Structural consequences of base modifications and metal iron interactions on the anticodon stem-loop from tRNA(Phe) (Escherichia coli)
Nikonowicz, Edward P.
Doctor of Philosophy thesis
The structural effects of naturally modified bases of RNA are mostly uncharacterized. The structural effects of two base modifications and of cations in the anticodon stem loop of tRNAPhe (Escherichia coli) were studied on a 17 mer RNA (ACSLPhe) by NMR spectroscopy. The anticodon region in fully modified tRNAs is proposed to be in a U-turn conformation. Solution NMR studies of unmodified tRNA anticodon stem-loops suggest that base modifications and divalent cations have a minor impact on the structure of the anticodon loop. The unmodified ACSL Phe contains two extra base pairs, 32--38 and 33--37, precluding a U-turn conformation. ACSLPhe contains a tri-nucleotide loop-composed of the anticodon residues. The attachment of a dimethylallyl group at the amino nitrogen of A37 (i6A37) has been proposed to increase the stacking ability of the anticodon residues and residue 37. In the i6A37 containing molecule (i6 A37-ACSLPhe), base pair 33--37 is broken, and base pair 32--38 is destabilized. Several loop resonances enter a regime of intermediate exchange. The presence of Mg2+ has been proposed to stabilize the U-turn in other studies. Mg2+ opens the loop region of ACSLPhe and makes it multi-conformational. Mg 2+ causes a peak pattern that resembles that of i6A37-ACSL Phe and stabilizes the open conformation of i6A37-ACSL Phe. However, i6A37-ACSLPhe in the presence of Mg2+ does not form a stable U-turn. Pseudouridine at position 32 (Psi32) is another naturally occurring modification on tRNAPhe. Psi32 on ACSLPhe (Psi32-ACSL Phe) increases the stability of the stem and is base paired in anti conformation with A38. The structure of Psi32-ACSL Phe is essentially the same as that of ACSLPhe. Two ion probes for Mg2+ binding sites, Mn2+ and Co (NH3)63+, were tested on ACSL Phe and i6A37-ACSLPhe. Both ions bind to the 5' side of the stem regions and weakly to the loop regions. Co (NH3)63+ induces a U-turn conformation in i6A37-ACSLPhe and ACSL Phe but in the latter case a second conformation coexists. It is concluded that the dimethylallyl modification and cations act synergistically in the stabilization of the U-turn. It is proposed that the greater charge density of Co(NH3)63+ relative to Mg2+ is responsible for the formation of a stable U-turn.
Molecular biology; Biochemistry; Biophysics