Ferritin mRNA: interactions of iron regulatory element with translational regulator protein P-90 and the effect on base-paired flanking regions.
The ferritin iron regulatory element (IRE), a conserved sequence of 28 nucleotides in a hairpin loop, is a conserved mRNA-specific translational regulatory element; flanking the IRE are regions of varying sequence, which form 9-17 base pairs close to the 5' cap. P-90 is a ferritin mRNA-specific translation regulatory protein purified from animal liver and reticulocytes. To study the P-90-RNA interaction, protein nucleases (RNase S1 and T1) and chemical nucleases FeEDTA and/or 1,10-phenanthroline-Cu were used as probes of an oligonucleotide (n = 55), containing the IRE and flanking regions (FL), and natural ferritin mRNA. Footprints and "toeprints" showed that P-90 binding was confined to the stem and loop of the IRE itself. However, P-90 altered the structure of the flanking region by increasing base stacking or helicity (RNase V1 sensitivity). Comparison of the reactivity of the IRE and flanking regions in natural mRNA and the 55-mer showed that long-range interactions included protecting bulges, single-stranded, and stacked regions from protein nucleases as well as stabilizing the P-90-RNA interaction. Structural integration of the IRE with the base-paired flanking regions was indicated by common features of reactivity (periodic hypersensitivity to FeEDTA) and changes in the FL region caused by P-90. The increased secondary structure of the IRE flanking regions caused by P-90 binding to the IRE provides a likely mechanism for blocking initiation of ferritin mRNA translation, since the combined structure (IRE + FL) is so close (8-17 nucleotides) to the cap.
Harrell
C M
CM
Department of Biochemistry, North Carolina State University, Raleigh 27695-7622.
McKenzie
A R
AR
Patino
M M
MM
Walden
W E
WE
Theil
E C
EC
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004492:Edetic Acid; D005290:Ferric Compounds; D005293:Ferritins / Q000235:genetics*; D005786:Gene Expression Regulation; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D010618:Phenanthrolines; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D012045:Regulatory Sequences, Nucleic Acid; D006163:Ribonuclease T1 / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism
D005290:Ferric Compounds; D010618:Phenanthrolines; D011506:Proteins; D012333:RNA, Messenger; D005293:Ferritins; D004492:Edetic Acid; D007501:Iron; D012260:Ribonucleases; D006163:Ribonuclease T1; C022542:ribonuclease S; C019179:Fe(III)-EDTA; C025205:1,10-phenanthroline