Identification and characterization of a novel transcription factor participating in the expression of eukaryotic initiation factor 2 alpha.
We have recently cloned and characterized the promoter region of the gene encoding eukaryotic initiation factor 2 alpha (eIF-2 alpha) to identify regulatory elements of this housekeeping gene. We compared the location of DNase I-hypersensitive (HS) sites with the distribution of protein-binding sites as revealed by footprint analysis. The eIF-2 alpha promoter contains four upstream DNase I-HS sites extending from -650 to -40 base pairs and a fifth downstream site near the first intron-exon junction. In vitro DNase I footprint analysis shows eight distinct DNA-protein interactions organized into clusters that correspond well with the distribution of the five HS sites. None of the protected regions, however, shares obvious sequence homology with the binding sites of known regulatory factors. To initiate our analysis of factors required for eIF-2 alpha expression, selected a CAP-proximal element shown by in vivo methylation protection analysis to bind a potential regulatory factor. A striking feature of this element is its palindrome sequences and eight-base pair direct repeats. We have purified to near homogeneity a 66-68-kDa protein that binds to this region and have designated it alpha-PAL. The alpha-PAL-binding site extends from -74 to -10. By methylation protection analysis and mobility shift assay, the alpha-PAL-binding site is shown to be two adjacent sites, one with high and one with lower affinity, which bind alpha-PAL in a noncooperative manner. When the high affinity binding site is cloned upstream of the adenovirus 2 core promoter, in vitro transcription is stimulated 2-3-fold. When linked to a CAT reporter gene, activity of the eIF-2 alpha promoter shows an approximate 2-fold dependence on the alpha-PAL element.
Jacob
W F
WF
Laboratory of Molecular Hematology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Silverman
T A
TA
Cohen
R B
RB
Safer
B
B
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D003850:Deoxyribonuclease I; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics*; D015870:Gene Expression; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D010957:Plasmids; D015183:Restriction Mapping; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D004273:DNA, Neoplasm; D015852:Eukaryotic Initiation Factor-2; D015345:Oligonucleotide Probes; D014157:Transcription Factors; D003850:Deoxyribonuclease I