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Figure 1.
Figure 1. The crystal structure of free Vts1 SAM and the
NMR structure of SRE-bound Vts1 SAM are essentially identical. A
gene fragment encompassing the SAM domain (residues 407-523) of
S. cerevisiae VTS1 was amplified by PCR methods and inserted
into a modified pGEX vector. The resulting
glutathione-S-transferase fusion protein was purified by
affinity chromatography from soluble extracts of E. coli
BL21(DE3) grown in a BioFlow 110 fermentor at 25 °C.
Isotopically labeled Vts1^407-526 was obtained from
fermentations in minimal M9 medium containing 1.0 g/l [U-98%]
[15N]ammonium chloride and 4.0 g/l [U-99%] [13C]glucose as the
sole sources of nitrogen and carbon, respectively. Vts1^407-523
was cleaved from the fusion protein with TEV protease. Proteins
were further purified and buffer-exchanged using S-100
gel-filtration chromatography. For crystallography, the 13 kDa
Vts1 fragment was digested with thermolysin to produce a minimal
10 kDa fragment, Vts1^436-523, that was purified further by
heparin chromatography. A 19 nt SRE RNA
(5'-GGAGGCUCUGGCAGCUUUC-3') was prepared in milligram quantities
for NMR spectroscopy by phage T7 RNA polymerase-driven, in vitro
transcription.17 The SRE RNA was purified by denaturing 20% PAGE
and electroeluted. Renaturation of the SRE RNA hairpin was
achieved by rapidly chilling a solution that was preheated to 94
°C. (a) A fluorescence polarization binding assay7 of the
thermolysin-resistant Vts1 SAM domain demonstrates high-affinity
binding for the SRE (5'-AGGCUCUGGCAGUCU-3') but not a mutant SRE
bearing substitutions at the first and third loop positions. (b)
A modest decrease in binding affinity is observed when either 5
mM EDTA or 10 mM Ca^2+ is present in the binding buffer. (c)
Superposition of the free Vts1 SAM domain crystal structure with
the bound NMR structure reveals no significant conformational
changes. The SRE binding site is indicated in red. Superposition
of the crystal structures of the Vts1 SAM domain with the (d)
EphA4 SAM domain and the (e) Smaug SAM domain. (f) Superposition
of the 20 lowest energy NMR structures of the RNA-bound Vts1 SAM
domain.
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