The role of 5-methylcytidine in the anticodon arm of yeast tRNAPhe: site-specific magnesium binding and coupled conformational transition in DNA analogs

Abstract

The tDNA(Phe)AC, d(CCAGACTGAAGAU13m5C14U15GG), with a DNA sequence similar to that of the anticodon stem and loop of yeast tRNA(Phe), forms a stem and loop structure and has an Mg(2+)-induced structural transition that was not exhibited by an unmodified tDNA(Phe)AC d(T13C14T15) [Guenther, R. H., Hardin, C. C., Sierzputowska-Gracz, H., Dao, V., & Agris, P. F. (1992) Biochemistry (preceding paper in this issue)]. Three tDNA(Phe)AC molecules having m5C14, tDNA(Phe)AC d(U13m5C14U15), d(U13m5C14T15), and d(T13,5C14U15), also exhibited Mg(2+)-induced structural transitions and biphasic thermal transitions (Tm approximately 23.5 and 52 degrees C), as monitored by CD and UV spectroscopy. Three other tDNA(Phe)AC, d(T13C14T15), d(U13C14U15), and d(A7;U13m5C14U15) in which T7 was replaced with an A, thereby negating the T7.A10 base pair across the anticodon loop, had no Mg(2+)-induced structural transitions and only monophasic thermal transitions (Tm of approximately 52 degrees C). The tDNA(Phe)AC d(U13m5C14U15) had a single, strong Mg2+ binding site with a Kd of 1.09 x 10(-6) M and a delta G of -7.75 kcal/mol associated with the Mg(2+)-induced structural transition. In thermal denaturation of tDNA(Phe)AC d(U13m5C14U15), the 1H NMR signal assigned to the imino proton of the A5.dU13 base pair at the bottom of the anticodon stem could no longer be detected at a temperature corresponding to that of the loss of the Mg(2+)-induced conformation from the CD spectrum. Therefore, we place the magnesium in the upper part of the tDNA hairpin loop near the A5.dU13 base pair, a location similar to that in the X-ray crystal structure of native, yeast tRNA(Phe).(ABSTRACT TRUNCATED AT 250 WORDS)

Posttranscriptional modification of tRNA in psychrophilic bacteria

“…5-Methylcytidine occurs in a number of archaea and eukaryotes (19) but until now has been identified only in bacteria, in thermophiles such as Thermotoga maritima (34) which are deeply branched in the universal phylogenetic tree. 5-Methylcytidine is a modified nucleoside known to play a role in Mg 2ϩ binding to tRNA (11). Its presence in tRNA from psychrophilic bacteria suggests a role in maintenance of the integrity of Mg 2ϩ binding domains in a relatively flexible molecule (see below).…”

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.…”

“…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).…”

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

A strategy for development of electrochemical DNA biosensor based on site-specific DNA cleavage of restriction endonuclease

“…Ethylenediaminetetraacetic acid (EDTA), mercaptohexanol (MCH) and tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (USA). The buffer solutions are as follows: hybridization buffer was the mixture of 100 mM NaCl and 10 mM TE (pH 8.0), buffers for DNA immobilization buffer are the mixture of 10 mM TE, 10 mM TCEP, 100 mM NaCl and 10 mM MgCl 2 (pH 7.4), MgCl 2 was added into the electrolyte to induce the formation of the hairpin structure of cDNA assembled onto the electrode surface, as reported previously (Dai et al, 1998;Dao et al, 1992;Gao et al, 1999;Ramsing et al, 1989). Washing buffer was the mixture of 0.1 M NaCl and 10 mM PB (pH 7.4).…”