Fig 1.
Functional and non-functional activation domains in Gal4 activator.
The Gal4—LexA hybrid constructs (BTM116 backbone) were assayed in L40 strain for activation of transcription. To distinguish activity of the N- and C-terminal activation domains in the Gal4 protein, a set of the Gal4 constructs were generated. The average value of the β-galactosidase activities from three independent experiments is presented as a percentage of the reference with standard deviation (means and plusmn; SD; n = 3). We standardized all results to previously reported Gal4 construct HaY including merely the activation domain 9aaTAD with the activity set to 100% [34]. The LexA is E.coli DNA binding domain and the Gal4 DBD is S.cerevisiae Gal4 DNA binding domain, both generally used for the generation of fusion hybrids. The regions of Gal4 protein in the constructs are noted and graphically presented. Single dot means end of protein sequence, tree points mean continuing of the sequence, which is no more shown.
Fig 2.
Artificial peptides in the original Gal4 constructs activated transcription.
To distinguish natural and artificial activity, a sets of LexA-Gal4 hybrid constructs were generated with and without artificial peptides presented in original constructs, an unintended consequence of cloning [24]. The LexA-Gal4 hybrid constructs assayed in L40 strain for transactivation activity are shown. The average value of the β-galactosidase activities from three independent experiments is presented as a percentage of the reference with standard deviation (means and plusmn; SD; n = 3). We standardized all results to previously reported Gal4 construct HaY including merely the activation domain 9aaTAD with the activity set to 100% [34]. The protein sequences are partially given by amino acid single letter code. Single dot means end of protein sequence, tree points mean continuing of the sequence, which is not more shown. The regions of Gal4 protein in the constructs are noted and graphically presented.
Fig 3.
The activation domain AD-III did not activate transcription.
The LexA-Gal4 hybrid constructs with activation domains AD-III, 9aaTAD and their artificial variants were assayed in L40 strain for activation of transcription. Gal4 DBD is Gal4 DNA binding domain. The red marks part of the artificial activation domain 9aaTAD (1/2 9aaTAD) and the blue marks part of the natural activation domain 9aaTAD (1/2 9aaTAD). Both these parts generated strong activators in the fusion with the Gal4 DNA binding domain, Gal4 DBD (83–100 aa), which served as other part of the artificial activation domain 9aaTAD (the second 1/2 9aaTAD). The artefact explained previous cloning errors of the reported construct pRJR200 with activation domain AD-III [31]. The impact of proximal amino acids on the function of the activation domain 9aaTAD was tested. The related constructs with and without activation domains AD-III and 9aaTAD are shown again to overview their activities (G81, G49, G59, G42, G405). The average value of the β-galactosidase activities from three independent experiments is presented as a percentage of the reference with standard deviation (means and plusmn; SD; n = 3). We standardized all results to previously reported Gal4 construct HaY including merely the activation domain 9aaTAD with the activity set to 100% [34]. The regions of Gal4 protein in the constructs are noted and graphically presented. The artificial activation domain AD-II is not more shown in this figure. The protein sequences are partially given by amino acid single letter code. Single dot means end of protein sequence, tree points mean continuing of the sequence, which is not more shown.
Fig 4.
Schema of the Gal4 regulation.
Competition of Gal4 inhibitor Gal80 with mediators of transcription for the 9aaTAD domain. The peptides from S.c.Gal4 and K.l.Gal9 (Gal4 ortholog) interacting with Gal80 are shown (structural data for peptides interaction at PDB accession code 3E1K and 3BTS). The positions of activation domains 9aaTAD in the Gal80 binding peptides are highlighted (14 amino acid long Gal4 region is needed for maximal activation of transcription, construct H577, including the nine amino acid long 9aaTAD motif and four adjacent amino acids to the N-terminus and one to the C-terminus of the activation domain 9aaTAD). The artificial activation domains AD-I, AD-II and AD-III are not more shown in this figure.