The C-terminal subdomain makes an important contribution to the DNA binding activity of the Pax-3 paired domain.
The recognition of DNA targets by Pax-3 is achieved through the coordinate use of two distinct helix-turn-helix-based DNA-binding modules: a paired domain, composed of two structurally independent subdomains joined by a short linker, and a paired-type homeodomain. In mouse, the activity of the Pax-3 paired domain is modulated by an alternative splicing event in the paired domain linker region that generates isoforms (Q+ and Q-) with distinct C-terminal subdomain-mediated DNA-binding properties. In this study, we have used derivatives of a classical high affinity paired domain binding site (CD19-2/A) to derive an improved consensus recognition sequence for the Pax-3 C-terminal subdomain. This new consensus differs at six out of eight positions from the C-terminal subdomain recognition motif present in the parent CD19-2/A sequence, and includes a 5'-TT-3' dinucleotide at base pairs 15 and 16 that promotes high affinity binding by both Pax-3 isoforms. However, with a less favorable guanine at position 15, only the Q- isoform retains high affinity binding to this sequence, suggesting that this alternative splicing event might serve to stabilize binding to suboptimal recognition sequences. Finally, mutagenic analysis of the linker demonstrates that both the sequence and the spacing in this region contribute to the enhanced DNA-binding properties of the Pax-3/Q- isoform. Altogether, our studies establish a clear role for the Pax-3 C-terminal subdomain in DNA recognition and, thus, provide insights into an important mechanism by which Pax proteins achieve distinct target specificities.
Vogan
K J
KJ
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Gros
P
P
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005136:Eye Proteins; D018398:Homeodomain Proteins; D051379:Mice; D016297:Mutagenesis, Site-Directed; D000072041:PAX3 Transcription Factor; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000072041:PAX3 Transcription Factor; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D014157:Transcription Factors; C068686:Pax3 protein, mouse; D004247:DNA