Expression of the carboxypeptidase E gene: characterization of the initiator-binding proteins.
Several of the genes for enzymes involved in peptide hormone processing, such as carboxypeptidase E (CPE), do not contain a TATA box. The region surrounding the major transcription initiation site of the CPE gene has sequence homology with the 'initiator' (Inr) elements of the TATA-less terminal deoxynucleotidyltransferase (TdT) gene, and the adenovirus major late (AdML) and other promoters. To investigate the promoter region of the CPE gene, GH4C1 cells were transiently transfected with constructs containing the luciferase reporter gene attached to various portions of the rat CPE gene (-395 to +45). Positive regulator elements were detected in positions -84 to -12 and +30 to +47. However, the Inr-like element of the CPE gene (-12 to +20) produced detectable luciferase activity in the absence of upstream and downstream sequences. This region of the CPE gene was much more active when expressed in the normal (sense) orientation than when expressed in the antisense orientation. A mutation within the consensus sequence between CPE and other Inr elements was much less active than the wild-type sequence. Interestingly, deletion of the Inr and surrounding sequences produced a large increase in the transcription from upstream sites, suggesting that proteins which bind at, or near, the Inr sequence suppress transcription from other sites. To characterize GH4C1 nuclear proteins which bind to the CPE gene, Southwestern blotting, UV cross-linking, and gel shift analyses were performed. The Southwestern analysis showed that the CPE and AdML Inr sequences labeled several proteins of similar sizes which are distinct from the transcription factor USF; this factor has been previously reported to bind to the AdML Inr sequence. A CPE Inr-binding protein co-purifies with an AdML Inr-binding protein on a CPE Inr affinity column. Gel shift assays showed that with some binding conditions, the proteins that bind to the CPE sequence also bind to the TdT and AdML Inr elements. Taken together, these results indicate that the -12 to +20 region of the CPE gene has the properties of an Inr element which binds some, but not all, of the factors which bind to other Inr elements.
Jung
Y K
YK
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461.
Fricker
L D
LD
D000818:Animals; D001483:Base Sequence; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000096:biosynthesis*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D013379:Substrate Specificity; D016385:TATA Box; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D002268:Carboxypeptidases; D043423:Carboxypeptidase H