Characterization of the human ADP-ribosylation factor 3 promoter. Transcriptional regulation of a TATA-less promoter.
The 5'-flanking region of the human ADP-ribosylation factor 3 gene contains the features of a housekeeping gene. It lacks a TATA or CAAT box, has several GC boxes within a highly GC-rich region, and utilizes multiple transcription initiation sites. The cis-acting elements involved in regulating expression of the gene were identified by transient transfections of IMR-32 neuroblastoma cells. Reporter plasmids were modified to facilitate construction of defined promoter deletions linked to chloramphenicol acetyltransferase or luciferase using ligation-independent cloning. Transfection analyses indicated that sequences within 58 base pairs of the transcription initiation site were necessary for full expression, in particular a sequence containing the 10-base pair palindrome TCTCGCGAGA. Electrophoretic mobility shift assays performed with IMR-32 nuclear extracts demonstrated that a DNA-binding protein, termed TLTF, bound to an oligonucleotide containing this palindrome. Competition experiments showed that mutations within the core of the palindrome abolished in vitro binding and that the same protein bound to a 5'-proximal sequence. Expression of the promoter containing a mutated palindrome was reduced dramatically, consistent with the conclusion that this region functions in vivo to control expression of the ARF3 gene.
Haun
R S
RS
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Moss
J
J
Vaughan
M
M
D020727:ADP-Ribosylation Factors; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D016385:TATA Box; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004247:DNA; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors