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Volume 14
Number 11 1986
14 Number 11 1986
Volume
Nucleic Acids Research
Nucleic Acids Research
TFIIIA and homologous genes. The 'finger' proteins
Alain Vincent
Institut Jacques Monod, CNRS and Universite Paris VII, 2, place Jussieu, 75251 Paris Cedex 05,
France
Received 3 April 1986; Accepted 2 May 1986
ABSTRACT
Differential regulation of gene expression , in a precise temporal and spatial
pattern during development, is thought to be partly mediated by site specific DNA
binding proteins which promote a selective activation of qene transcription (1).
From studies on XenopusTFl IA, a factor selectively required for transcription
of 5 S ribosomal RNA genes, Miller etal. (2) proposed a novel structural model of
interaction between DNA and DNA binding protein. The striking homology of TFI I IA
with several recently sequenced Drosophi/aand yeast gene products suggests
that multiple regulatory proteins may have evolved from a small ancestra DNA
binding protein domain and that the characteristic features of TFIIIA and
TFIIIA-5S DNA interactions may be of general significance.
TFI I IA and the develoDmental control of 5 5 RNA gene exDression in XenoDus
5 5 ribosomal RNA gene transcription In Xenopus provides a model system for
studying differential gene regulation during development. Initiation of
transcription of 5 S genes by polymerase III requires the formation of a "stable
transcription complex" including DNA in the internal control region (ICR) and at
least three transcription factors, TFIIIA, B and C (3), the specific Interaction
between TFIIIA and the internal control region being the primary event in the
activation of the genes (4). The establishment of stable active complexes within
the otherwise transcriptionally silent chromatin has been postulated to provide a
means for maintaining the selective expression of a specific set of activated
genes; the repressed genes not complexed with transcription factors are
prevented from recruiting these factors by a chromatin structure dependent on
histoneHI (5).
Both oocyte type and somatic type 5 S RNA genes (20 000 and 400 genes per
haploid genome, respectively) are transcribed during oogenesis. In somatic cells,
however, more than 95% of the 5 S RNAs are transcribed from the somatic type
genes, which corresponds to a greater than 1000 fold preferential transcription
of somatic over oocyte type genes on a per gene basis. Much of this preference for
somatic 5 S gene transcription is attributed to the concentration of the positive
© I R L Press Limited, Oxford, England.
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Nucleic Acids Research
transcription factor, TFIIIA, and to differences in binding constant of TFIIIA to
the 50 bp long ICR of the two types of genes (the two ICR differ by 3 bp ) (6).
TFIIIA is a single protein of 38500 dalton which interacts also with 5 S RNA to
form 7 S ribonucleoprotein particles that is a storage form of 5 S RNAs during
oogenesis. Early in oogenesis, TFIIIA is present in excess, ensuring thereby the
activation of both types of 5 S RNA genes. As the cellular concentration of TFIIIA
decreases during late oogenesis and embryogenesis it becomes a limiting factor.
This results in the preferential formation of stable active complexes with
somatic 5 5 genes. The imposition of complete oocyte 5 S gene repression could
occur gradually over a few cell divisions (7).
Binding of TFI II A to the 5S RNA gene: "the finger Drotein model".
The intact TFI I IA factor binds to the ICR as a monomer and protects about 50
nucleotides in the center of the 5 5 RNA gene from digestion by DNAse 1. Three
functional domains are separable by proteolytic cleavage. At the C terminus of
the protein is a domain required for efficient RNA transcription but not for
binding to DNA. A second domain binds to the 5' end of the ICR, and, together with
the third which binds only to the 3' side of the control region but Is Inactive In
promoting transcription, partly supports transcription (8). Computer analysis of
the predicted TFIIIA aminoacid sequence (9) revealed a continuous run of nine
similar units, each of about 30 residues (residues 13 to 276), covering the ICR
binding domain (2,10). The sequence information, the high zinc content of the 75
ribonucleo- protein particle, and more detailed studies on proteolytic digestion
led Miller et 8f (2) to propose that TFIIIA structure includes nine loop like
domains, the "fingers", formed by interaction of two invariant pairs of cysteines
and histidines with an atom of zinc. The key feature of the suggested structure Is
that the "fingers" (schematically drawn on figure 1B), which are the proposed
DNA binding regions, are independent units linked by flexible joints; the Phe and
Leu residues may form a hydrophobic core within each finger, and the fingers may
bind to nucleic acids by interaction of positively charged DNA-binding amino
acids with the phosphate backbone of DNA (1 1). Such a structure, consistent
with the asymetry of the TFIIIA DNA binding site, could explain : 1) how a
relatively small protein could bind to a long stretch of double helical DNA,
because each "finger" interacts with roughly half a DNA period, and 2) how a
"stable" transcription complex is able to wlthstand the continuous cycling of RNA
polymerase through it because some fingers remain in contact with the DNA
while others release this contact as polymerase passes by. A three dimensional
model of a triple complex formed between TFIIIA, the histone octamer and the 5S
RNA gene is further proposed by Rhodes (12) to explain how RNA polymerase III
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Nucleic Acids Research
a
1
Xen.TFINA
Dros.Kr
Dros. sry
3
6
10
.0.
.©
.
16
23
19
29
®..
. ®
-<13
0* e ........)®
C)..e.©..C
8)®
.(®*
c).
Yeast ADRI
D.D
0
©
.
.* . .
.
. - --*c- *(o
*60 09
.(.
.
- ®
*C)...(®0. .(D
-
.
..
29 a.a.
- - - -
. so*
.® .
*
30a.a.
28 a.a.
.
®(
28 a.a.
b
Zn
Xen.
Dros.
Figure 1. Alignment of repeats present in the TFIIIA (ref.2), Ar (ref.14),
beta
and delto (ref.13), and ADRI (ref.30) polypeptides is shown. The second sr,y
csteine
of the cysteine pair is arbitrarily placed at position 6 of the repeat. Onl the best
conserved residues are indicated. The potential DNA-binding loop (finger) is
formed by residues 7 to 18 indicated bu larger black dots (see ref.2).
Folding scheme for the A17nopilu,s and Drzisopbi/ repeated domains: the finger
structure, drawn according to the model proposed bu Miller et al. (2). Each domain
is centered on a tetrahedral arrangement of metal (Zn) ligands. The marked
residues are the conserved amino acids which include the Cys and His metal
ligands and the three hydrophobic residues that may form a structural core.
could interact simultaneously with transcription factors bound at the internal
control region of the gene and the start point of transcription and how histone HI
could repress transcription of 5S RNA genes by preventing TFIIIA binding.
Prosobih,g
fingeproteins.
Sequence analysis of the Drosopbe/5 genes Serendipity (s.rg) beta and delta (13),
and Kruppel (At-) (14) revealed that the finger motif has been conserved during
evolution. Figure IA shows the characteristic features of the 30 residues TFIIIA
repeated domain preserved in the DrosopbiJ'A repeat- the Cys 3,Cys 6, Phe 10, Leu
16, His 19 and His 23 - , i.e., the precise spacing between Cys 6 and His 19, the
putative DNA binding domain.
The Cys-X2-Cys motif and additional Cys and His residues are found at regularly
spaced positions in the DNA binding region of various nucleic acids binding
proteins whith no close homology with TFIIIA. . Examples are proteins encoded
by the gag-pol region of retroviruses, the [rcsqsop4l/y copia element and the
cauliflower mosaic Yirus (15,16), as well as the recently sequenced human and
avian cestrogen receptors and the human glucocorticoid receptor (17,20). In the
latter examples, three Cys-X2-Cys units and additional Cys and Lys residues are
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Nucleic Acids Research
found at equivalent positions in a 66 amino acids domain -the putative DNA
binding site- highly homologous between the three proteins. This suggests that
the DNA binding domain of these proteins may include one or more independent
folding units, but markedly different from the TFIIIA finger (20). The
metallothionein family of proteins represents another group of proteins which
contain repeated Cys residues (21), but is not a good analogy forTFIIIA (2).
The extensive homology of Drosopbil5 s,rg beta, sr,y delta and At with TFI I A
raises the strong possibility that -.r,y and A', code for DNA (or RNA) binding
proteins whith some properties similar to those of TFIIIA. This is of considerable
interest since the Ar gene falls within the class of Dr.csap4lJ/M segmentation
genes required for the establishment of adjacent groups of segments (22) and
presumably acts on the expression of other (sets of) specific genes (14). No
obvious morphological defect has yet been associated with mutations at the sry
complex locus; embryos carrying a delta mutation over a deficiency die before
hatching (J. Lengyel, personal communication). It is then remarkable that a yeast
regularory protein, ADRI, which is required for the transcriptional activation
(derepression) of the alcohol dehydrogenase (ADH2) gene also shows amino acid
sequence homology with the repeated domain of TFI I IA ((30), see fig. 1A). Two
fingers are found in the amino terminal third of ADRI, a domain which is as
active as the entire protein in ADH2 derepression.
4, 6 and 7 "fingers" are found in the Kr7 sri' beta and sry delta polypeptides,
respectively (figure 2). One complete finger has been found in yet another
Drosqopfl/l blastoderm specific gene (J. Lenggel, personal communication ). The
finger region is the only region of homology between the Drosopbi/8 finger
proteins, suggesting that partial and independent gene duplication and/or
conversion events have been at the origin of finger protein coding genes. Within
the A;:7 ;r/& beta and ,y delta polypeptides, each "finger tip" displays a unique
sequence of "DNA binding amino acids". This suggests that the contacts between
DNA and these proteins do probably not involve a stretch of repeated short
sequences (see, for example, the 5S-TFIIA contact region). Although beta and
delta most likely arose from a duplication event after the multiplication of the
internal repeat the degree of conservation between fingers at homologous
positions in the beta and delta proteins is variable. This might reflect unequal
evolutionary constraints on the different fingers perhaps because DNA
recognition and DNA binding strength are not carried by the same and all fingers.
The invariance of the amino acid stretch between His 23 and Cys 3 (the
inter-finger region, Fig. 1) in Kr suggests that this portion of the repeat is
important for the folding of that protein. The portions of the TFIIIA, Kr or srb,
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TFIIIA
000 0 0
1H_2
Kr
NH
2
2COOH
2
33~2
~~~~222
1
NH
00
0
sry beta
344
0
0
529
CH
0
166351
1
sry delta
N1
193
4
3
ADR i
1 98 (1' " 5155
NH8
1323
COOH
Figure 2. Scheme for a linear arrangement of fingers in the TFIIIA (344 a.a.), A
(529 a.a.), srg beta (351 a.a.), s,y delta (430 a.a.) and ADR1 (1323 a.a.)
polypeptides. Orientation of each polypeptide is amino (left) to carboxy terminus
polypeptides which are adjacent to the finger region and are characteristic of
each polypeptide might in turn be involved in specific protein-protein
interactions.
DISCUSSION
The finding that several Drasap/Ai/ and one yeast protein contain a repeated
"finger" motif supports the prediction (2) that various regulatory proteins may
have evolved from a small DNA binding ancestral domain. Furthermore, it predicts
that variations in the DNA (or RNA) binding strength of each finger -by variations
in the sequence at each finger tip- and in the number of fingers could establish a
highly specific recognition system for nucleic acid segments.
Such a system of DNA recognition is clearly different from the classical
bacterial system - a DNA binding-domain made of two alpha helices (CZ' and "3)
connected by a beta turn with alpha helix '3" lying into the major groove of DNA (23), or the presumably analogous system in eucaryotes, carried by regulatory
protei-ns such as the yeast- mat a 1 and alpha 2 and the Drosop,t'// homeo-box
containing gene products (24,25). Miller at &I (25) propose that eucaryotic
DNA-binding proteins which recognise the same DNA sequence , such as mat a 1
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and alpha 2 or, possibly, several homeo-domain containing OrosopbfAie proteins,
could perform different functions because of the different protein-protein
interactions they can make. The finger proteins may constitute a separate class
of regulatory proteins. Their characteristics may include the variable length and
asymetry of their DNA binding sites, (their binding sites might often lie within
the transcribed DNA (enhancers?), multiple fingers circumventing the passing
polymerase problem) the I to 1 stoechiometry of binding, and the possibility to
induce DNA gyration and form dynamic chromatin, a reaction that appears to be
the key step -formation of dynamic chromatin- in the activation of the 5S RNA
gene (26).
DraSOpA//i "finger' proteins and development; chromosomal regulation of gene
expression?
The Kruppel gene function is required for the formation of thoracic and abdominal
segments in the DrmopsV1/Ae embryo. Kr gene expression is strictly zygotic, and
restricted to the blastoderm and early gastula stages in the portions of the
embryo corresponding to the anlage of the thoracic and anterior abdominal
segments missing in a strong Ar mutant (27). SIry gene expression is also
developmentally regulated. Sry beta and delta mRNA accumulate during
oogenesis, are abundant in polysomes of early embryos but (similarly to TFIIIA
RNA in A%inqp/s) are present at a very reduced level during the rest of the fly life
cycle (a).
Pattern formation and cell differentiation are thought to result from the
coordinate expression of specific combinations of regulatory genes. The
combinatorial aspect of control might be reflected at the level of chromatin
structure by the fact that a given gene, depending on the cell looked at, displays a
particular array of DNase I hypersensitive sites which are thought to correlate
with the presence of trans-acting control elements (29). From the data
summarised above, and with reference to the concept of chromosomal regulation
of gene expression (7,29), it is tempting to propose that 'finger" proteins include
RNA pol ii associated positive transcription factors whose mode of action is
analogous to that of TFIIIA. These factors would act primarily by antagonising
the binding of histone HI to the chromatin and promoting the selective formation
of "activated transcription complexes" at specific gene sites thus 'determining
these genes. Autocatalytic activation of a gene coding for a positive
transcription factor might then depend on the affinity of the protein for its own
gene control region. Finally, exists the interesting possibility that finger
proteins other than TF IIA are capable of binding to specific RNA, and mediate an
autoregulation of gene expression (14).
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Nucleic Acids Research
The multi-finger structure of the proteins considered here may be typical of
many others present in eucearyotic cells that recognise specific DNA sequences
and help to determine the temporal and spatial pattern of gene expression during
development. Isolation of several such proteins might represent an important
step towards the understanding of how DNA binding proteins act to regulate gene
expression.
ACKNOWLEDGEMENTS
thank Michael Rosbash and Frangois Schweisguth for helpful discussions and
assistance with computer searches and colleagues in J.-A. Lepesent's laboratory
for critical reading of the manuscript.
REFERENCES
1. Dynan, W.S. and Tijan, R. (1985) Nature, 313, 284-209.
2. Miller, J., McLachlan, A.D. and Kluq, A. (1985) EMBO J. 4, 1609-1614.
3. Lassar, A.B., Martin, P.L. and Roeaer, R.G. (1983) Science, 222, 740-748.
4. Bogenhaqen, D.F., Wormington, W.M. and Brown, D.D. (1982) Cell 28, 413-421.
5. Sclisse , M.S. and Brown, D.D. (1984) Cell, 37, 903-913.
6. Brown, D.D. and Schlissel, M.S. (1985) Cell 42, 759-767.
7. Brown, D.D. (1984) Cell 37,359-365.
8. Smith, D.R., Jackson, I.J. and Brown, D.D. (1984) Cell, 37, 645-652.
9. Ginsberg, A.M., King, B.O. and Roeder, R.G. (1984) Cell, 39, 479-489.
10. Brown, R.S., Sander, C. and Argos, P. ( 1985) FEBS Lett., 186, 271-274.
11. Olhendorf, D.H. and Matthews, O.W. (1983) Annu. Rev. Biophys. Bioeng., 12,
259-284.
12. Rhodes, D. (1985) EMBO J., 4, 3473-3482.
13. Vincent, A., Colot, H.V., and Rosbash, M. (1 985) J. Mol. Biol.., 186, 149-166.
14. Rosenberg, U.B., SchrOder, C., Preiss, A., Kienlin, A., Cote, S., Riede, I. and
Jackle, H. (1986) Nature, 319, 336-339.
15. Mount, S.M. and Rubin, G.M. ( 1985) Mol. Cell. Biol., 5, 1630-1638.
16. Covey, S.N. (1966) Nucl. Acids Res., 14J 623-633.
17. Hollenberg, S.M., Weinberger, C., Ong, E.S., CerellI, G., Oro, A., Lebo, R.,
Thompson, E.B., Rosenfeld, 1l.G. and Evans, R.M. ( 1985) Nature, 318, 635-641.
18. Green, G.L., Gilna, Pl., Waterfield, M., Baker, A., Hort, Y. and Shine, J. (1986)
Science, 231J 1150-1154.
19. Green, S., Walter P., Kumar, V., Krust, A., Bornert, J.M., Argos, P. and
Chambon; P. (19866 Nature, 320J,134-140.
20. Krust, A., Green, S., Argos, P., Kumar, V., Walter, P., Bornert, J.M., and
Chambon, P. ( 1 986) EMBO J.J in press.
21. Kagi, J.H.R. and Nordberg, M. (1979) Metallothionein (Birkhauser Verlag,
Basel).
22. Nusslein-Volhard, C. and Wieschaus, E. (1980) Nature, 287, 283-295.
23. Pabo, C.O. and Sauer, R.T. ( 1984) X. Rev. Biochem., 52, 293-32 1.
24. Loughton, A. and Scott, M.P. ( 1984) Nature, 310, 25-31.
25. Miller, A.M.., MacKay, V.L. and Nasmyth, K.A. (1985) Nature, 314, 598-603.
26. Kmiec, E.B. and Worcel, A. (1965) Cell, 41, 945-953.
27. Knipple, D.C., Seifert, E., Rosenberg, U.B., Preiss, A. and J8ckle, H. (1985)
Nature, 317, 40-44.
28. Vincent, A., O'Connell, P.O.C., Gray, M. and Rosbash, M. (1984) EMBO J.,
3,1003-1013.
29. Weintraub, H. (1985) Cell, 42, 705-71 1.
30. Hartshorne, T.A., Blumberg, H. and Young, E.T. (1986) Nature, 320 ,283-287.
4391