A magnesium-induced conformational transition in the loop of a DNA analog of the yeast tRNAPhe anticodon is dependent on RNA-like modifications of the bases of the stem

Abstract

Two single-stranded DNA heptadecamers corresponding to the yeast tRNA(Phe) anticodon stem-loop were synthesized, and the solution structures of the oligonucleotides, d(CCAGACTGAAGATCTGG) and d(CCAGACTGAAGAU-m5C-UGG), were investigated using spectroscopic methods. The second, or modified, base sequence differs from that of DNA by RNA-like modifications at three positions; dT residues were replaced at positions 13 and 15 with dU, and the dC at position 14 with d(m5C), corresponding to positions where these nucleosides occur in tRNA(Phe). Both oligonucleotides form intramolecular structures at pH 7 in the absence of Mg2+ and undergo monophasic thermal denaturation transitions (Tm = 47 degrees C). However, in the presence of 10 mM Mg2+, the modified DNa adopted a structure that exhibited a biphasic "melting" transition (Tm values of 23 and 52 degrees C) whereas the unmodified DNA structure exhibited a monophasic denaturation (Tm = 52 degrees C). The low-temperature, Mg(2+)-dependent structural transition of the modified DNA was also detected using circular dichroism (CD) spectroscopy. No such transition was exhibited by the unmodified DNA. This transition, unique to the modified DNA, was dependent on divalent cations and occurred most efficiently with Mg2+; however, Ca2+ also stabilized the alternative conformation at low temperature. NMR studies showed that the predominant structure of the modified DNA in sodium phosphate (pH 7) buffer in the absence of Mg2+ was a hairpin containing a 7-nucleotide loop and a stem composed of 3 stable base pairs. In the Mg(2+)-stabilized conformation, the loop became a two-base turn due to the formation of two additional base pairs across the loop.(ABSTRACT TRUNCATED AT 250 WORDS)

The Escherichia coli tRNA-Guanine Transglycosylase Can Recognize and Modify DNA

“…For example, DNA analogues of the yeast tRNA Phe anticodon stem loop containing modifications such as deoxyuracil, 5-methylcytidine (m 5 C), and 1-methylguanine (m 1 G) are able to bind Mg ϩ2 . This binding is dependent upon the presence of the modified bases (15). The Mg ϩ2 is bound in the upper part of the DNA hairpin (16) in a position that is analogous to that seen in the x-ray crystal structure of yeast tRNA Phe (19).…”

“…Additional studies have revealed that the presence of modified bases further increases the capability of DNA to mimic RNA (15)(16)(17)(18). For example, DNA analogues of the yeast tRNA Phe anticodon stem loop containing modifications such as deoxyuracil, 5-methylcytidine (m 5 C), and 1-methylguanine (m 1 G) are able to bind Mg ϩ2 .…”

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).…”