AUA as a translation initiation site in vitro for the human transcription factor Sp3.
Sp3 is a bifunctional transcription factor that has been reported to stimulate or repress the transcription of numerous genes. Although the size of Sp3 mRNA is 4.0 kb, the size of the known Sp3 cDNA sequence is 3.6 kb. Thus, Sp3 functional studies have been performed with an artificially introduced start codon, and thus an aminoterminus that differs from the wild-type. Ideally, full-length cDNA expression vectors with the appropriate start codon should be utilized for these studies. Using 5'rapid amplification of cDNA ends, a full-length Sp3 cDNA clone was generated and the sequence verified in nine cell lines. No AUG initiation codon was present. However, stop codons were present in all three frames 5' to the known coding sequence. In vitro translation of this full-length cDNA clone produced the expected three isoforms-one at 100 kDa and two in the mid 60 kDa range. Electrophoretic mobility shift assays showed that the protein products had the ability to bind to the Sp1/3 consensus sequence. In vitro studies, using our Sp3 clone and site directed mutagenesis, identified the translation initiation site for the larger isoform as AUA. AUA has not been previously described as an endogenous initiation codon in eukaryotes.
Hernandez
Eric Moore
EM
Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20007, USA.
Johnson
Anna
A
Notario
Vicente
V
Chen
Andrew
A
Richert
John R
JR
D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018387:Codon, Initiator / Q000235:genetics*; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis / Q000235:genetics*; D011817:Rabbits; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D018387:Codon, Initiator; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C497121:SP3 protein, human; D014157:Transcription Factors; D051705:Sp3 Transcription Factor