THE IMPORTANCE OF A SINGLE-G IN THE HAIRPIN LOOP OF THE IRON-RESPONSIVE ELEMENT (IRE) IN FERRITIN MESSENGER-RNA FOR STRUCTURE - AN NMR-SPECTROSCOPY STUDY

Noncoding sequences regulate the function of mRNA and DNA. In animal mRNAs, iron responsive elements (IREs) regulate the synthesis of proteins for iron storage, uptake and red cell heme formation. Folding of the IRE was indicated previously by reactivity with chemical and enzymatic probes. H-1- and P-31-NMR spectra now confirm the IRE folding; an atypical P-31-spectrum, differential accessibility of imino protons to solvents, multiple long-range NOEs and heat stable subdomains were observed. Biphasic hyperchromic transitions occurred (52 and 73 degrees C). A G-C base pair occurs in the hairpin loop (HL) (based on dimethylsulfate, RNAase T-1 previously used, and changes in NMR imino proton resonances typical of G-C base pairs after G/A substitution). Mutation of the hairpin loop also decreased temperature stability and changed the P-31-NMR spectrum; regulation and protein (IRP) binding were previously shown to change. Alteration of IRE structure shown by NMR spectroscopy, occurred at temperatures used in studies of IRE function, explaining loss of IRP binding. The effect of the HL mutation on the IRE emphasizes the importance of HL structure in other mRNAs, viral RNAs (e.g. HIV-TAR), and ribozymes.