Abstract
The transcriptional factor GATA-6 gene produces two translational isoforms from a single mRNA through ribosomal leaky scanning. L-type GATA-6 has an extension of 146 amino acid residues at its amino terminus. In the extension, there is a unique PEST sequence (Glu31-Cys46), which is composed of an amino terminal Pro-rich segment and a carboxyl terminal Ser-cluster. Substitution of either half of the PEST sequence with Ala residues by cassette mutagenesis reduced the apparent molecular size of L-type GATA-6 on SDS-polyacrylamide gel-electrophoresis. However, the effect of substitution of the Pro-rich segment was much more significant; the mobility increase of the Pro-rich segment on the gel was 13% while that of the Ser-cluster was 8%. Substitution of each amino acid residue demonstrated that the effect of Pro substitution is greater than that of the Ser and Thr residues. Such increased mobility of L-type GATA-6 in the presence of a detergent may apparently correlate with the decrease in transcription activity in vivo as determined by means of luciferase reporter gene assay. The activity of ΔAla (with Ala residues instead of the PEST sequence) was reduced to one fifth of that of ΔA (with the PEST sequence). These results suggest that the PEST sequence of L-type GATA-6 does not function as a constitutive protein degradation signal, but rather plays structural and functional roles in the activation of gene expression on the GATA responsive promoter.
Highlights
The GATA DNA-binding proteins recognize a canonical DNA motif (A/T) GATA (G/A), and regulate the expression of various genes required for developmental processes and tissue-specific functions [1] [2]
Construction of a Control Expression Plasmid GATA-6 mRNA has an additional initiation codon located in frame upstream of the canonical one [7], the latter being located at a similar position in the GATA proteins [4]
It was further demonstrated that both species are not produced through proteolysis [9], or that the CTG codons located in the intra-coding region of L-type GATA-6 mRNA does not function as translational initiation sites [7]
Summary
The GATA DNA-binding proteins recognize a canonical DNA motif (A/T) GATA (G/A), and regulate the expression of various genes required for developmental processes and tissue-specific functions [1] [2]. Six GATA proteins that contain a characteristic zinc finger region having two tandem zinc fingers separated by 29 amino acid residues (CVNC-X17-CNAC)-X29-(CXNC-X17-CNAC) have been found [1] [3] [4]. This zinc finger region is commonly located on the amino-terminal side of the last half of the primary structure of GATA proteins [1]-[4]. The carboxyl-terminal zinc finger of GATA proteins is required for specific DNAbinding, whereas the amino-terminal zinc finger may interact with protein cofactors [2]. The transcriptional activation domains are located within the amino terminus of a protein [6]
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