Structure of the 5' untranslated regulatory region of ferritin mRNA studied in solution.
Ferritin mRNAs are the first eukaryotic mRNAs for which a conserved, translational regulatory sequence has been identified. The sequence of twenty-eight nucleotides, called the IRE (iron regulatory element), is found in the 5'-noncoding region and is required for enhanced translation of ferritin mRNA by excess cellular iron; regulation occurs at initiation. The prediction of secondary structure in the IRE is a hairpin loop. We now report an analysis of the IRE structure in solution studied in natural ferritin mRNAs [H and H'(M) subunits] by primer extension, after modification or cleavage by dimethyl sulfate, RNAases T1 and V1, and the chemical nuclease 1, 10-phenanthroline-copper (OPCu) which cleaves single-stranded and bulged regions of RNA. Overall, the structure in solution of the ferritin mRNA regulatory region is a hairpin loop, with magnesium-sensitive features, in which half the stem is provided by the IRE and half by flanking regions; only secondary structure is conserved in the flanking regions. Predicted bulges or internal loops along the stem were clearly detected by OPCu but were missed by the more bulky probe RNAase T1, indicating the efficacy of OPCu in probing subtle features of RNA structure. Magnesium-dependent deviations from the predicted structure were observed in the stem between the hairpin loop and the bulge at C6. The location of the IRE in relation to the initiator AUG or the cap is variable in different ferritin mRNAs. However, the number of nucleotides in the base-paired flanking regions of known ferritin mRNAs is proportional to the distance of the IRE from the cap and places the secondary/tertiary structure 8-10 nucleotides from the cap where interference with initiation is likely.
Wang
Y H
YH
Department of Biochemistry, North Carolina State University, Raleigh 27695-7622.
Sczekan
S R
SR
Theil
E C
EC
D001483:Base Sequence; D005293:Ferritins / Q000235:genetics*; D007438:Introns; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012330:RNA, Double-Stranded; D012333:RNA, Messenger / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012996:Solutions
D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012996:Solutions; D005293:Ferritins; D008274:Magnesium