NMR resonance assignments of the archaeal ribosomal protein L7Ae in the apo form and bound to a 25 nt RNA
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The archaeal protein L7Ae forms part of a protein complex in the ribosome that specifically recognizes and binds to kink-turn RNA. In this complex, L7Ae directly interacts with the oligonucleotide and creates a functional arrangement for site-specific 2′-O-methylation. We report the solution NMR backbone assignment of Methanocaldococcus jannaschii L7Ae (117 residues, 12.7 kDa) in the ligand-free state and when bound to a 25 nucleotide C/D box kink-turn mimic RNA.
KeywordsNMR Assignment L7Ae Kink-turn Ribosomal protein
The archaeal ribosomal protein L7Ae of Methanocaldococcus jannaschii belongs to a family of proteins that recognize and bind kink-turn motifs in ribosomal and box C/D as well as box H/ACA RNAs. In the crystal structure of L7Ae bound to a kink-turn derived from an archaeal box H/ACA sRNA the protein folds into a compact globular domain comprising a four-stranded central β-sheet that is surrounded by a total of five α-helices and a short 310-helix (Suryadi et al. 2005). L7Ae interacts with RNA by docking into its major groove, which stabilizes the kink-turn conformation of the oligonucleotide and creates the functional three-dimensional arrangement that is required for site-specific 2′-O-methylation by fibrillarin (Huang and Lilley 2013). Crystallographic analysis of ligand-free L7Ae from M. jannaschii showed that only minimal conformational differences between ligand-free and RNA-bound L7Ae exist (Hamma and Ferré-D’Amaré 2004).
Here we report the solution NMR backbone and partial side chain assignment of the M. jannaschii protein L7Ae (117 residues, 12.7 kDa) in the ligand-free state and when bound to a 25 nucleotide C/D box kink-turn mimic RNA. Our assignments lay the foundation for NMR studies of protein dynamics and binding interactions between L7Ae and RNA.
Methods and experiments
Protein expression and purification
A pET-28a vector encoding N-terminal His6-tagged L7Ae protein carrying kanamycin resistance was kindly provided by Keith Gagnon (University of Texas, Southwestern Medical Center, Dallas, TX). Expression of 15N/13C labeled samples was carried out in M9 minimal medium (containing 25 µg/ml kanamycin) with 15NH4Cl and 13C6-glucose as sole nitrogen and carbon sources, respectively, using E. coli BL21 cells. Overexpression was induced with 0.5 mM IPTG. Because L7Ae is a nucleic acid binding protein, after harvesting the cells by centrifugation at 4,000 rpm, 4 °C, the cell pellet was re-suspended in denaturing buffer A (20 mM TrisHCl pH 7.5, 250 mM NaCl, 10 mM imidazole, 6 M urea). The cells were lysed by sonication and the lysate was passed through a 45 µm filter before loading on a 5 ml HisTrap excel preloaded Ni-column (GE Healthcare). The protein was eluted within a 20 ml gradient from 0 to 100 % buffer B (same as buffer A but with 500 mM imidazole). Subsequently, the fractions containing L7Ae were loaded onto a size exclusion column (320 ml Superdex 75 26/600, GE Healthcare) and eluted with 50 mM potassium phosphate and 25 mM NaCl (KP buffer). L7Ae was concentrated to 1 ml using Amicon Ultra Centrifugal Filters with 3 kDa cutoff (Millipore) and the His-tag was cleaved with thrombin (5 U, Merck Millipore) overnight at room temperature. The protein was further purified by size exclusion chromatography (320 ml Superdex 75 26/600 column, GE Healthcare) with KP buffer. Fractions of pure L7Ae eluting at ca. 198 ml were concentrated to a final protein concentration of approximately 1 mM using Amicon Ultra Centrifugal Filters with 3 kDa cutoff (Millipore). The buffer was exchanged to 10 mM sodium cacodylate, pH 6.5, 50 mM NaCl with 10 % D2O by repeated dilution/concentration.
RNA production and purification
RNA samples were prepared by solid phase synthesis with standard 2′-O-TOM protected building blocks (ChemGenes). The 25 nt sequence 5′-GCUCUGACCGAAAGGCGUGAUGAGC-3′ was synthesized on an Applied Biosystems (ABI) 391 PCR Mate using an in-house written synthesis cycle. Custom primer support PS 200 (GE Healthcare) was used with an average loading of 80 µmol/g. Amidites (0.1 M) and activator (BTT, 0.3 M) solutions were dried overnight using freshly activated molecular sieves. The removal of protecting groups and cleavage from solid support were conducted by treatment with aqueous methylamine (40 %, 700 µl) and ammonia solution (33 % in water, 700 µl) at 40 °C for 90 min. After evaporation of the alkaline solvents, 2′-O-protecting groups were removed by dissolving the crude RNA in 1 M TBAF in THF (1.2 ml). After 14 h at 33 °C, the reaction was quenched by adding the same volume 1 M TEAA buffer (1.2 ml, pH 7.0, triethylammonium acetate). The volume of the solution was reduced to approximately 1 ml and applied to a 50 ml HiPrep 26/10 desalting column (GE Healthcare). The crude RNA was eluted with water, evaporated to dryness and re-dissolved in 1 ml of deionized water. The quality of the crude sequence was checked via anion exchange chromatography on a ThermoFisher DNAPac PA-100 column (4 × 250 mm). Purification was achieved by applying the crude RNA on a semipreparative ThermoFisher DNAPac PA-100 column (9 × 250 mm). The fractions containing the desired RNA were pooled, diluted with 0.1 M TEABC (triethylammonium bicarbonate) buffer and loaded onto a C18 SepPak cartridge (Waters). The RNA was eluted with acetonitrile/water (1:1) as the triethylammonium salt and lyophilized overnight.
NMR experiments were carried out on 600 MHz Bruker Avance II+ and 500 MHz Agilent DirectDrive spectrometers at 25 °C. For backbone resonance assignment we used 1H-15N-HSQC, 1H-13C-HSQC, and HNCO, HN(CO)CA, HNCA, HNCACB, CBCA(CO)NH and 15N-HSQC-TOCSY (50 ms, 90 ms mixing time) triple resonance experiments. Data were processed using NMRPipe (Delaglio et al. 1995) and analyzed with CcpNmr (Vranken et al. 2005).
Assignment and data deposition
The protein chemical shift assignments of ligand-free and RNA-bound L7Ae and have been deposited at the Biological Magnetic Resonance Data Bank (http:www.bmrb.wisc.edu) with BMRB accession numbers 19907 (L7Ae) and 19908 (L7Ae-RNA complex).
This work was supported by the Austrian Science Fund FWF (P22735 to MT and I844 to CK).
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