Supplementary Information

Transcription

1 Supplementary Information Structural aspects of messenger RNA maintenance by the ribosome Lasse B. Jenner 1,2, Natalia Demeshkina 1,3, Gulnara Yusupova 1,3* and Marat Yusupov 1,3*. 1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Département de Biologie et de Génomique Structurales, Illkirch, F-67400, France 2 INSERM, U964, Illkirch, F-67400, France 3 CNRS, UMR7104, Illkirch, F-67400, France *To whom correspondence should be addressed. gula@igbm c.fr, marat@igbm c.fr

2 Supplementary Figures Supplementary Figure 1 The 70S elongation and initiation complexes. Top view of the 70S ribosome complex in (a) the elongation state and (b) the initiation state. Sequences of messenger RNA used for (a) elongation and (b) initiation complexes shown: SD sequence in green with core adenosine shown in pink, E, P and A codons are shown in blue, red and yellow respectively.

3 Supplementary Figure 2 Stabilization of mrna-trna interactions by ms 2 i 6 A37. (a) 2F o F c density contoured at 2.0 σ showing the cross-strand stacking interaction of the methylthio group of hypermodified nucleoside 2-methylthio-N6 isopentenyl adenosine (ms 2 i 6 A37) of trna Phe GAA on the first base of the codon in the P site. (b) 2F o F c density contoured at 1.5 σ for the environment around mrna nucleotide at position 1, which flips out and stacks directly on nucleotide G926 of 16S rrna.

4 Supplementary Figure 3 Positional shift of the Shine-Dalgarno duplex. Movement of the messenger RNA upon transition from initiation (red) to the elongation state (yellow) gives rise to a positional shift of the SD duplex. 5 and 3 -ends of the Shine-Dalgarno sequence are marked in (a) magenta - initiation and (b) teal - elongation.

5 Supplementary Methods Purification of ribosomes We isolated Thermus thermophilus 70S ribosomes from the strain HB8 following large-scale fermentation as previously described 1. We performed all of the following procedures at 4 C. We washed cells (100 g) with 1 L of buffer A (150 mm MgCl 2, 500 mm NH 4 Cl, 40 mm Tris- HCl ph 7.5, 1.5 mm EDTA-Na 2, 1 mm DTT) and suspended them in 100 ml of the same buffer. Then we added DNase to 1 U ml 1 together with PMSF 1 µg/ml (final). We disrupted the cells in a French Press and removed the debris by 30 min centrifugation at 30,000 g (4 C). We layered the supernatant on cushion 1 (29 ml S30 on 7 ml of cushion 1: 1.5 M sucrose, 0.68 M CsCl, 150 mm MgCl 2, 20 mm Tris-HCl ph 7.5, 1.5 mm EDTA-Na 2, 1 mm DTT) and centrifuged in a SW28 rotor at 100,000 g during 20 h at 4 C. We collected a fraction of around 5 ml from the bottom of the cushion and diluted it three times with buffer B (50 mm MgCl 2, 150 mm NH 4 Cl, 20 mm Tris-HCl ph 7.5, 0.5 mm EDTA-Na 2, 1 mm DTT). We then layered it on Cushion 2 (29 ml S30 on 7 ml of cushion 2: 1.8 M sucrose, 0.8 M CsCl, 150 mm MgCl 2, 20 mm Tris-HCl ph 7.5, 1.5 mm EDTA-Na 2 ) and centrifuged it in a SW28 rotor at 100,000 g (40 h at 4 C). We collected a 4 ml fraction from the bottom of the cushion and dialyzed it against buffer B. We added 4 M (NH 4 ) 2 SO 4 to the dialyzed solution to a final concentration of 1M. We then loaded the ribosomes (500 mg) on a 200 ml column of Toyopearl Butyl 650S equilibrated in buffer C (10 mm MgCl 2, 400 mm NaCl, 20 mm Tris- HCl ph 7.5, 0.5 mm EDTA-Na 2, 1 mm DTT) containing 1 M (NH 4 ) 2 SO 4. We washed the column by two volumes of buffer C with 0.8 M (NH 4 ) 2 SO 4, and eluted the ribosomes by 900 ml of a reverse gradient of (NH 4 ) 2 SO 4 (from 80 % to 40 %), keeping the other components of buffer C constant. The flow rate was 6 ml min 1, and the fraction volume was 12 ml. We collected the 70S peak and adjusted the concentrations of (NH 4 ) 2 SO 4 and MgCl 2 to 1 M and 50 mm respectively. We then further concentrated the peak by step-wise elution from 200 ml Toyopearl Butyl 650S equilibrated in buffer C containing 1 M (NH 4 ) 2 SO 4. Finally, we dialyzed against buffer D (10 mm Mg(Ac) 2, 50 mm KCl, 10 mm NH 4 Cl, 1 mm DTT, 5 mm HEPES, ph 7.5) and stored the 70S ribosome preparation in small aliquots at 80 C. Supplementary References 1. Gogia, Z.V., Yusupov, M.M., Spirina, T.N.. Structure of Thermus thermophilus ribosomes. Method of isolation and purification of the ribosomes. Molekul. Biol. (USSR) 20, (1986).