Crystallization and preliminary X-ray diffraction analysis of a tRNA^Seracceptor-stem microhelix

Abstract

In order to understand elongator tRNA Ser and suppressor tRNA Sec identity elements, the respective acceptor-stem helices have been synthesized and crystallized in order to analyse and compare their structures in detail at high resolution. The synthesis, crystallization and preliminary X-ray diffraction results for a seven-base-pair tRNA Ser acceptor-stem helix are presented here. Diffraction data were collected to 1.8 Å , applying synchrotron radiation and cryogenic cooling. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 36.14, b = 38.96, c = 30.81 Å , = 110.69 .

Crystallization and preliminary X-ray diffraction analysis of anEscherichia colitRNAGlyacceptor-stem microhelix

“…Here, we present the crystallization and preliminary X-ray diffraction data of an E. coli tRNA Gly acceptor-stem microhelix with the aim of attempting to compare the high-resolution structures of the respective prokaryotic and eukaryotic segments in detail. This study supplements a small but growing number of investigations aiming at determining the three-dimensional structures of tRNA acceptor-stem microhelices reflecting alternative amino-acid specificities Ramos & Varani, 1997;Seetharaman et al, 2003;Fö rster et al, 1999Fö rster et al, , 2006.…”

tRNASeracceptor stem: conformation and hydration of a microhelix in a crystal structure at 1.8 Å resolution

“…We chose the two seryl-tRNA isoacceptors with the highest possible sequence similarity to the selenocysteine-specific tRNA Sec for structural analysis in order to compare the specific identity elements for aminoacylation with serine (Fö rster et al, 2006), but only that presented here produced crystals suitable for X-ray analysis.…”

“…The synthesis, purification and crystallization of the tRNA Ser microhelix have been described previously (Fö rster et al, 2006). Following hybridization of the two complementary RNA strands 5 0 -GGAGAGA-3 0 and 5 0 -UCUCUCC-3 0 to generate the tRNA Ser microhelix, crystallization occurred at a concentration of 0.5 mM RNA in 50 mM sodium cacodylate pH 6.0, 15 mM magnesium sulfate and 1.8 M lithium sulfate at 293 K in 24-well Linbro Plates (ICN Biomedicals Inc., Ohio, USA) using the hanging-drop vapour-diffusion technique.…”

“…The tRNA Ser acceptor-stem microhelix with sequence r(GGAGAGA)Ár(UCUCUCC) from Escherichia coli was chemically synthesized, purified and crystallized as described previously (Fö rster et al, 2006) and the structure was examined by X-ray crystallography. Crystallographic and X-ray diffraction parameters are summarized in Table 1.…”