Abstract. Two similarities among transcriptional activat- ing regions of many eukaryotic transcription factors, like those from GAL4, GCN4, and VPI 6, are that they ...
Chromosoma (1992) 101 : 342-348
CHROMOSOMA 9 Springer-Verlag 1992
Activating regions of yeast transcription factors must have both acidic and hydrophobic amino acids Douglas M. Ruden* Department of Biochemistryand Molecular Biology,Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA ReceivedAugust 4, 1991 Accepted August 10, 1991 by H. J/ickle Abstract. Two similarities among transcriptional activating regions of many eukaryotic transcription factors, like those from GAL4, GCN4, and VPI 6, are that they have a net negative charge, and that many of them can potentially form amphipathic e-helices with acidic amino acids on the hydrophilic face. Based on these similarities, E. Giniger ar/d M. Ptashne previously designed a short peptide (AH) which is predicted to have the potential to form a negatively charged amphipathic e-helix; AH was able to mediate transcription activation in yeast when it was attached to the D N A binding and dimerization portion of GAL4 [GAL4(l-147)]. This paper describes screening of a pool of AH derivatives containing randomized amino acids fused to GAL4(1-147) and to an analogous region of LexA [LexA(1-87)] in yeast strains. Results suggest that both acidic and hydrophobic amino acids are critical features of activating regons these results are consistent with the model that activating regions often form amphipathic e-helices. This work is novel because hydrophobic amino acids are also shown to be important in activating regions of yeast transcription factors.
Introduction Transcriptional activators in eukaryotes are composed of two components: a sequence-specific DNA-binding region (generally a dimer) and an activating region which is thought to interact with a general transcription factor (reviewed by Ptashne 1988; Mitchell and Tjian 1989; Ptashne and Gann 1990). GAL4, an 881 amino acid protein, is an activator of the galactose metabolism genes in yeast (reviewed by Johnston 1987). The D N A binding and dimerization region of GAL4 is located within residues 1 147, and the activating region within residues 148-881 (Carey et al. 1990; Ma and Ptashne * Present address: Max-Planck-Institut ffir biophysikalische Chemie, Abt. 170, Postfach 2841, W-3400 G6ttingen, Federal Republic of Germany
1987 a). When the activating region of GAL4 is attached to the DNA binding domain of LexA [LexA [LexA(l87)+GAL4(74-881)], this protein activates transcription in yeast provided that a LexA operator is present in a yeast promoter (Brent and Ptashne 1985). It has been demonstrated that activating regions can be readily generated by fusing Escherichia coli genomic D N A fragments to GAL4 [GAL4(1-I47)] or LexA [LexA(1-202)] (Ma and Ptashne 1987b; Ruden et al. 1991). The E. coli-derived activating regions can also functionally replace the activating region of the developmentally important Drosophila protein bicoid (Driever et al. 1989). A similarity among the E. coli-derived activating regions isolated from GAL4 and LexA fusion proteins and many natural activating regions, such as those from yeast activators GAL4 and GCN4 and Herpes viral activator VP16, are that they have a net negative charge (Ma and Ptashne 1987a, Hope and Struhl 1986; Cress and Triezenberg 1991). A second similarity among activating regions is that many can potentially form amphipathic e-helices with the acidic amino acids on the hydrophilic face (Ma and Ptashne 1987b; Hope et al. 1988). Based on these similarities, Giniger and Ptashne (1987) designed an acidic peptide (AH), NH2-[(Glu-Leu-Gln)3-Ala-Leu2-Glna]-COOH, which is predicted to form such an amphipathic e-helix. When attached to GAL4(1-147), AH mediated the activation of transcription of the GALI gene which had four GAL4 binding sites in an upstream activating sequence [ UAS~]. An AH derivative that consisted of the same amino acids as AH but in a scrambled order failed to mediate the activation of transcription in vivo, again suggesting the importance of structure in activating regions (Giniger and Ptashne 1987). In this study, by randomizing the sequence in AH, I provide evidence that yeast activators must have both acidic and hydrophobic amino acids in their activating region, presumably to form amphipathic e-helices. The advantage of this study over previous studies is that statistical arguments are made showing not only the importance of acidic but also hydrophobic amino acids in yeast transcription factors.
343 Materials and methods
Strains and media. Yeast strain [YT6:pRYI71(MATa gal4-542 gal80-538 ura3-52 his3-200 ade2-101 adel lys2-801 trpl-901 arol leu2-3-112, mell, Met-), Himmelfarb et al. 1990] contained, integrated at the URA3 locus, a GALI-lacZ fusion gene. Strain YT6:LexAl(OPx2,+36) contained, integrated at the URA3 locus, a GALI-lacZ fusion gene in which the GALl UAS~ was replaced with two LexA operators (Ruden et al. 1991). Yeast were grown in standard minimal media lacking uracil and histidine and containing 3% (v/v) glycerol and 2% (w/v) galactose and fl-galactosidase activity was measured as previously described (Ma and Ptashne 1987a). Randomized AH derivatives that were able to activate transcription in yeast were screened by transforming pools of clones into the appropiate yeast strain, and plating on yeast minimal plates lacking histidine and uridine and containing 2% (w/v) gluocose (Sherman et al. 1983). The yeast colonies were then replica-plated onto yeast minimal plates lacking histidine and uridine and containing 1 x BU [0.1 M phosphate buffer, pH 6], 3% (v/v) glycerol, 2% (w/v) galactose, and 200 I~g/ml X-gal (5-bromo4-chloro-3-indol-fl-o-galactoside;(Ma and Ptashne 1987b). If the AH derivative mediated the activation of transcription, the colony containing that derivative turned blue within 1 to 3 days. Plasmid constructions. The randomized AH cassettes shown in Fig. 1 were ligated into the yeast expression vectors MA424 and LexA (1-87)+ PL; which had been previously digested with restriction endonucleases EcoRI and BamHI; these generated GAL4(1147) and LexA(l-87) fusions, respectively. All oligonucleotides were synthesized and HPLC purified by Operon Technology, unless otherwise indicated. MA424 has been described previously (Ma and Ptashne 1987b); LexA(1-87)+PL' is identical to MA424 except that sequences encoding the first 147 amino acids of GAL4 have been replaced with sequences encoding the first 87 amino acids of LexA. These plasmids express the proteins from the strong ADHI yeast promoter and transcription is terminated from the ADH1 terminator. The DNA sequence of LexA(1-87)+ PL' from the codon for the 87th amino acid of LexA (italicized) is: 5' CCT CGA CCG [GAATTC] R CCGG [GGATCC] B [GTCGAC] s [CTGCAG]P; where ' R ' is the recognition sequence for the restriction endonuclease EcoRI, ' B ' for BamHI, ' S ' for SalI, and ' P ' for Pstl; as with the parent vector MA424, the EcoRI, BamHI and SalI sites are unique (Ma and Ptashne 1987b). The protein sequence of LexA(1 87)+ AH from amino acid 87 to the carboxyl-
terminus, inclusive, is: NHz-Pro-[Arg-Pro-Glu-Phe-Pro-Glu-Ile](Glu-Leu-Gln)3-Ala-Leuz-Gln3-COOH; the seven amino acids in brackets are encoded by polylinker sequences and are present in all LexA + AH derivatives.
Results
T h e f o l l o w i n g e x p e r i m e n t s were d o n e to d e t e r m i n e the i m p o r t a n c e o f the v a r i o u s a m i n o acids in A H . F o r instance, if the acidic a m i n o acids were i m p o r t a n t for activ a t i n g f u n c t i o n , t h e n t h r o u g h screening for a c t i v a t o r s t h a t h a v e all o f the acidic a m i n o acids in A H r a n d o m ized, o n l y t h o s e derivatives t h a t h a v e acidic a m i n o acids s h o u l d be i s o l a t e d (Fig. 1 A). Likewise, if the h y d r o p h o b ic a m i n o acids o f A H were i m p o r t a n t , t h e n t h r o u g h screening for a c t i v a t o r s t h a t h a v e the h y d r o p h o b i c amin o acids r a n d o m i z e d , o n l y t h o s e derivatives t h a t h a v e h y d r o p h o b i c a m i n o acids s h o u l d be i s o l a t e d (Fig. 1 B).
Analysis o f A H derivatives To test the a b o v e t w o ideas, t w o classes o f r a n d o m i z e d cassettes e n c o d i n g deriviates o f A H were m a d e . T h e class I cassettes e n c o d e d A H d e r i v a t i v e s t h a t h a v e the sequence NHz-[(X-Leu-Gln)3-Ala-Leu2-Glrla]-COOH , a n d the class II cassettes e n c o d e d A H derivatives t h a t have the sequence N H 2 - [ ( G l u - X - X ) a - A l a - L e u 2 - G l n 3 ] C O O H , w h e r e X r e p r e s e n t s the r a n d o m i z e d a m i n o acids (in " w i l d - t y p e " A H X = G l u in the class I cassette, a n d X - X = L e u - G l n in the class II cassette). A t o t a l o f f o u r libraries were m a d e t h a t c o n t a i n these cassettes b y fusing each o f the t w o cassettes to L e x A ( l - 8 7 ) a n d G A L 4 ( 1 147) (see Fig. l). To investigate the a m i n o a c i d requirem e n t f o r a c t i v a t i o n , the a m i n o acids f o u n d 'in a n unscreened set o f cassettes were c o m p a r e d with t h o s e f o u n d in a set o f cassettes screened for the ability to a c t i v a t e t r a n s c r i p t i o n in yeast. A c t i v a t o r s were i s o l a t e d f r o m all
A. Class I Cassettes S'-AATTCCC6666ATCI"~~ 3'............. GGGCCCCTAGII
CTT c A G F ~
CTC C A G r ~ C T 6 CAA 6CT TTG TTG CAA CAA CAA T ...........3'
I II 6AA GTCII I II GAG GTCI I I IIGAC GTT CGA AAC AAC GZZ GTT 6TT ACTA6-5' X
Leu Gin
X
Leu Gin
X Leu Sln Ale Leu Leu Gin Gin Girl Stop
Fig. 1A, B. Cassette mutagenesis of AH. Class I
B. Class II Cassettes S'-AATTCCCGGGGATCGAS ~GAG
~
GAG~
GCTTTGrrG CASCAACAGV...........~'
3'. ............. GSGCCCCTAGCTC I I t I I I qCTC [!, I I I I tlCTC I ~ ~ ~ ~ ~ IIC6A AAC AAC GVC GTT GTC ACT AG-S' Glu
X
X
Glu
X
X
GIu
X
X
Ala
Leu
Leu Gln Gin Gln Stop
I ADH 1(terminator) EeORI
BarnHI
and Class II cassettes are described in the text. The boxed regions are the randomized portions, which can encode any of the 20 amino acids. N indicates that all four standard deoxynucleotides (G, A, T, C) were incorporated in that position, S indicates that equal amounts of G and C were incorporated at that position, and I indicates that deoxyinosine was incorporated at that position. These oligonucleotides were ligated to yeast expression vectors digested with EcoRI and BamHI restriction enzymes and made either LexA(1-87) or GAL4(1-147) fusion proteins (see Materials and methods), pADH and ADHI (terminator) are the yeast ADH1 promoter and terminator, respectively
344 Table 1. Class I cassettes that were not screened for in yeast
Name 1. LexA(1-87) + I(101) 2. LexA(1-87) + I(102) 3. LexA(1-87) + I(103) 4. LexA(1-87) + I(104) 5. LexA(1-87) + I(105) 6. LexA(1-87) + I(106) 7. LexA(1-87) + I(107) 8. LexA(l-87) + I(108) 9. LexA(l-87) + I(109) 10. LexA(1-87) + I(110) 11. LexA(1-87) + I(l 11) 12. LexA(1-87) + I(112) 13. LexA(1-87) + I(113)
Charge 0 0 +1 -- 1 0 0 -- 1 0 +2 0 0 +1 -2
Activity
Amino acid sequence
