Characterization of an antisense Inr element in the eIF-2 alpha gene.
We recently discovered an opposing initiator promoter (Inr) downstream of the sense promoter region of the eIF-2 alpha gene (Silverman, T., Noguchi, M., and Safer, B. (1992) J. Biol. Chem. 267, 9738-9742). By reverse transcriptase/polymerase chain reaction analysis of G0 and activated (G1) T-lymphocyte RNAs, overlapping sense and antisense transcripts are now identified. Sense transcription of the eIF-2 alpha gene proceeds from left to right to generate alpha-mRNA; antisense transcription proceeds from right to left to generate RNA, having a sequence complementary to eIF-2 alpha mRNA. Upstream indicates a position 5' relative to the transcription start site. Using DNase I footprint analysis and EMSA, we have found a potential cis-regulatory sequence immediately upstream of the Inr which binds a 43-kDa protein. In addition to conferring protection against DNase I (+457 to +474), the factor also generates hypersensitive sites directly over the Inr (+447 to +457). Insertion of the Inr footprint region into a luciferase reporter gene construct increases expression 150-fold. While mutation of the Inr conserved sequence decreases luciferase activity by 50%, mutation of the 43-kDa factor binding site inhibits luciferase activity by 20%. Sense orientation of the Inr footprint region decreases activity by 80%. The 43-kDa Inr-associated binding protein may be involved in allowing access of RNA polymerase II transcription complexes ot the initiation site of this TATA-less gene. A model for the regulation of eIF-2 alpha expression involving the rapid degradation of dsRNA generated by the relative activities of the two overlapping and opposing promoters is proposed.
Noguchi
M
M
Molecular Hematology Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Miyamoto
S
S
Silverman
T A
TA
Safer
B
B
D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D003850:Deoxyribonuclease I; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid
D017931:DNA Primers; D015852:Eukaryotic Initiation Factor-2; D016372:RNA, Antisense; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D003850:Deoxyribonuclease I