Cys/Cys zinc fingers in this protein are evolving at an extremely slow rate, comparable to some histone genes (14). Earlier work documented high degrees of conservation in homeo box domains of segmentation genes (15,16). Species variation in TFIHA was previously examined at the macromolecular level and differences were observed in the immunological, DNA binding, and transcription promotion properties of Xenopus and Rana TFIHIA (17). In the present study, the sequences of fulllength, TFIIHA cDNAs from X. borealis and R. catesbeiana are reported. Similarities and differences in these amino acid sequences provide insight into TFiIA structure and function and indicate a high rate of evolution for the TFIA-type zinc finger.
METHODS AND MATERIALS RNA isolation and northern blotting Poly(A) RNA was isolated from the ovarian tissue of immature Xenopus borealis (4-5 cm frogs, Nasco, WI) and Rana catesbeiana (6-7 cm frogs, W. Lemberger, Oshkosh, WI) by the guanidinium isothiocyanate-SDS-proteinase K method (18) utilizing the Invitrogen (La Jolla, CA) Fast Track mRNA isolation system. The tissue was homogenized in lysis buffer and the homogenate was applied directly to an oligo(dT) cellulose column for selection of poly(A) RNA. Poly(A)+ RNA was eluted off the column in low ionic strength buffer, ethanol precipitated, and quantitated by absorbance at 260 nm. Approximately 100 ,ug of
2118 Nucleic Acids Research, Vol. 18, No. 8
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2
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4
Isolation of cDNA inserts and DNA sequence analysis Lambda gtlO bacteriophage clones containing cDNA inserts that cross-hybridized with the X. laevis TFIIIA cDNA were purified as described previously (23). Phage DNAs were digested with EcoRI or NotI and electrophoresed through a 1 % agarose gel. Insert cDNAs were electroeluted and ligated into Phagescript M13 bacteriophage RF (Stratagene, La Jolla, CA) that had been digested with EcoRI or NotI. Single-stranded phage DNA containing inserts were purified and partially sequenced by the dideoxy chain termination method (24) using a modified form of T7 DNA polymerase (Sequenase, United States Biochemical, Cleveland, OH). One cDNA insert was sequenced completely on both strands utilizing a series of complementary oligonucleotides as primers (obtained from K. Jackson, St. Francis Research Facility of Tulsa, OK). Nucleotide and amino acid sequences were analyzed with software packages from Genetics Computer Group (Madison, WI, ref. 25) and Beckman Instruments (Palo Alto, CA, ref. 26).
poly(A)+ selected)
RNA was isolated from 1 g ovarian tissue. Total RNA (not was also isolated from Xenopus laevis ovarian tissue and liver. 5 jig of the respective RNAs were glyoxylated and electrophoresed on 1 % agarose gels and transferred to a nylon membrane (19,20). Xenopus laevis TFIIJA cDNA (a generous gift from William Jack and Robert Roeder) was nick-translated with [&32P] dATP to a specific activity of 108 cpm4Lg DNA. Probe hybridization to the northern filter was performed in 0.1IM PIPES, 0.8M NaCl, 5 X Denhardt's, 0.1I% sarkosyl, and 200 ug/mi denatured salmon sperm DNA at 550C for 24 hours. After washing in 0. 2 X SSC, 0. 05 % sarkosyl, and 0.02 % sodium pyrophosphate for 2 hours at 50'C the filters were exposed to Kodak X-OMAT AR5 film.
RESULTS Identification of TFIIIA mRNA from X. borealis and R. catesbeiana Ovarian tissue RNA from immature X. borealis and R. catesbeiana was examined for the presence of transcripts which cross-hybridize with the 32P-labeled Xenopus laevis TFIIIA cDNA (6). Fig. 1 is a northern blot of X. laevis liver RNA (lane 1) or ovarian tissue RNA (lane 2), X. borealis ovarian tissue RNA that had been poly(A)+ selected (lane 3), and R. catesbeiana poly(A) + RNA (lane 4) all probed with X. laevis TFIIIA cDNA. X. borealis ovarian tissue contains a major poly(A)+ RNA species (lane 3) migrating in similar fashion to the 1.5 kb X. laevis TFIIIA mRNA present in total ovarian RNA (lane 2). R. catesbeiana ovarian tissue also contains a similarly migrating mRNA that cross-hybridizes with this TFIIIA probe (lane 4). The X. laevis TFIIIA probe did not detect under these conditions any cross-hybridizing RNA species in X. laevis liver (lane 1) because of the high selectivity of this probe and the extremely low abundance of TFIIIA mRNA in this organ compared to ovarian tissue. The greater abundance of the TFIIIA mRNA in the X. borealis ovarian RNA compared with X. laevis ovarian RNA is due to poly(A)+ enrichment. The weakly hybridizing R. catesbeiana mRNA is most likely due to sequence diversity.
cDNA library construction and screening cDNA was synthesized from X. borealis and R. catesbeiana poly(A)+ RNA using a modified version (Invitrogen's Lambda Librarian) of the Gubler and Hoffman procedure (21). After first strand synthesis by reverse transcriptase, the RNA template was degraded with RNase H and second strand synthesis was performed by E. coli DNA polymerase I and ligase. Following ligation of EcoRI-NotI adapters, the cDNA was size-fractionated by electrophoresis through a 1 % agarose gel. The 0.5-3 kb fraction was electroeluted and ligated to EcoRI-digested lambda gtlO DNA (22) and packaged with Packagene extract (Promega, Madison, WI). The unamplified library was plated directly on C600hfl E. coli cells and 1.2 x 105 recombinants were screened with nick-translated X. laevis TFIIIA cDNA under conditions similar to that described for the northern analysis. Subsequent secondary and tertiary screenings of initial positives were performed in a similar fashion.
Sequence of X. borealis and R. catesbeiana TFIIIA cDNA cDNA libraries were constructed from the same preparations of X. borealis or R. catesbeiana polyA+ RNA electrophoresed in Fig. 1. After screening about 1 x 105 lambda gtlO recombinants from each library with the X. laevis TFIIIA cDNA probe, seven positive X. borealis clones and two positive R. catesbeiana clones were isolated. Four of the X. borealis clones and two of the R. catesbeiana clones contained inserts of about 1.4 kb, the approximate size of the respective TFIIIA mRNAs (Fig. 1). One clone from each species was chosen for complete sequencing on both strands using a series of complementary oligonucleotides as sequencing primers. The sequences of the non-coding strands (minus the majority of the poly(A) tail) and the predicted amino acid sequence (numbered on left margin) are given in Figs. 2 and 3. The X. borealis cDNA (Fig. 2) consists of 1363 bp plus a EcoRI-NotI linker at the 5' end and a poly(A) tail. An open reading frame, coding for a protein of 339 amino acids, begins at position 67 and extends to a TAA termination codon at position
Fig. 1. Identification of Xenopus borealis and Rana cat~esbeiana TFIIIA mRNA. RNA isolation and glyoxylation, agarose gel electrophoresis, northern transfer and hybridization with nick-translated Xenopus laevis TFIIIA cDNA were performed as described in METHODS AND MATERIALS. 5 icg of total RNA from X. laevis liver or ovarian tissue were electrophoresed in lanes 1 and 2 respectively; 5 itg of poly(A) + RNA from X. borealis or R. catesbeiana ovarian tissue were electrophoresed in lanes 3 or 4. Electrophoretic migrations of X. laevis 18S and 28S ribosomal RNAs are indicated.
Nucleic Acids Research, Vol. 18, No. 8 2119 10 20 30 40 50 60 GAATTCGCGG CCGCAGTTGC GAGGAAGCCA TAGAAAGTCA TAGGAAGTTT AGTTGCAGAA CGAGCG 70 80 90 100 110 120 ATG GGG GAG AAG GCA CTG CCG GTG GTT TAT AAG CGG TAC ATC TGC TCC TTC GCC GAC TGC 1 Met Gly Glu Lys Ala Leu Pro Val Val Tyr Lys Arg Tyr Ile Cys Ser Phe Ala Asp Cys 130 140 150 160 170 180 GGC GCT TCT TAT AAC AAG AAC TGG AAG CTG CGG GCG CAT CTG TGC AAA CAC ACG GGA GAG
21 Gly Ala Ser Tyr Asn Lys Asn Trp Lys Leu Arg Ala His Leu Cys Lys His Thr Gly Glu 190 200 210 220 230 240 AAA CCG TTT CCA TGT AAG GAA GAA GGA TGT GAT AAA GGA TTT ACC TCG TTG CAT CAC TTA 41 Lys Pro Phe Pro Cys Lys Glu Glu Gly Cys Asp Lys Gly Phe Thr Ser Leu His His Leu 250 260 270 280 290 300 ACC CGC CAT TCA ATT ACT CAT ACC GGC GAG AAA AAC TTC AAA TGT GAC TCT GAC AAA TGT 61 Thr Arg His Ser Ile Thr His Thr Gly Glu Lys Asn Phe Lys Cys Asp Ser Asp Lys Cys 310 320 330 340 350 360 GAC TTG ACA TTT ACT ACA AAG GCA AAC ATG AAG AAG CAC TTT AAC AGA TTC CAT AAT CTC 81 Asp Leu Thr Phe Thr Thr Lys Ala Asn Met Lys Lys His Phe Asn Arg Phe His Asn Leu 370 380 390 400 410 420 CAA CTC TGT GTC TAT GTG TGC CAC TTC GAG GGC TGT GAC AAG GCA TTC AAG AAA CAT AAT 101 Gln Leu Cys Val Tyr Val Cys His Phe Glu Gly Cys Asp Lys Ala Phe Lys Lys His Asn 430 440 450 460 470 480 CAA TTA AAG GTT CAC CAG TTT ACT CAT ACG CAG CAG CTG CCA TAC AAG TGC CCT CAT GAA 121 Gln Leu Lys Val His Gln Phe Thr His Thr Gln Gln Leu Pro Tyr Lys Cys Pro His Glu
490 500 510 520 530 540 GGA TGT GAC AAG AGT TTT TCT GTG CCT TCC TGT TTA AAA CGT CAT GAG AAA GTC CAT GCA 141 Gly Cys Asp Lys Ser Phe Ser Val Pro Ser Cys Leu Lys Arg His Glu Lys Val His Ala 550 560 570 580 590 600 GGC TAT CCC TGC AAA AAG GAT GAT TCT TGC TTG TTT GTT GGA AAG ACC TGG ACA TTG TAC 161 Gly Tyr Pro Cys Lys Lys Asp Asp Ser Cys Leu Phe Val Gly Lys Thr Trp Thr Leu Tyr 610 620 630 640 650 660 TTG AAA CAT GTG AAA GAA TGC CAT CAG GAG CCA GTA ATG TGT GAT GAG TGT AAA AGA ACA 181 Leu Lys His Val Lys Glu Cys His Gln Glu Pro Val Met Cys Asp Glu Cys Lys Arg Thr 670 680 690 700 710 720 TTC AAG CAC AAA GAT TAC CTG AGG AAT CAT AAG AAA ACG CAC AAA AAA GAG AGA ACT GTG 201 Phe Lys His Lys Asp Tyr Leu Arg Asn His Lys Lys Thr His Lys Lys Glu Arg Thr Val 730 740 750 760 770 780 TAT TGC TGC CCT CGA GAT GGC TGT GAG CGG TCC TAT ACC ACT GAA TTC AAT CTT CAA AGT 221 Tyr Cys Cys Pro Arg Asp Gly Cys Glu Arg Ser Tyr Thr Thr Glu Phe Asn Leu Gln Ser
790 800 810 820 830 840 CAC ATG CAA TCA TTT CAT GAG GAA CAG AGA CCT TTT GCT TGT GAG CAT GCT GAA TGC GGG 241 His Met Gln Ser Phe His Glu Glu Gln Arg Pro Phe Ala Cys Glu His Ala Glu Cys Gly 850 860 870 880 890 900 AAA TCC TTT GCA ATG AAA AAA AGC CTG GAG AGG CAT TCA GTT GTA CAT GAT CCA GAG AAG 261 Lys Ser Phe Ala Met Lys Lys Ser Leu Glu Arg His Ser Val Val His Asp Pro Glu Lys 910 920 930 940 950 960 AGG AAG CTC AAG GAG AAA TGC CCT CGC CCA AAG AGA AGC CTT GCC TCT CGC CTC TCT GGA 281 Arg Lys Leu Lys Glu Lys Cys Pro Arg Pro Lys Arg Ser Leu Ala Ser Arg Leu Ser Gly 970 980 990 1010 1000 1020 TGT GCA CCC CCC AAG AGC AAA GAA AAG AGT GCA GCA AAG GCA ACA GAA AAG ACT GGT TCA 301 Cys Ala Pro Pro Lys Ser Lys Glu Lys Ser Ala Ala Lys Ala Thr Glu Lys Thr Gly Ser 1030 1040 1050 1060 1070 1080 GTT GTG AAA AAT AAG CCC TCT GGC ACT GAA ACC AAA GGC TCT CTG GTT ATA GAG AAA 321 Val Val Lys Asn Lys Pro Ser Gly Thr Glu Thr Lys Gly Ser Leu Val Ile Glu Lys
1100 1140 1090 1110 1120 1130 1150 TAAACTT TACAGCAATA ACTAAAAGAA CATGAATGTA TATATTTTGT TAAAATTGCC TCGGGGTGGT 1170 1180 1160 1190 1200 1210 1220 TTAACCTACT TGGTTGGGTT TTTTTTTTTT TCCTTAAACC AGGGCTTTTG TTTTATAGTT TTCTTTTAAT
1230
1240
1250
1260
1270
1280
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TTATTTGTTT GTCTACTATA ACAAAAGGAA TCTGTTCTAG ACATGTTAAT TTTGTTTTAT AAACTGCAGT
1310 1320 1330 1340 1350 1300 1360 ATTAGCCATG CCTACAGGTA AAGGCACTGT GTTAATGGCT ACATACCACA ATTCTACCAC ATTCTGCTAI 1370 1380 TAAAGATGAG ATGCAGCAAAn
Fig. 2. The nucleotide and deduced amino acid sequences of Xenopus borealis TFIIIA cDNA. cDNA synthesis, cloning, and sequencing were performed as described in METHODS AND MATERIALS. Partial sequences of two other independent clones were in complete agreement with this sequence. A putative polyadenylation signal is located at nucleotides 1359-1364.
2120 Nucleic Acids Research, Vol. 18, No. 8 10 GCGGCCGCGG GTGGGGAAG 60 70 40 50 30 ATG GGT GAG AAG GCG CCA CCG GCC GTG TAT AAA CGT TAT ATC TGC TCC TTC TCG GAT TGC 1 Met Gly Glu Lys Ala Pro Pro Ala Val Tyr Lys Arg Tyr Ile Cys Ser Phe Ser Asp Cys
20
120 130 90 80 100 110 AGT GCC TCC TAC AAC AAG AAC TGG AAG CTG CAG GCT CAT CTC TGC AAA CAC ACC GGG GAG 21 Ser Ala Ser Tyr Asn Lys Asn Trp Lys Leu Gln Ala His Leu Cys Lys His Thr Gly Glu 180 190 150 160 170 AGA CCT TTC CCC TGC ACG TTT GAA GGA TGC AGT AAA GGC TTT GTG ACT TTG TTT CAC CTG 41 Arg Pro Phe Pro Cys Thr Phe Glu Gly Cys Ser Lys Gly Phe Val Thr Leu Phe His Leu
140
200
210
220
230
240
250
ACC CGT CAT TCT ATG ACA CAT ACT GGG GAG AAG CCT TGC AAA TGT GAT GCT CCA GAT TGT 61 Thr Arg His Ser Met Thr His Thr Gly Glu Lys Pro Cys Lys Cys Asp Ala Pro Asp Cys
260
270
280
290
300
310
GAC TTG TCA TTT ACC ACA ATG ACC AAT ATG AGG AAA CAT TAT CAA CGA GCT CAT CTT TCT 81 Asp Leu Ser Phe Thr Thr Met Thr Asn Met Arg Lys His Tyr Gln Arg Ala His Leu Ser
320
330
340
350
360
370
CCA AGC CTT ATT TAT GAG TGT TAC TTT GCA GAT TGT GGG AAA ACC TTT AAG AAA CAC AAT 101 Pro Ser Leu Ile Tyr Glu Cys Tyr Phe Ala Asp Cys Gly Lys Thr Phe Lys Lys His Asn
380
390
400
410
420
430
CAA CTA AAA ATT CAT CAA TAC ATC CAT ACT AAC CAG CAG CCA TAC AAA TGT ACT CAT GAG
121 Gln Leu Lys Ile His Gln Tyr Ile His Thr Asn Gln Gln Pro Tyr Lys Cys Thr His Glu
440
450
460
470
480
490
GGA TGT GAC AAA AGC TTT TCC TCA CCT TCT AGA CTT AAA CGC CAT GAA AAA GTT CAT GCA 141 Gly Cys Asp Lys Ser Phe Ser Ser Pro Ser Arg Leu Lys Arg His Glu Lys Val His Ala
500
510
520
530
540
550
GGA TAT CCA TGC CAA AAG GAC AGC ACT TGC TCA TTT GTG GGA AAA ACC TGG ACA GAG TAC 161 Gly Tyr Pro Cys Gln Lys Asp Ser Thr Cys Ser Phe Val Gly Lys Thr Trp Thr Glu Tyr
560
570
580
590
600
610
ATG AAA CAC TTG GCA GCT AGT CAT TCA GTT TGC TGT ACA GAG CCA GCC ATC TGC GAT GTA 181 Met Lys His Leu Ala Ala Ser His Ser Val Cys Cys Thr Glu Pro Ala Ile Cys Asp Val
620
630
640
650
660
670
TGT AAT CGC AAG TTT AGG AAC AAA ACT CAC CTG AAG GAC CAT AAG AGA ACC CAC GAA GAA 201 Cys Asn Arg Lys Phe Arg Asn Lys Thr His Leu Lys Asp His Lys Arg Thr His Glu Glu
680
690
700
710
720
730
GAG CGG GTG GTA TAC AAA TGC CCC AGG GAT GGA TGT GAT CGA ACG TAT ACC AAA AAG TTT 221 Glu Arg Val Val Tyr Lys Cys Pro Arg Asp Gly Cys Asp Arg Thr Tyr Thr Lys Lys Phe
740
750
760
770
780
790
GGT CTC CAG AGT CAT ATC CTT TCC TTT CAT GAG GAG TCA CGT CCA TTT GCC TGT GGG CAT 241 Gly Leu Gln Ser His Ile Leu Ser Phe His Glu Glu Ser Arg Pro Phe Ala Cys Gly His
800
810
820
830
840
850
CCT GGA TGT GGC AAG ACC TTT GCA ATG AAG CAA AGT CTT GAT AGA CAT GCA AAT ACC CAT 261 Pro Gly Cys Gly Lys Thr Phe Ala Met Lys Gln Ser Leu Asp Arg His Ala Asn Thr His
860 870 880 890 900 910 281 GAC CCA GAG AAG AAA AAG ATG AAG AAG CCA CGT CCA AAG AAA AGT CTT GCC TCC CGC CTC Asp Pro Glu Lys Lys Lys Met Lys Lys Pro Arg Pro Lys Lys Ser Leu Ala Ser Arg Leu 920
930
940
950
960
970
TCT GGA TAC AAT CCA AAG AAA TTG TCT AAA ACA CCA AAG TCT GTA TCT GAA TTG AGA AAA 301 Ser Gly Tyr Asn Pro Lys Lys Leu Ser Lys Thr Pro Lys Ser Val Ser Glu Leu Arg Lys
980
990 1000 1010 1020 1030 1040 CTG CCT CCA GAT ACT GCT ACA GCA ATG CAG AAT CTC AGC ATT AAA TAACAT GGTAGCACCT 321 Leu Pro Pro Asp Thr Ala Thr Ala Met Gln Asn Leu Ser Ile Lys 1050 1060 1070 1080 1090 1100 1110 GTTACCGTAA AGCAATTTAT ATTTTATGTT AAAATTTTAC CTCAAGAGAA GTAACCCATT TTTATGGGAG 1120 1130 1140 1150 1160 1170 1180 TTTTTGCTGT GAGGGTTTCT AATGGTTTCA GTTCCTGAGT CAGGGTATTT ATTTTCTTTT ATCTGTTTCA 1190 1200 1210 1220 1230 1240 1250 TTTATATGAA TGTACTGCTC CTGCCTGTTT GGTACTATAA AAGTTTAATG TCTTTGCAAT GATTAAAGGA 1260 GGAAAGCAAAn
Fig. 3. The nucleotide and deduced amino acid sequence of Rana catesbeiana TFIIIA cDNA. cDNA synthesis, cloning, and sequencing were performed as described in the METHODS. A partial sequence from one other independent clone was in complete agreement with this sequence. A putative polyadenylation signal is located at nucleotides 1242-1247.
Nucleic Acids Research, Vol. 18, No. 8 2121
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Sequence
Nucleic Acids Research, Vol. 18, No. 8 2117
variation in transcription factor IIIA
Chris J.Gaskins and Jay S.Hanas Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA Received November 28, 1989; Revised and Accepted March 13, 1990
ABSTRACT Previous studies characterized macromolecular differences between Xenopus and Rana transcription factor IIIA (TFIIIA) (Gaskins et al.,1989, Nucl. Acids Res. 17, 781 - 794). In the present study, cDNAs for TFIIIA from Xenopus borealis and Rana catesbeiana (American bullfrog) were cloned and sequenced in order to gain molecular insight into the structure, function, and species variation of TFIIIA and the TFIIIAtype zinc finger. X. borealis and R. catesbeiana TFIIIAs have 339 and 335 amino acids respectively, 5 and 9 fewer than X. laevis TFIIIA. X. borealis TFIIIA exhibited 84% sequence homology (55 amino acid differences) with X. laevis TFIIIA and R. catesbeiana TFIIIA exhibited 63% homology (128 amino acid changes) with X. Iaevis TFIIIA. This sequence variation is not random; the Cterminal halves of these TFIIIAs contain substantially more non-conservative changes than the N-terminal halves. In particular, the N-terminal region of TFIIIA (that region forming strong DNA contacts) is the most conserved and the C-terminal tail (that region involved in transcription promotion) the most divergent. Hydropathy analyses of these sequences revealed zinc finger periodicity in the N-terminal halves, extreme hydrophilicity in the C-terminal halves, and a different C-terminal tail hydropathy for R. catesbeiana TFIIIA. Although considerable sequence variation exists in these TFIIIA zinc fingers, the Cys/His, Tyr/Phe and Leu residues are strictly conserved between X. laevis and X. borealis. Strict conservation of only the Cys/His motif is observed between X. laevis and R. catesbeiana TFIIIA. Overall, Cys/His zinc fingers in TFIIIA are much less conserved than Cys/Cys fingers in erythroid transcription factor (Eryf 1) and also less conserved than homeo box domains in segmentation genes. The collective evidence indicates that TFIIIA evolved from a common precursor containing up to 12 finger domains which subsequently evolved at different rates.
INTRODUCTION Transcription of 5S ribosomal RNA genes in Xenopus oocytes requires RNA polymerase HI and at least three additional factors (1,2). One of these proteins, transcription factor IHA (TFIIHA), has a mass of about 40,000 Daltons and promotes transcription by binding to the 50 bp internal control region (ICR) of the 5S
RNA gene (1,3,4). TFIHA contains zinc and requires the metal for function (5). The amino acid sequence of Xenopus laevis TFIIIA (6) contains at least nine repetitive domains (fingers), each with the potential to coordinate a zinc ion between two cysteine and two histidine residues (7,8). In vitro mutagenesis studies suggest a region comprising the N-terminal fingers has a major role in forming DNA contacts with the 3' portion of the 5S RNA gene ICR (9,10,1 1) whereas the C-terminal tail has a major role in promoting transcription (9). Although TFIIIA was the first DNA binding protein shown to require zinc for function and contain zinc fingers, many other gene regulatory proteins containing amino acid repeats similar to those found in TFIIIA have since been described in a wide variety of organisms (12). Types of zinc fingers found in eukaryotic gene regulatory proteins include the Cys/His motif exemplified by TFIIIA and the Cys/Cys motif exemplified by steroid hormone receptors (13) and the erythroid transcription factor (14). Zinc fingers are common evolutionary threads connecting major classes of eukaryotic DNA binding proteins. Recently, the examination of species variation in the erythroid transcription factor revealed that the Cys/Cys zinc fingers in this protein are evolving at an extremely slow rate, comparable to some histone genes (14). Earlier work documented high degrees of conservation in homeo box domains of segmentation genes (15,16). Species variation in TFIHA was previously examined at the macromolecular level and differences were observed in the immunological, DNA binding, and transcription promotion properties of Xenopus and Rana TFIHIA (17). In the present study, the sequences of fulllength, TFIIHA cDNAs from X. borealis and R. catesbeiana are reported. Similarities and differences in these amino acid sequences provide insight into TFiIA structure and function and indicate a high rate of evolution for the TFIA-type zinc finger.
METHODS AND MATERIALS RNA isolation and northern blotting Poly(A) RNA was isolated from the ovarian tissue of immature Xenopus borealis (4-5 cm frogs, Nasco, WI) and Rana catesbeiana (6-7 cm frogs, W. Lemberger, Oshkosh, WI) by the guanidinium isothiocyanate-SDS-proteinase K method (18) utilizing the Invitrogen (La Jolla, CA) Fast Track mRNA isolation system. The tissue was homogenized in lysis buffer and the homogenate was applied directly to an oligo(dT) cellulose column for selection of poly(A) RNA. Poly(A)+ RNA was eluted off the column in low ionic strength buffer, ethanol precipitated, and quantitated by absorbance at 260 nm. Approximately 100 ,ug of
2118 Nucleic Acids Research, Vol. 18, No. 8
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2
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Isolation of cDNA inserts and DNA sequence analysis Lambda gtlO bacteriophage clones containing cDNA inserts that cross-hybridized with the X. laevis TFIIIA cDNA were purified as described previously (23). Phage DNAs were digested with EcoRI or NotI and electrophoresed through a 1 % agarose gel. Insert cDNAs were electroeluted and ligated into Phagescript M13 bacteriophage RF (Stratagene, La Jolla, CA) that had been digested with EcoRI or NotI. Single-stranded phage DNA containing inserts were purified and partially sequenced by the dideoxy chain termination method (24) using a modified form of T7 DNA polymerase (Sequenase, United States Biochemical, Cleveland, OH). One cDNA insert was sequenced completely on both strands utilizing a series of complementary oligonucleotides as primers (obtained from K. Jackson, St. Francis Research Facility of Tulsa, OK). Nucleotide and amino acid sequences were analyzed with software packages from Genetics Computer Group (Madison, WI, ref. 25) and Beckman Instruments (Palo Alto, CA, ref. 26).
poly(A)+ selected)
RNA was isolated from 1 g ovarian tissue. Total RNA (not was also isolated from Xenopus laevis ovarian tissue and liver. 5 jig of the respective RNAs were glyoxylated and electrophoresed on 1 % agarose gels and transferred to a nylon membrane (19,20). Xenopus laevis TFIIJA cDNA (a generous gift from William Jack and Robert Roeder) was nick-translated with [&32P] dATP to a specific activity of 108 cpm4Lg DNA. Probe hybridization to the northern filter was performed in 0.1IM PIPES, 0.8M NaCl, 5 X Denhardt's, 0.1I% sarkosyl, and 200 ug/mi denatured salmon sperm DNA at 550C for 24 hours. After washing in 0. 2 X SSC, 0. 05 % sarkosyl, and 0.02 % sodium pyrophosphate for 2 hours at 50'C the filters were exposed to Kodak X-OMAT AR5 film.
RESULTS Identification of TFIIIA mRNA from X. borealis and R. catesbeiana Ovarian tissue RNA from immature X. borealis and R. catesbeiana was examined for the presence of transcripts which cross-hybridize with the 32P-labeled Xenopus laevis TFIIIA cDNA (6). Fig. 1 is a northern blot of X. laevis liver RNA (lane 1) or ovarian tissue RNA (lane 2), X. borealis ovarian tissue RNA that had been poly(A)+ selected (lane 3), and R. catesbeiana poly(A) + RNA (lane 4) all probed with X. laevis TFIIIA cDNA. X. borealis ovarian tissue contains a major poly(A)+ RNA species (lane 3) migrating in similar fashion to the 1.5 kb X. laevis TFIIIA mRNA present in total ovarian RNA (lane 2). R. catesbeiana ovarian tissue also contains a similarly migrating mRNA that cross-hybridizes with this TFIIIA probe (lane 4). The X. laevis TFIIIA probe did not detect under these conditions any cross-hybridizing RNA species in X. laevis liver (lane 1) because of the high selectivity of this probe and the extremely low abundance of TFIIIA mRNA in this organ compared to ovarian tissue. The greater abundance of the TFIIIA mRNA in the X. borealis ovarian RNA compared with X. laevis ovarian RNA is due to poly(A)+ enrichment. The weakly hybridizing R. catesbeiana mRNA is most likely due to sequence diversity.
cDNA library construction and screening cDNA was synthesized from X. borealis and R. catesbeiana poly(A)+ RNA using a modified version (Invitrogen's Lambda Librarian) of the Gubler and Hoffman procedure (21). After first strand synthesis by reverse transcriptase, the RNA template was degraded with RNase H and second strand synthesis was performed by E. coli DNA polymerase I and ligase. Following ligation of EcoRI-NotI adapters, the cDNA was size-fractionated by electrophoresis through a 1 % agarose gel. The 0.5-3 kb fraction was electroeluted and ligated to EcoRI-digested lambda gtlO DNA (22) and packaged with Packagene extract (Promega, Madison, WI). The unamplified library was plated directly on C600hfl E. coli cells and 1.2 x 105 recombinants were screened with nick-translated X. laevis TFIIIA cDNA under conditions similar to that described for the northern analysis. Subsequent secondary and tertiary screenings of initial positives were performed in a similar fashion.
Sequence of X. borealis and R. catesbeiana TFIIIA cDNA cDNA libraries were constructed from the same preparations of X. borealis or R. catesbeiana polyA+ RNA electrophoresed in Fig. 1. After screening about 1 x 105 lambda gtlO recombinants from each library with the X. laevis TFIIIA cDNA probe, seven positive X. borealis clones and two positive R. catesbeiana clones were isolated. Four of the X. borealis clones and two of the R. catesbeiana clones contained inserts of about 1.4 kb, the approximate size of the respective TFIIIA mRNAs (Fig. 1). One clone from each species was chosen for complete sequencing on both strands using a series of complementary oligonucleotides as sequencing primers. The sequences of the non-coding strands (minus the majority of the poly(A) tail) and the predicted amino acid sequence (numbered on left margin) are given in Figs. 2 and 3. The X. borealis cDNA (Fig. 2) consists of 1363 bp plus a EcoRI-NotI linker at the 5' end and a poly(A) tail. An open reading frame, coding for a protein of 339 amino acids, begins at position 67 and extends to a TAA termination codon at position
Fig. 1. Identification of Xenopus borealis and Rana cat~esbeiana TFIIIA mRNA. RNA isolation and glyoxylation, agarose gel electrophoresis, northern transfer and hybridization with nick-translated Xenopus laevis TFIIIA cDNA were performed as described in METHODS AND MATERIALS. 5 icg of total RNA from X. laevis liver or ovarian tissue were electrophoresed in lanes 1 and 2 respectively; 5 itg of poly(A) + RNA from X. borealis or R. catesbeiana ovarian tissue were electrophoresed in lanes 3 or 4. Electrophoretic migrations of X. laevis 18S and 28S ribosomal RNAs are indicated.
Nucleic Acids Research, Vol. 18, No. 8 2119 10 20 30 40 50 60 GAATTCGCGG CCGCAGTTGC GAGGAAGCCA TAGAAAGTCA TAGGAAGTTT AGTTGCAGAA CGAGCG 70 80 90 100 110 120 ATG GGG GAG AAG GCA CTG CCG GTG GTT TAT AAG CGG TAC ATC TGC TCC TTC GCC GAC TGC 1 Met Gly Glu Lys Ala Leu Pro Val Val Tyr Lys Arg Tyr Ile Cys Ser Phe Ala Asp Cys 130 140 150 160 170 180 GGC GCT TCT TAT AAC AAG AAC TGG AAG CTG CGG GCG CAT CTG TGC AAA CAC ACG GGA GAG
21 Gly Ala Ser Tyr Asn Lys Asn Trp Lys Leu Arg Ala His Leu Cys Lys His Thr Gly Glu 190 200 210 220 230 240 AAA CCG TTT CCA TGT AAG GAA GAA GGA TGT GAT AAA GGA TTT ACC TCG TTG CAT CAC TTA 41 Lys Pro Phe Pro Cys Lys Glu Glu Gly Cys Asp Lys Gly Phe Thr Ser Leu His His Leu 250 260 270 280 290 300 ACC CGC CAT TCA ATT ACT CAT ACC GGC GAG AAA AAC TTC AAA TGT GAC TCT GAC AAA TGT 61 Thr Arg His Ser Ile Thr His Thr Gly Glu Lys Asn Phe Lys Cys Asp Ser Asp Lys Cys 310 320 330 340 350 360 GAC TTG ACA TTT ACT ACA AAG GCA AAC ATG AAG AAG CAC TTT AAC AGA TTC CAT AAT CTC 81 Asp Leu Thr Phe Thr Thr Lys Ala Asn Met Lys Lys His Phe Asn Arg Phe His Asn Leu 370 380 390 400 410 420 CAA CTC TGT GTC TAT GTG TGC CAC TTC GAG GGC TGT GAC AAG GCA TTC AAG AAA CAT AAT 101 Gln Leu Cys Val Tyr Val Cys His Phe Glu Gly Cys Asp Lys Ala Phe Lys Lys His Asn 430 440 450 460 470 480 CAA TTA AAG GTT CAC CAG TTT ACT CAT ACG CAG CAG CTG CCA TAC AAG TGC CCT CAT GAA 121 Gln Leu Lys Val His Gln Phe Thr His Thr Gln Gln Leu Pro Tyr Lys Cys Pro His Glu
490 500 510 520 530 540 GGA TGT GAC AAG AGT TTT TCT GTG CCT TCC TGT TTA AAA CGT CAT GAG AAA GTC CAT GCA 141 Gly Cys Asp Lys Ser Phe Ser Val Pro Ser Cys Leu Lys Arg His Glu Lys Val His Ala 550 560 570 580 590 600 GGC TAT CCC TGC AAA AAG GAT GAT TCT TGC TTG TTT GTT GGA AAG ACC TGG ACA TTG TAC 161 Gly Tyr Pro Cys Lys Lys Asp Asp Ser Cys Leu Phe Val Gly Lys Thr Trp Thr Leu Tyr 610 620 630 640 650 660 TTG AAA CAT GTG AAA GAA TGC CAT CAG GAG CCA GTA ATG TGT GAT GAG TGT AAA AGA ACA 181 Leu Lys His Val Lys Glu Cys His Gln Glu Pro Val Met Cys Asp Glu Cys Lys Arg Thr 670 680 690 700 710 720 TTC AAG CAC AAA GAT TAC CTG AGG AAT CAT AAG AAA ACG CAC AAA AAA GAG AGA ACT GTG 201 Phe Lys His Lys Asp Tyr Leu Arg Asn His Lys Lys Thr His Lys Lys Glu Arg Thr Val 730 740 750 760 770 780 TAT TGC TGC CCT CGA GAT GGC TGT GAG CGG TCC TAT ACC ACT GAA TTC AAT CTT CAA AGT 221 Tyr Cys Cys Pro Arg Asp Gly Cys Glu Arg Ser Tyr Thr Thr Glu Phe Asn Leu Gln Ser
790 800 810 820 830 840 CAC ATG CAA TCA TTT CAT GAG GAA CAG AGA CCT TTT GCT TGT GAG CAT GCT GAA TGC GGG 241 His Met Gln Ser Phe His Glu Glu Gln Arg Pro Phe Ala Cys Glu His Ala Glu Cys Gly 850 860 870 880 890 900 AAA TCC TTT GCA ATG AAA AAA AGC CTG GAG AGG CAT TCA GTT GTA CAT GAT CCA GAG AAG 261 Lys Ser Phe Ala Met Lys Lys Ser Leu Glu Arg His Ser Val Val His Asp Pro Glu Lys 910 920 930 940 950 960 AGG AAG CTC AAG GAG AAA TGC CCT CGC CCA AAG AGA AGC CTT GCC TCT CGC CTC TCT GGA 281 Arg Lys Leu Lys Glu Lys Cys Pro Arg Pro Lys Arg Ser Leu Ala Ser Arg Leu Ser Gly 970 980 990 1010 1000 1020 TGT GCA CCC CCC AAG AGC AAA GAA AAG AGT GCA GCA AAG GCA ACA GAA AAG ACT GGT TCA 301 Cys Ala Pro Pro Lys Ser Lys Glu Lys Ser Ala Ala Lys Ala Thr Glu Lys Thr Gly Ser 1030 1040 1050 1060 1070 1080 GTT GTG AAA AAT AAG CCC TCT GGC ACT GAA ACC AAA GGC TCT CTG GTT ATA GAG AAA 321 Val Val Lys Asn Lys Pro Ser Gly Thr Glu Thr Lys Gly Ser Leu Val Ile Glu Lys
1100 1140 1090 1110 1120 1130 1150 TAAACTT TACAGCAATA ACTAAAAGAA CATGAATGTA TATATTTTGT TAAAATTGCC TCGGGGTGGT 1170 1180 1160 1190 1200 1210 1220 TTAACCTACT TGGTTGGGTT TTTTTTTTTT TCCTTAAACC AGGGCTTTTG TTTTATAGTT TTCTTTTAAT
1230
1240
1250
1260
1270
1280
1290
TTATTTGTTT GTCTACTATA ACAAAAGGAA TCTGTTCTAG ACATGTTAAT TTTGTTTTAT AAACTGCAGT
1310 1320 1330 1340 1350 1300 1360 ATTAGCCATG CCTACAGGTA AAGGCACTGT GTTAATGGCT ACATACCACA ATTCTACCAC ATTCTGCTAI 1370 1380 TAAAGATGAG ATGCAGCAAAn
Fig. 2. The nucleotide and deduced amino acid sequences of Xenopus borealis TFIIIA cDNA. cDNA synthesis, cloning, and sequencing were performed as described in METHODS AND MATERIALS. Partial sequences of two other independent clones were in complete agreement with this sequence. A putative polyadenylation signal is located at nucleotides 1359-1364.
2120 Nucleic Acids Research, Vol. 18, No. 8 10 GCGGCCGCGG GTGGGGAAG 60 70 40 50 30 ATG GGT GAG AAG GCG CCA CCG GCC GTG TAT AAA CGT TAT ATC TGC TCC TTC TCG GAT TGC 1 Met Gly Glu Lys Ala Pro Pro Ala Val Tyr Lys Arg Tyr Ile Cys Ser Phe Ser Asp Cys
20
120 130 90 80 100 110 AGT GCC TCC TAC AAC AAG AAC TGG AAG CTG CAG GCT CAT CTC TGC AAA CAC ACC GGG GAG 21 Ser Ala Ser Tyr Asn Lys Asn Trp Lys Leu Gln Ala His Leu Cys Lys His Thr Gly Glu 180 190 150 160 170 AGA CCT TTC CCC TGC ACG TTT GAA GGA TGC AGT AAA GGC TTT GTG ACT TTG TTT CAC CTG 41 Arg Pro Phe Pro Cys Thr Phe Glu Gly Cys Ser Lys Gly Phe Val Thr Leu Phe His Leu
140
200
210
220
230
240
250
ACC CGT CAT TCT ATG ACA CAT ACT GGG GAG AAG CCT TGC AAA TGT GAT GCT CCA GAT TGT 61 Thr Arg His Ser Met Thr His Thr Gly Glu Lys Pro Cys Lys Cys Asp Ala Pro Asp Cys
260
270
280
290
300
310
GAC TTG TCA TTT ACC ACA ATG ACC AAT ATG AGG AAA CAT TAT CAA CGA GCT CAT CTT TCT 81 Asp Leu Ser Phe Thr Thr Met Thr Asn Met Arg Lys His Tyr Gln Arg Ala His Leu Ser
320
330
340
350
360
370
CCA AGC CTT ATT TAT GAG TGT TAC TTT GCA GAT TGT GGG AAA ACC TTT AAG AAA CAC AAT 101 Pro Ser Leu Ile Tyr Glu Cys Tyr Phe Ala Asp Cys Gly Lys Thr Phe Lys Lys His Asn
380
390
400
410
420
430
CAA CTA AAA ATT CAT CAA TAC ATC CAT ACT AAC CAG CAG CCA TAC AAA TGT ACT CAT GAG
121 Gln Leu Lys Ile His Gln Tyr Ile His Thr Asn Gln Gln Pro Tyr Lys Cys Thr His Glu
440
450
460
470
480
490
GGA TGT GAC AAA AGC TTT TCC TCA CCT TCT AGA CTT AAA CGC CAT GAA AAA GTT CAT GCA 141 Gly Cys Asp Lys Ser Phe Ser Ser Pro Ser Arg Leu Lys Arg His Glu Lys Val His Ala
500
510
520
530
540
550
GGA TAT CCA TGC CAA AAG GAC AGC ACT TGC TCA TTT GTG GGA AAA ACC TGG ACA GAG TAC 161 Gly Tyr Pro Cys Gln Lys Asp Ser Thr Cys Ser Phe Val Gly Lys Thr Trp Thr Glu Tyr
560
570
580
590
600
610
ATG AAA CAC TTG GCA GCT AGT CAT TCA GTT TGC TGT ACA GAG CCA GCC ATC TGC GAT GTA 181 Met Lys His Leu Ala Ala Ser His Ser Val Cys Cys Thr Glu Pro Ala Ile Cys Asp Val
620
630
640
650
660
670
TGT AAT CGC AAG TTT AGG AAC AAA ACT CAC CTG AAG GAC CAT AAG AGA ACC CAC GAA GAA 201 Cys Asn Arg Lys Phe Arg Asn Lys Thr His Leu Lys Asp His Lys Arg Thr His Glu Glu
680
690
700
710
720
730
GAG CGG GTG GTA TAC AAA TGC CCC AGG GAT GGA TGT GAT CGA ACG TAT ACC AAA AAG TTT 221 Glu Arg Val Val Tyr Lys Cys Pro Arg Asp Gly Cys Asp Arg Thr Tyr Thr Lys Lys Phe
740
750
760
770
780
790
GGT CTC CAG AGT CAT ATC CTT TCC TTT CAT GAG GAG TCA CGT CCA TTT GCC TGT GGG CAT 241 Gly Leu Gln Ser His Ile Leu Ser Phe His Glu Glu Ser Arg Pro Phe Ala Cys Gly His
800
810
820
830
840
850
CCT GGA TGT GGC AAG ACC TTT GCA ATG AAG CAA AGT CTT GAT AGA CAT GCA AAT ACC CAT 261 Pro Gly Cys Gly Lys Thr Phe Ala Met Lys Gln Ser Leu Asp Arg His Ala Asn Thr His
860 870 880 890 900 910 281 GAC CCA GAG AAG AAA AAG ATG AAG AAG CCA CGT CCA AAG AAA AGT CTT GCC TCC CGC CTC Asp Pro Glu Lys Lys Lys Met Lys Lys Pro Arg Pro Lys Lys Ser Leu Ala Ser Arg Leu 920
930
940
950
960
970
TCT GGA TAC AAT CCA AAG AAA TTG TCT AAA ACA CCA AAG TCT GTA TCT GAA TTG AGA AAA 301 Ser Gly Tyr Asn Pro Lys Lys Leu Ser Lys Thr Pro Lys Ser Val Ser Glu Leu Arg Lys
980
990 1000 1010 1020 1030 1040 CTG CCT CCA GAT ACT GCT ACA GCA ATG CAG AAT CTC AGC ATT AAA TAACAT GGTAGCACCT 321 Leu Pro Pro Asp Thr Ala Thr Ala Met Gln Asn Leu Ser Ile Lys 1050 1060 1070 1080 1090 1100 1110 GTTACCGTAA AGCAATTTAT ATTTTATGTT AAAATTTTAC CTCAAGAGAA GTAACCCATT TTTATGGGAG 1120 1130 1140 1150 1160 1170 1180 TTTTTGCTGT GAGGGTTTCT AATGGTTTCA GTTCCTGAGT CAGGGTATTT ATTTTCTTTT ATCTGTTTCA 1190 1200 1210 1220 1230 1240 1250 TTTATATGAA TGTACTGCTC CTGCCTGTTT GGTACTATAA AAGTTTAATG TCTTTGCAAT GATTAAAGGA 1260 GGAAAGCAAAn
Fig. 3. The nucleotide and deduced amino acid sequence of Rana catesbeiana TFIIIA cDNA. cDNA synthesis, cloning, and sequencing were performed as described in the METHODS. A partial sequence from one other independent clone was in complete agreement with this sequence. A putative polyadenylation signal is located at nucleotides 1242-1247.
Nucleic Acids Research, Vol. 18, No. 8 2121
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