Online Supplementary Material. Messenger RNA Interactions in the Decoding Center Control the Rate of Translocation

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1 Online Supplementary Material Messenger RNA Interactions in the Decoding Center Control the Rate of Translocation Prashant K. Khade and Simpson Joseph

2 Supplementary Figure 1 Dissociation of the f[ 35 S]Met-Phe-tRNA Phe from ribosomal A-site determined using the tripeptide formation assay. Ribosomes contained trna fmet in the P site and f[ 35 S]Met-Phe-tRNA Phe in the A site. Formation of f[ 35 S]Met-Phe-Ala tripeptide was analyzed by electrophoretic TLC (etlc). Time course of tripeptide formation with: (a) control mrna, (b) mrna+4d, (c) mrna+5d, and (d) mrna+6d. (e) Plot showing the time course of dissociation of f[ 35 S]Met-Phe-tRNA Phe for control mrna( ), mrna+4d ( ), mrna+5d ( ), and mrna+6d ( ). The error bars represent s.d. from three experiments. (f) Table showing the dissociation rate constants (k off ). Mean ± s.d. from three experiments are shown.

3 c mrna k (s 1 ) Control 0.8 ± D 0.8 ± D 0.9 ± D 1.4 ± F 0.5 ± F 0.9 ± F 0.5 ± 0.1 Supplementary Figure 2 Kinetics of translocation measured by the tripeptide formation assay. (a) Time course showing the formation of f[ 35 S]Met-Phe-Ala tripeptide by ribosomes with 2 - deoxynucleotide substituted mrnas. Symbols: control mrna ( ), mrna+4d ( ), mrna+5d ( ), and mrna+6d ( ). (b) Time course showing the formation of f[ 35 S]Met-Phe-Ala tripeptide by ribosomes with 2 -fluoro substituted mrnas. Symbols: control mrna ( ), mrna+4f ( ), mrna+5f ( ), and mrna+6f ( ). The amount of tripeptide formed with respect to the total amount of f[ 35 S]Met-tRNA fmet bound to the P site are shown. The actual extent of f[ 35 S]Met-Phe dipeptide converted to f[ 35 S]Met-Phe-Ala tripeptide ranged between 60% to 70%. The error bars represent s.d. from three experiments. (c) Table showing the rate of tripeptide formation with the 2 -deoxynucleotide and 2 -fluoro substituted mrnas. Mean ± s.d. from three experiments are shown.

4 Supplementary Figure 3 Translocation of mrnas with single 2 -deoxynucleotide substitutions. Ribosomes contained trna fmet in the P site and fmet-phe-trna Phe in the A site. (a) Time course showing translocation of ribosomes programmed with control mrna (trace 1) and mrnas having a single 2 -deoxynucleotide substitution at positions 3D (trace 2), 2D (trace 3), and 1D (trace 4). (b) Time course showing translocation of ribosomes programmed with control mrna (trace 1) and mrnas having a single 2 -deoxynucleotide substitution at positions +1D (trace 2), +2D (trace 3), and +3D (trace 4). (c) Time course showing translocation of ribosomes programmed with control mrna (trace 1) and mrnas having a single 2 -deoxynucleotide substitution at positions +4D (trace 2), +5D (trace 3), and +6D (trace 4). Note the time on the x- axis has been extended to 40 seconds. (d) Time course showing translocation of ribosomes programmed with control mrna (trace 1) and mrnas having a single 2 -deoxynucleotide substitution at positions +7D (trace 2), +8D (trace 3), and +9D (trace 4). The maximum fluorescence intensity was normalized to 1 and shown in arbitrary units (AU).

5 Supplementary Figure 4 Kinetics of trna dissociation from the A site with 2 -fluoro substituted mrnas. (a) Time course showing the dissociation of f[ 35 S]Met-Phe-tRNA Phe from the A site. Symbols: control mrna ( ), mrna+4f ( ), mrna+5f ( ), and mrna+6f ( ). The dissociation of f[ 35 S]Met-tRNA fmet from the P site is also shown as a control ( ). (b) Time course showing the dissociation of N-acetylated-[ 14 C]Phe-tRNA Phe from the A site. Symbols: control mrna ( ), mrna+4f ( ), mrna+5f ( ), and mrna+6f ( ). (c) Bar graph showing the extent of spontaneous translocation of f[ 35 S]Met-Phe-tRNA Phe from A to P-site in 30 minutes. Spontaneous translocation was measured by the puromycin reaction as described in supplementary methods. The extent of spontaneous translocation was greater with mrna having a 2 -deoxynucleotide or 2 - fluoro substitution at position +5. (d) Time course showing the dissociation of N-acetylated-[ 14 C]Phe-tRNA Phe from the A site with double and triple 2 -fluoro substituted mrnas. Symbols: control mrna ( ), mrna+5f+6f ( ), and mrna+4f+5f+6f ( ). In all cases, the error bars represent s.d. from three experiments.

6 Supplementary Figure 5 Dissociation of the f[ 35 S]Met-Phe-tRNA Phe from ribosomal A-site determined by the tripeptide formation assay. Ribosomes contained trna fmet in the P site and f[ 35 S]Met-Phe-tRNA Phe in the A site. Formation of f[ 35 S]Met-Phe-Ala tripeptide was analyzed by electrophoretic TLC (etlc). Time course of tripeptide formation with: (a) control mrna, (b) mrna+4f, (c) mrna+5f, and (d) mrna+6f. (e) Plot showing the time course of dissociation of f[ 35 S]Met-Phe-tRNA Phe for control mrna( ), mrna+4f ( ), mrna+5f ( ), and mrna+6f ( ). The error bars represent s.d. from three experiments. (f) Table showing the dissociation rate constants (k off ). Mean ± s.d. from three experiments are shown.

7 Supplementary Figure 6 Effect of viomycin on the extent of translocation. The extent of translocation was determined by the tripeptide formation assay. Ribosome with f[ 35 S]MettRNA fmet in the P site was mixed with EF-Tu GTP Phe-tRNA Phe, EF-Tu GTP Ala-tRNA Ala, and EF-G GTP. The reaction was incubated for 1 min at room temperature and quenched by adding KOH. One round of translocation of the mrna will place the alanine codon in the A site and allow the binding of EF-Tu GTP Ala-tRNA Ala ternary complex resulting in the formation of f[ 35 S]Met-Phe-Ala tripeptide by the ribosome. In the presence of viomycin, the extent of tripeptide formed will be reduced due to inhibition of translocation. Top panel shows formation of tripeptide analyzed by etlc for the indicated mrnas. Bottom bar graph shows the percentage of dipeptide converted to tripeptide by the ribosome with the indicated mrnas in the absence of viomycin (striped bars) and in the presence of viomycin (black bars). The error bars represent s.d. from three experiments.

8 Supplementary Table 1 Dissociation rate constants of A site trna mrna k a off (min 1 ) k b off (min 1 ) Control 0.04 ± ± D 0.07 ± ± D 0.27 ± ± D 0.19 ± ± F 0.03 ± ± F 0.08 ± ± F 0.08 ± ± F+6F ND ± F+5F+6F ND ± a; Dissociation of f[ 35 S]Met-Phe-tRNA Phe. b; Dissociation of N-Ac[ 14 C]Phe-tRNA Phe. ND; not determined. Mean ± s.d. from three experiments are shown.

9 Supplementary Table 2 Translocation rates of A site N-acetylated-Phe-tRNA Phe mrna k 1 (s 1 ) k 2 (s 1 ) A 1 / A 1 + A 2 k av (s 1 ) Control 7.5 ± ± ± ± D 9.4 ± ± ± ± D 15.2 ± ± ± ± D 9.0 ± ± ± ± F 11.2 ± ± ± ± F 11.8 ± ± ± ± F 16.3 ± ± ± ± F+6F 48.8 ± ± ± ± F+5F+6F 43.6 ± ± ± ± 3.1 Ribosomes contained trna Met in the P-site and N-acetylated-Phe-tRNA Phe in the A-site. Translocation rates were determined as described in supplementary methods. k 1 and k 2 are the apparent rate constants for the fast and slow phases, respectively. A 1 and A 2 are the amplitudes for the fast and slow phases, respectively. Weighted average rate constant (k av ) were calculated as the sum of k 1 and k 2 normalized with respect to their amplitude: k av = (k 1 A 1 + k 2 A 2 ) / (A 1 + A 2 ). Mean ± s.d. from three experiments are shown.

10 Supplementary Table 3 Translocation rates of A site fmet-phe-trna Phe mrna k 1 (s 1 ) k 2 (s 1 ) A 1 / A 1 + A 2 k av (s 1 ) Control 13.3 ± ± ± ± 0.7-3D 12.5 ± ± ± ± 0.3-2D 12.4 ± ± ± ± 0.1-1D 12.4 ± ± ± ± D 14.8 ± ± ± ± D 14.2 ± ± ± ± D 14.4 ± ± ± ± D 12.2 ± ± ± ± D 12.6 ± ± ± ± D 15.8 ± ± ± ± 0.5 Ribosomes contained trna fmet in the P-site and fmet-phe-trna Phe in the A-site. Translocation rates were determined as described in supplementary methods. k 1 and k 2 are the apparent rate constants for the fast and slow phases, respectively. A 1 and A 2 are the amplitudes for the fast and slow phases, respectively. Weighted average rate constant (k av ) were calculated as the sum of k 1 and k 2 normalized with respect to their amplitude: k av = (k 1 A 1 + k 2 A 2 ) / (A 1 + A 2 ). Mean ± s.d. from three experiments are shown.

11 Supplementary Discussion The reason for using N-acetylated-Phe-tRNA Phe in the A site. To determine the rate of translocation using the fluorescence-based kinetic assay, we formed pre-translocation complexes with deacylated trna fmet in the P site and fmet-phetrna Phe in the A site. Control mrna and most of the mrnas with a single 2 -deoxynucleotide substitution showed a rapid decrease in fluorescence intensity indicating no inhibition in the rate of translocation (Supplementary Figure 3 and Supplementary Table 3). In contrast, mrna+5 and mrna+6 showed a very slow decrease in fluorescence intensity suggesting severe inhibition of translocation (k obs = 0.05 ± 0.01 s 1 ) (Supplementary Figure 3c). The reason for this slow rate of translocation became clear after we performed additional experiments as described below. Our studies showed that fmet-phe-trna Phe dissociates more rapidly from pretranslocation complex programmed with mrnas having a single 2 -deoxynucleotide substitution in the A site codon (Figure 1c and Supplementary Table 1). Therefore, the slow rate of translocation can be explained by the dissociation of the fmet-phe-trna Phe from the A site followed by the competitive binding of Phe-tRNA Phe present in 2-fold excess in the reaction. The fmet-phe-trna Phe in the A site will be eventually replaced by Phe-tRNA Phe because it binds more stably resulting in the slow rate of translocation. This hypothesis is consistent with a previous study that showed that Phe-tRNA Phe has a 1,500-fold higher affinity for the A site and are translocated 130-fold slower than fmet-phe-trna Phe 1. We verified this hypothesis by forming pre-translocation complex with a deacylated trna fmet in the P site and a Phe-tRNA Phe in the A site and determined the rate of translocation. The decrease in fluorescence intensity was similar to the change observed with the single 2 -deoxynucleotide substitution at position +5 and the rate of translocation was identical (k obs = 0.05 ± 0.01 s 1 ) (data not shown). This strongly

12 supports the idea that dissociation of fmet-phe-trna Phe from the A site followed by the rebinding of Phe-tRNA Phe is the reason for the slow rate of translocation with the single 2 - deoxynucleotide substitutions in the A site codon. These considerations prompted us to determine the kinetics of translocation with pre-translocation complexes containing a deacylated trna Met in the P site and a N-acetylated-Phe-tRNA Phe in the A site. N-acetylated-Phe-tRNA Phe is an analog of peptidyl trna and is translocated efficiently by the ribosome 2,3. The use of N- acetylated-phe-trna Phe avoids the competition for binding to the A site by a different trna. Effect of viomycin on the extent of translocation We used the tripeptide assay to determine the extent of translocation in the presence of viomycin. The extent of translocation was >80% for all the mrnas in the absence of viomycin (Supplementary Fig. 6). In the presence of viomycin, the extent of translocation was reduced with control mrna and with mrnas having either a 2 -deoxynucleotide or 2 -fluoro substitution at position +4. Whereas, mrnas having either a 2 -deoxynucleotide or 2 -fluoro substitution at positions +5 had a similar extent of translocation with or without viomycin ( 80% translocation). mrnas with 2 -deoxynucleotide or 2 -fluoro substitution at position +6 showed a slightly reduced extent of translocation with viomycin (50-60% translocation). The double and triple fluoro substituted mrnas showed the same extent of translocation with or without viomycin ( 80% translocation). These results show that the translocation of control mrna and mrnas with a 2 -deoxynucleotide or a 2 -fluoro substitution at position +4 are more strongly inhibited by viomycin, whereas mrnas with a 2 -deoxynucleotide or a 2 -fluoro substitution at positions +5 or +6 are translocated to a greater extent, even in the presence of viomycin, because of their improved rate of translocation.

13 Supplementary Methods Preparation of ribosomes, mrnas and trnas Tight-couple ribosomes were purified from E. coli MRE600 as described previously 4. Synthetic mrnas with 2 -deoxynucleotide or 2 -fluoro substitutions were purchased from Dharmacon. Aminoacylation of trna fmet and trna Phe were performed using purified E. coli histidine-tagged synthetase, essentially as described 4. Formylation of initiator trna fmet and N-acetylation of trna Phe was performed as described 4. The aminoacylated trnas were purified by HPLC on a C18 reverse phase column 4. The extent of aminoacylation was verified by acid gel electrophoresis, and the level of aminoacylation was greater than 95%. Elongator trna Met, trna Ala were prepared by in vitro transcription 4. E. coli histidine-tagged AlaRS was purified as described 5. f[ 35 S]Met-tRNA fmet was prepared as described 6. N-acetylated-[ 14 C] Phe-tRNA Phe was prepared as described 7 and purified by HPLC as described earlier. Dissociation kinetics of f[ 35 S]Met-Phe-tRNA Phe determined by filter binding assay All experiments were performed in buffer A [20 mm KHepes (ph 7.6), 150 mm NH 4 Cl, 6 mm MgCl 2, 4 mm β-mercaptoethanol, 2 mm spermidine and 50 µm spermine] 8 unless noted otherwise. Initiation complex was formed by incubating activated 70S ribosome (0.5 µm), mrna (0.75 µm) and f[ 35 S]Met-tRNA fmet (0.75 µm) 6. EF-Tu GTP Phe-tRNA Phe ternary complex was prepared by incubating EF-Tu (5 µm), GTP (1 mm), phosphoenol pyruvate (3 mm) and pyruvate kinase (0.25 µg µl 1 ) at 37 C for 30 min in buffer B (20 mm KHepes ph 7.6, 6 mm MgCl 2, 150 mm NH 4 Cl). Then Phe-tRNA Phe (6 µm) was added and the incubation continued for another 30 min. EF-Tu GTP Phe-tRNA Phe ternary complex was purified by gel filtration as described 9 and quantified by the Bradford assay. Spermine (50 µm), spermidine (2

14 mm) and β-mercaptoethanol (4 mm) were added to make their final concentration as in buffer A. The concentration of the ternary complex was adjusted to 2 µm by adding buffer A. We used at least a 4-fold excess of EF-Tu GTP Phe-tRNA Phe ternary complex over the initiation complex to prevent the rebinding of dissociated f[ 35 S]Met-Phe-tRNA Phe. 50 µl of initiation complex was mixed with 50 µl ternary complex and incubated at room temperature. Then 10 µl aliquots were removed from the reaction at different time intervals, diluted by adding 100 µl of ice-cold buffer C (50 mm KHepes ph 7.5, 15 mm MgCl 2, 100 mm NH 4 Cl, and 1 mm DTT) and immediately filtered through nitrocellulose filters (0.45 µm HA, Millipore). Filters were washed two times with 4 ml ice-cold buffer C. Filters were dried, dissolved in scintillation cocktail (30% ScintiSafe, Fisher) and counted. In parallel, aliquots of the reaction were removed, quenched by adding 0.5 M KOH and analyzed by etlc to determine the extent of f[ 35 S]Met-Phe dipeptide formed. The experiments were repeated three times. The dissociation rate constant (k off ) was determined by fitting the data to a single exponential equation: Y = A 1 *exp(-k 1 *x) using GraphPad Prism software (San Diego, CA). The extent of spontaneous translocation during the dissociation experiment was measured by puromycin reaction (5 mm final conc. of puromycin). A 10 µl aliquot of the ribosome mix was mixed with puromycin and the reaction was quenched after 10 seconds with 0.5 M KOH and analyzed by etlc. The formation of f[ 35 S]Met-Phe-Puromycin was analyzed by etlc as described previously 4. Dissociation kinetics of f[ 35 S]Met-Phe-tRNA Phe determined by the tripeptide formation assay

15 Initiation complexes were prepared in buffer A by incubating activated 70S ribosomes (1 µm) with mrna (1.5 µm) at 37 C for 10 min followed by the addition of f[ 35 S] Met-tRNA fmet (1.5 µm) and the incubation was continued at 37 C for 10 min. Unbound f[ 35 S] Met-tRNA fmet was removed by ultra-filtration using Microcon Centrifugal Filter Devices (Amicon; 100,000 MWCO) and by washing two times with 300 µl of buffer A. EF-Tu GTP Phe-tRNA Phe ternary complex was formed by incubating 4 µm trna Phe, 1 mm phenylalanine, 20 µg PheRS, 3 mm ATP, 1 mm GTP, 3 mm phosphoenol pyruvate, 0.25 µg µl 1 of pyruvate kinase, and 8 µm EF- Tu at 37 C for 1 hour in buffer A. The concentration of EF-Tu GTP Phe-tRNA Phe ternary complex was adjusted to 2 µm by adding buffer A. EF-Tu GTP Ala-tRNA Ala was formed by incubating trna Ala (1.5 µm), alanine (1 mm), AlaRS (30 µg), ATP (1 mm), GTP (1 mm), phosphoenol pyruvate (3 mm), pyruvate kinase (0.25 µg µl 1 ) and EF-Tu (4.5 µm) at 37 C for 1 hour. EF-G (2.5 µm) was then added to the AlaRS reaction. Equal volumes of initiation complex and EF-Tu GTP Phe-tRNA Phe ternary complex were mixed to form f[ 35 S]Met-PhetRNA Phe in the A site. Then 10 µl aliquots of the reaction were removed at various time points and mixed with 10 µl of EF-Tu GTP Ala-tRNA Ala having EF-G GTP. The reaction was quenched after 30 seconds by adding KOH (0.5 M final concentration). The formation of tripeptide and dipeptide were analyzed by etlc as described previously 4. The dissociation rate constant (k off ) was determined by fitting the data to a single exponential equation as described above. Dissociation of N-acetylated-[ 14 C]Phe-tRNA Phe from the A site Initiation complexes were prepared in buffer A by incubating activated 70S ribosomes (3.3 µm), mrna (10 µm) and trna Met (23 µm) as described above in the dissociation kinetics of

16 f[ 35 S]Met-Phe-tRNA Phe. N-acetylated-[ 14 C]Phe-tRNA Phe (1 µm) was added to the ribosome complex and incubated for 2 hours at room temperature to allow binding to A-site. Dissociation of N-acetylated-[ 14 C]Phe-tRNA Phe from A was initiated by mixing 30 µl of ribosome complex with 270 µl of buffer A containing trna Phe (3.3 µm) as chase. 20 µl Aliquots were removed from the reaction at different time intervals, diluted by adding 100 µl of ice-cold buffer C and passed through nitrocellulose filter and washed as described above in dissociation kinetics of f[ 35 S]Met-Phe-tRNA Phe determined by filter binding. Experiments to measure the dissociation rate constant of trna from the A site in the presence of viomycin was carried out by adding viomycin (200 µm final conc.) to the initiation complex. The dissociation rate constant (k off ) was determined by fitting the data to a single exponential equation as described above. Rapid-kinetic assay for translocation Pre-transloaction complex was formed using 70S ribosomes (0.5 µm), mrna (0.75 µm), fmettrna fmet (0.75 µm), Phe-tRNA Phe (1 µm) in buffer A as described 6. Pre-translocation complex (0.25 µm after mixing) was rapidly mixed with equal volume of EF-G GTP (final conc. after mixing was 1.25 µm EF-G and 1mM GTP in buffer A) using a stopped-flow instrument (µsfm- 20, Bio-Logic). More than five shots were performed per experiment and the experiments were repeated at least three times with different batches of ribosomes, EF-G and synthetic mrnas. Translocation time courses were analyzed by fitting to the Biokine double exponential equation: Y=a*x+b+A1*exp(-k1*x)+A2*exp(-k2*x). Stopped-flow experiments with N-acetylated-PhetRNA Phe in the A site were performed similarly except pre-translocation complex were formed with trna Met (3.5 µm) and N-acetylated-Phe-tRNA Phe (1 µm). Experiments with viomycin were performed by adding 200 µm viomycin (final conc. after mixing) to the pre-translocation

17 complex and incubating for 10 min before transferring to the stopped-flow instrument. To determine the maximum rate of translocation (k trans ), translocation rates were measured at increasing concentrations of EF-G. k trans was obtained by using the equation: k obs = k trans [EF- G]/K 1/2 +[EF-G]. K 1/2 is the concentration of EF-G where half the maximum rate of translocation was observed. Kinetics of tripeptide formation by quench-flow The tripeptide formation assay is based on the fact that the mrnas used in this study have a phenylalanine codon (UUU) and an alanine codon (GCU) at the second and third positions, respectively (Fig. 1b). One round of translocation will place the alanine codon in the A site and allow the binding of EF-Tu GTP Ala-tRNA Ala ternary complex resulting in the formation of f[ 35 S]Met-Phe-Ala tripeptide by the ribosome. Initiation complex containing activated 70S ribosome (0.5 µm), mrna (0.75 µm) and f[ 35 S]Met-tRNA fmet (0.6 µm) were prepared and purified by Microcon as described above except f[ 35 S]Met-tRNA fmet charging mixture prepared in buffer A (0.6 µm trna fmet, 3 mm ATP, 0.7 µm [ 35 S]L-Methionine, 0.4 mm N 10 - formyltertahydrofolic acid, 10 µg formyl transferase and 10 µg MetRS) was added directly to the ribosome-mrna complex. EF-Tu GTP Phe-tRNA Phe and EF-Tu GTP Ala-tRNA Ala were formed by incubating, trna Ala (1.5 µm), alanine (1 mm), AlaRS (20 µg), ATP (1 mm), GTP (1 mm), phosphoenol pyruvate (3 mm), pyruvate kinase (0.25 µg µl 1 ), and EF-Tu (7.5 µm) at 37 C for 30 min followed by the addition of Phe-tRNA Phe (1 µm) and EF-G (2.5 µm). Then the EF-Tu ternary complex reaction mix was incubated for another 30 min. To determine the rate of tripeptide formation 15 µl of 70S initiation complex was rapidly mixed with 15 µl of the reaction mix containing EF-Tu GTP Phe-tRNA Phe, EF-Tu GTP Ala-tRNA Ala and EF-G GTP and

18 quenched with 15 µl of 1M KOH in a quench-flow instrument (QFM-400, Bio-Logic). Tripeptide was resolved by etlc and quantified as described previously 4.

19 Supplementary References 1 Semenkov, Y. P., Rodnina, M. V. & Wintermeyer, W. Nat Struct Biol 7, , (2000). 2 Dorner, S., Brunelle, J. L., Sharma, D. & Green, R. Nat Struct Mol Biol 13, , (2006). 3 Feinberg, J. S. & Joseph, S. J Mol Biol 364, , (2006). 4 Feinberg, J. S. & Joseph, S. RNA 12, , (2006). 5 Lovato, M. A., Swairjo, M. A. & Schimmel, P. Mol Cell 13, , (2004). 6 Garcia-Ortega, L., Stephen, J. & Joseph, S. Mol Cell 32, , (2008). 7 Walker, S. E. & Fredrick, K. Methods 44, 81-86, (2008). 8 Bartetzko, A. & Nierhaus, K. H. Methods Enzymol 164, , (1988). 9 Rodnina, M. V. & Wintermeyer, W. Proc Natl Acad Sci U S A 92, , (1995).