5-Methylcytidine is required for cooperative binding of magnesium(2+) and a conformational transition at the anticodon stem-loop of yeast phenylalanine tRNA

Abstract

The role of modified nucleosides in tRNA structure and ion binding has been investigated with chemically synthesized RNAs corresponding to the yeast tRNA(Phe) anticodon stem and loop (tRNA(ACPhe). Incorporation of d(m5C) at position 14 of the stem of tRNA(ACPhe)-d(m5C14), CCAGACUGAAGAU-d(m5C14)-UGG, analogous to m5C40 in native tRNA(Phe), introduced a strong Mg2+ binding at a site distant from the m5C. A Mg(2+)-induced structural transition, detected by circular dichroism spectroscopy, was similar to that observed for the DNA analog of tRNA(ACPhe) (Guenther et al., 1992; Dao et al., 1992). In contrast, Mg2+ had little effect on unmodified tRNA(ACPhe)-rC14 or tRNA(ACPhe)-d(C14). Modified tRNA(ACPhe)-d(m5C14) bound two Mg2+ ions, and the binding was cooperative. The dissociation constant of the two Mg2+ ions from tRNA(ACPhe)-d(m5C14), 2.5 x 10(-9) M2, is the result of an RNA structure significantly stabilized by Mg2+ binding, delta G = -11.7 kcal/mol. The tRNA(ACPhe)-d(m5C14) structure, investigated by 1H NMR, had a double stranded stem of five base pairs and two additional base pairs across what was a seven membered loop in the unmodified tRNA(Phe)AC. Methylation of cytidine in the yeast tRNA(ACPhe) enables the molecule to form more than one conformation through a process regulated by Mg2+ concentration. Thus, the simplest of posttranscriptional modifications of tRNA, a methylation, is involved in a somewhat distant, internal-site Mg2+ binding and stabilization of tRNA structure, especially that of the anticodon stem and loop.

Ribosome binding of DNA analogs of tRNA requires base modifications and supports the "extended anticodon".

“…When equal in concentration to tRNAPhe, both tRNAPhl-(m'G11, m5C14) and tRNAPhe-(m1G11) were able to inhibit more than 50% of the tRNAPhe from binding the 30S subunit (Fig. 3) and an open 7-membered loop (6)(7)(8). However, tRNAAhC-(mlG'1, m5C14), the best competitor of tRNAPhe in the ribosome binding assay (Fig.…”

“…However, tRNAAhC-(mlG'1, m5C14), the best competitor of tRNAPhe in the ribosome binding assay (Fig. 3 (6,8). In addition, A is substituted for T7 to disrupt the ThA10 base pair in tDNAhC*-d(A7, Ul3m5Cl4UU5) (Fig.…”

“…The introduction of these bases into tRNA is a post-transcriptional event involving specific enzymes. Although the functions of modified nucleosides in tRNA molecules are not yet well understood, several studies show that tRNA structure (12), metal ion binding (6)(7)(8), and interaction with cognate aminoacyl-tRNA synthetase are substantially influenced by the presence of modified nucleosides (13)(14)(15)(16). By directing the local or global structural changes in tRNA, these base modifications can affect the tRNA's interaction with different macromolecules (5).…”

“…Modified nucleoside phosphoramidites were synthesized from the corresponding modified nucleosides (A.M., B.N., E.S., A.K., and J.J., unpublished work). They were deprotected and purified as previously described (8). The anticodon domain were synthesized and purified as described (7,8).…”

“…They were deprotected and purified as previously described (8). The anticodon domain were synthesized and purified as described (7,8).…”