Mutational analysis of AREA, a transcriptional activator mediating nitrogen metabolite repression in Aspergillus nidulans and a member of the "streetwise" GATA family of transcription factors

Abstract

The transcriptional activator AREA is a member of the GATA family of transcription factors and mediates nitrogen metabolite repression in the fungus Aspergillus nidulans. The nutritional versatility of A. nidulans and its amenability to classical and reverse genetic manipulations make the AREA DNA binding domain (DBD) a useful model for analyzing GATA family DBDs, particularly as structures of two AREA-DNA complexes have been determined. The 109 extant mutant forms of the AREA DBD surveyed here constitute one of the highest totals of eukaryotic transcription factor DBD mutants, are discussed in light of the roles of individual residues, and are compared to corresponding mutant sequence changes in other fungal GATA factor DBDs. Other topics include delineation of the DBD using both homology and mutational truncation, use of frameshift reversion to detect regions of tolerance to mutational change, the finding that duplication of the DBD can apparently enhance AREA function, and use of the AREA system to analyze a vertebrate GATA factor DBD. Some major points to emerge from work on the AREA DBD are (i) tolerance to sequence change (with retention of function) is surprisingly great, (ii) mutational changes in a transcription factor can have widely differing, even opposing, effects on expression of different structural genes so that monitoring expression of one or even several structural genes can be insufficient and possibly misleading, and (iii) a mutational change altering local hydrophobic packing and DNA binding target specificity can markedly influence the behavior of mutational changes elsewhere in the DBD.

FIG. 1

Extant loss-of-function single-residue substitutions and single- or contiguous-residue deletions within residues 665 to 728 (numbered as in reference and EMBL entry X52491) of AREA (2, 19, 42, 45, 59, 60, 62, 63). The region of GATA factor homology is boldfaced. Zinc-chelating Cys residues are underlined. Short raised vertical lines indicate residues 675, 685, 695, 705, 715, and 725. Raised horizontal lines indicate the extents of β-strands 1 to 4, the alpha-helix, and the extended loop (76). Residues involved in DNA contacts (76) are indicated as follows: major groove, filled symbols; minor groove, open symbols; hydrophobic interactions, circles; electrostatic interactions, squares; hydrogen bonds, triangles; interactions involving backbone amide or alpha protons, diamonds. Single- or contiguous-residue substitutions or deletions (Δ) resulting in loss of AREA function are indicated below the sequence. The double-headed arrow from Arg-685 indicates that both R685L and R685Q substitutions abolish function. The quadruple-tailed arrow from residues 673 to 676 indicates that deletion of these four adjacent residues leads to loss of function. Certain of these sequence changes have been obtained in strains carrying other areA mutations from which they have not been separated and which might influence their phenotypes: C673R, R685L, and N695S in an areA1601 (75) background; Q691P and G698D in an L683V (7, 42, 45) background; and L693P in a ΔKTD713-715 (52) background. A696P results in loss of function in both wild-type (Leu-683) and L683V backgrounds (59).

FIG. 2

(A) Extant functional single-residue substitutions and single- or contiguous-residue deletions within residues 665 to 728 of AREA (45, 59, 65, 86). Notation and structural information are in the legend to Fig. 1. (B) Extant functional substitutions and deletions in an L683V background (59, 85). The sequence includes the mutant Val residue at position 683 with the wild-type Leu residue shown above.

FIG. 3

(A) Revertants of G698D in an L683V background (10): (B) revertants of Q691P in an L683V background (10); (C) revertants of P709L (59, 62). Notation follows that for Fig. 2B.

FIG. 4

(A) Revertants of A696P in a wild-type (Leu-683) background (59); (B) revertants of A696P in an L683V background (10, 42). Notation follows that for Fig. 2B.

FIG. 5

(A) Second-generation revertant of P682S L683V A696P (10); (B) Second- and third-generation revertants of L683V P692R A696P (10). The third-generation revertants are derived from the second-generation revertant L683M P692R A696P. Notation follows that for Fig. 2B.

FIG. 6

AREA sequence changes (boxed) resulting from first- and second-generation reversions of a −1 frameshift mutation in codon 688 (84). The bottom sequence is that for a second-generation revertant of the revertant whose sequence is directly above. Other second-generation revertants from the revertant in which LKE replaces PEGQ contain one of the following second-site suppressor substitutions (not shown): F700Y, V707M, or K713R. Structural information at the top is as in Fig. 1.

FIG. 7

(A) AREA sequence changes resulting from first- and second-generation reversions of a −1 frameshift mutation in codon 703 in an L683V background (84), displayed as in Fig. 6. Second-generation revertants of the L683V ΔL703 mutant are the L683V 700+V ΔL703, L683V ΔL703 H704R, and L683V ΔL703 K713R mutants plus the duplication mutant described in the text. Second-generation revertants of the L683V L701F L703V mutant are the L683V L701F, L683V F700V L701F L703V, N675Y L683V L701F L703V (not shown), and L683M L701F L703V (not shown) mutants. Second-generation revertants of L683V L703F H704T are the L683V F700Y L703F H704T and L683V L703F H704R mutants. N675I L683V L703V (not shown) is a second-generation revertant of L683V L703V. (B) Mutational changes (functional) in panel A separated from L683V (and therefore in a Leu-683 background) by genetic recombination (84), displayed as in Fig. 6. An N675I L703V double mutant was identified among progeny of a cross designed to separate N675I from L683V and L703V in the second-generation revertant N675I L683V L703V. It must have arisen either by a double-recombination event (involving intervals of 21 and 58 bp) or by reversion of the mutation responsible for the L683V change. The lack of recovery of the single change N675I, by what should have been a more frequent recombination event, suggests that N675I might result in a nonfunctional AREA protein, particularly as we have also been unable to obtain an N675I recombinant by outcrossing an N675I L703V strain in which the (single) relevant recombination interval would be 80 bp.