Determinants of GATA-1 binding to DNA: the role of non-finger residues.
Mammalian GATA transcription factors are expressed in various tissues in a temporally regulated manner. The prototypic member, GATA-1, is required for normal erythroid, megakaryocytic, and mast cell development. This family of DNA-binding proteins recognizes a consensus (A/T)GATA(A/G) motif and possesses homologous DNA binding domains consisting of two zinc fingers. The C-terminal finger of GATA-1 recognizes the consensus motif with nanomolar affinities, whereas the N-terminal finger shows a binding preference for a GATC motif, albeit with much reduced affinity (Kd approximately microm). The N-terminal finger of GATA-2 also shows a preference for an AGATCT binding site, with an increased affinity attributed to N- and C-terminal flanking basic residues (Kd approximately nm). To understand the differences in the binding specificities of the N- and C-terminal zinc fingers of GATA-1, we have constructed a series of swapped domain peptides. We show that the specificity for AGATAA over AGATCT arises from the C-terminal non-finger basic domain. Thus, the N-terminal finger binds preferentially to AGATAA once appended to the C-terminal arm of the C-terminal finger. We further show that this specificity arises from the highly conserved QTRNRK residues. The converse is, however, untrue in the case of the C-terminal finger; swapping of QTRNRK with the corresponding LVSKRA does not switch the DNA binding specificity from AGATAA to AGATCT. These results highlight the important role of residues adjacent to the CXXCX17CNAC zinc finger motif (i.e. non-finger residues) in the specific recognition of DNA residues.
Ghirlando
Rodolfo
R
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA. rodolfog@intra.niddk.nih.gov
Trainor
Cecelia D
CD
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D050989:GATA2 Transcription Factor; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050989:GATA2 Transcription Factor; D010455:Peptides; D014157:Transcription Factors; D004247:DNA