Kinetics of Ribosome-Catalyzed Polymerization Using Artificial

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ACS Chemical Biology

Kinetics of ribosome-catalyzed polymerization using artificial aminoacyl-tRNA substrates clarifies inefficiencies and improvements Jinfan Wang, Marek Kwiatkowski and Anthony C. Forster* Department of Cell and Molecular Biology, Uppsala University, Husargatan 3, Box 596, Uppsala 75124, Sweden *Corresponding Author A.C. Forster, tel: +46-18-4714618, e-mail: [email protected]

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Abstract Ribosomal synthesis of polymers of unnatural amino acids (AAs) is limited by low incorporation efficiencies using the artificial AA-tRNAs, but the kinetics have yet to be studied. Here, kinetics was performed on 5 consecutive incorporations using various artificial AA-tRNAs with all intermediate products being analyzed. Yields within a few seconds displayed similar trends to our prior yields after 30 minutes without preincubation, demonstrating the relevance of fast kinetics to traditional longincubation translations. Interestingly, the 2 anticodon swaps were much less inhibitory in the present optimized system, which should allow more flexibility in the engineering of artificial AA-tRNAs. The biggest kinetic defect was caused by the penultimate dC introduced from the standard, chemoenzymatic, charging method. This prompted peptidyl-tRNA drop off, decreasing processivities during 5 consecutive AA incorporations. Indeed 2 tRNA charging methods that circumvented the dC dramatically improved efficiencies of ribosomal, consecutive, unnatural AA incorporations to give near wild-type kinetics.


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Introduction Ribosomal synthesis of polymers of unnatural amino acids (AAs) in a purified translation system (1) is the basis of directed evolution in vitro of peptidomimetic drug leads from enormous libraries (2, 3). Incorporation of multiple unnatural AAs per translation product also has utility for mechanistic and biophysical studies (4, 5). Nevertheless, a major challenge is low incorporation efficiencies using the artificial AA-tRNAs. Several different methods have been developed for charging tRNAs with unnatural AAs (2, 6-8). The most widely used method over the years is also the most flexible in terms of the AA portion: enzymatic ligation of chemically-synthesized pdCpA-NVOC amino acids to unmodified tRNAs lacking their terminal CA dinucleotide (8). Using pdCpA-type substrates in purified translations incubated for 30 minutes, dramatic reductions were found in the yields of multiple incorporations of unnatural non-N-alkyl AAs compared with single incorporations (9). The unnatural substrate features causing the most severe reductions during five consecutive incorporations were (i) the anticodon swap and (ii) the penultimate dC rather than the unnatural AAs or lack of tRNA modifications (10). Effect (i) was possibly due to creating anticodon loop conformations unfavorable for initial codon recognition. Effect (ii) was surprising because the dC substitution had been shown to have minimal effects on single translation incorporations in crude (11) and purified (10, 12) systems. In order to solve problem (ii), we recently developed a facile N-NVOC-AA-pCpA synthesis method to facilitate the preparations of mischarged tRNAs lacking the dC (13). Kinetic studies of ribosomal single incorporation using artificial AA-tRNAs have identified mechanisms limiting incorporations and led to ways of improving incorporation efficiencies (12, 14-17). For example, kinetic studies of non-N-alkyl L-



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AA-tRNAs revealed that low incorporation efficiencies were mainly due to low binding affinities to elongation factor Tu (EF-Tu) that delivers them to the ribosomal A site. Hence, efficiencies were improved by increasing EF-Tu concentration, lowering temperature and using tRNA bodies that bind more tightly to EF-Tu (14, 15). On the other hand, incorporations of N-alkyl L-AAs were found to be limited by slow peptidyl transfer reactions (12, 17). Higher pH, increased Mg2+ ion concentration and tRNAPro were shown to hasten peptide bond formation with N-methyl AAs (12, 17). However these kinetic studies do not include translocation from the ribosomal A site to the P site or multiple unnatural AA incorporations where drops in yields are much more severe than for single incorporations. Here we studied the kinetics of five consecutive incorporations using these artificial AA-tRNAs (10) to investigate the mechanisms limiting polymer synthesis.


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Results and Discussion 1. Development of assay to measure rates of synthesis of every intermediate in hexapeptide synthesis In order to obtain detailed kinetic information of each step during five consecutive incorporations of various AAs, we utilized an in vitro-reconstituted, purified, E. coli translation system that was optimized for both efficiency and fidelity (18, 19). A control

experiment

was

first

done

with

native

Ala-tRNAAla

(termed

Ala-tRNA Ala _modified ). On a quench-flow apparatus, a ribosomal mix containing ugc 70S initiation complex programmed with mMAAAAAKT (mMA5KT) and [3H]fMettRNAfMet in the P site was rapidly mixed with a factor mix containing excess ternary complex (EF-Tu:GTP: Ala-tRNA Ala _modified ) and EF-G:GTP. EF-G was added to ugc 20 µM to achieve maximal translocation rate in the translation reaction (20). After quenching the reaction at desired time points, products were analyzed by RP-HPLC. Figure 1a shows an example of HPLC separation of every intermediate in fMAAAAA (fMA5) synthesis. By quantifying the fraction of the 3H signal from each intermediate out of total 3H signal at each assayed time point, we were able to follow the timeevolution of every intermediate product in the hexapeptide synthesis (Figure 1b). Note that, at the saturation time point, not all the intermediate products had incorporated into full-length fMA5 peptide. This was not unexpected because the processivity of translation has been reported to be incomplete for synthesis of the first few peptide bonds (21-23). We thus developed a data-fitting model to deduce the rate and processivity at each incorporation step (see Supplementary Figure 1a II, Supplementary Table 1 and Supplementary Appendix). The rate of the first incorporation

step

(fMA

dipeptide

formation

from


fMet-tRNAfMet

and


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Ala-tRNA Ala _modified ) was ~73 s-1. For the subsequent steps, the incorporation ugc rates, ~10 s-1, were measured as the overall rates for translocation and the following peptide bond formation. These rates were limited by the slower translocation reactions, and were comparable with earlier reported translocation rates that involved short open reading frames (20). The processivity at each incorporation step was measured to be > 90%. These results indicated that our translation system was robust for studying the kinetics of consecutive incorporations of AAs.

2. Kinetic effects of changes in substrates: most important is the penultimate 2’ OH group, not the anticodon swap Next, we studied the kinetics of five consecutive incorporations using AA-tRNAs with different substrate changes used for delivery of unnatural AAs (Figure 2a) (10). A control wild-type tRNAAla sequence lacking natural nucleoside modifications and lacking the dC substitution was prepared by either of two synthetic routes: charging of full-length transcript by Ala-tRNA synthetase or charging of transcript lacking the terminal CA by ligation to NVOC-Ala-pCpA and photodeprotection (termed

Ala-tRNA AlaB _enzymatic and Ala-tRNA AlaB _chemoenzymatic , respectively). Both ugc ugc preparations of this substrate exhibited wild-type rates (compare Figure 2b orange and yellow with red, Supplementary Figure 1b,e and Supplementary Table 1) and processivities (Figure 2c and Supplementary Table 1). The yields of fMA5 were ~80% in all three cases (Figure 2d and Supplementary Table 1). Hence, the lack of modifications, pre-charging and photodeprotection had only minor, if any, effects. In a further control, note that the presence or absence of E. coli total tRNA (tBulk) also did not affect the assay (Supplementary Figure 1c, Supplementary Table 1 and left


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black square in Figure 3). This also validated our new all-ribo synthetic chemistry for natural polymer synthesis (13). When a penultimate dC was introduced to the tRNA body in using the traditional chemoenzymatic synthesis ( Ala-tRNA AlaB _dC ), the incorporation rates ugc decreased by ~2-fold at each incorporation step (Figure 2b green, Supplementary Figure 1d and Supplementary Table 1). The processivities along the synthesis decreased by ~10-20% at each incorporation step (Figure 2c and Supplementary Table 1) and the yield of fMA5 hence decreased to only ~42% (Figure 2d and Supplementary Table 1). This result is consistent with the ~2-fold decrease in yield of full-length product in 30-minute batch reactions attributed to the dC (10). While it is formally possible that the effect of the dC was somehow potentiated by the lack of modifications, testing this is not experimentally tractable and such an effect seems unlikely because the lack of modifications had a minimal effect (orange and yellow in Figure 2d). When changed to the unnatural AAs aG or mS ( aG-tRNA AlaB _dC or ugc

mS-tRNA AlaB _dC ), incorporation was faster than from Ala-tRNA AlaB _dC (Figure 2b ugc ugc compare light and dark blue with green, Supplementary Figure 1f,i and Supplementary Table 1). While mS-tRNA AlaB _dC processivities were similar to those ugc of Ala-tRNA AlaB _dC , the processivities of aG-tRNA AlaB _dC were higher (Figure 2c, ugc ugc Supplementary Figure 1 and Supplementary Table 1). The yields of fMaG5 and fMmS5 syntheses were ~62% and ~40%, respectively (Figure 2d and Supplementary Table 1), consistent with the results reported for 30-minute translation reactions (10).



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When

the

wild-type

UGC

anticodon

was

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swapped

to

GGU

(

Ala-tRNA AlaB _enzymatic ), we found no apparent effect on the fMA5 yield (Figure 2d ggu dark purple, Supplementary Figure 1l and Supplementary Table 1). This was unexpected because this swap reduced the yield three fold in 30-minute translation reactions (10). However, an anticodon swap previously found to abolish fMA5 synthesis in 30-minute translation reactions ( Ala-tRNA AlaB _enzymatic (10)) had guu much slower kinetics: fMA formation was ~10-fold slower and the subsequent steps were ~3-fold slower (Figure 2b light purple, Supplementary Figure 1m and Supplementary Table 1). The processivity at each incorporation step was only slightly impeded by this anticodon swap and the fMA5 yield was ~67% (Figure 2c,d and Supplementary Table 1). These findings indicated that our kinetic translation system was more optimal for consecutive incorporations of AAs than the 30-minute incubation reaction system (10). Now that we have kinetic data on incorporation of multiple unnatural AAs in translation, our understanding of limiting features for multiple and single unnatural incorporations has become much clearer. Firstly, the summary of yields within 3 sec (Figure 2d) is strikingly similar to our yields after 30 min without preincubation obtained previously with very similar substrates and mRNAs (10). The only substantial differences between the two extensive sets of yields is that the two anticodon swaps were less inhibitory in the present system optimized for speed and accuracy, which should allow more flexibility in the design and engineering of artificial AA-tRNAs. The remarkable congruence of these two in vitro studies thus now clearly demonstrates the relevance of fast kinetics to traditional slow batch translations (and likely also to in vivo incorporations). The explanation is that the reactions competing with incorporation (shown below to be peptidyl-tRNA drop off) 8 Environment ACS Paragon Plus

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occur extremely fast, so long incubations are not helpful in overcoming them. In our earlier cases of single unnatural incorporations, rough correlations in yield between kinetics (12, 14, 15, 17) and slow batch incorporations were also seen for poorer substrates, such as N-methyl-AAs and bulky biotinyllysine (1, 24), but meaningful comparisons were not possible for the small non-N-alkyl AAs because they incorporated quantitatively under batch conditions (1, 9, 10). It thus seems likely that where incorporations of single unnatural AAs are inefficient either in vitro or in vivo, their incorporations could be improved rationally by factors that we found improve single incorporation kinetics (see Introduction). Secondly, in our fast kinetics studies, the biggest defect in ribosomal polymerization using artificial AA-tRNAs was caused by the penultimate dC introduced from the standard chemoenzymatic ligation method. This suggests that a tRNA-charging method that circumvents the penultimate dC such as ribozyme charging (7) or our new method for chemoenzymatic synthesis of N-NVOC-AApCpA (13) should dramatically improve yields of consecutive incorporations of unnatural AAs compared with the dC method. This hypothesis is tested in the following section.

3. Efficient incorporation of multiple unnatural AAs by charging with N-NVOCAA-pCpA or flexizyme Though syntheses of tRNAs charged by flexizyme ribozymes or our new chemoenzymatic synthesis of N-NVOC-AA-pCpA have been described, neither class of substrate had been assayed for kinetics of incorporation of multiple unnatural AAs in translation. It was thus important to rule out the possibility that the unnatural AAs might be inhibitory independent of the dC.



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As predicted in the section above, both dC-free charging methods indeed overcame the inhibitory effect of the dC in the setting of multiple unnatural AA incorporations to enable efficient yields (right-most 4 black squares in Figure 3) and kinetics (Supplementary Table 1) comparable to wild-type substrates.

4. The penultimate dC induces faster drop-off of peptidyl-tRNAs along the hexapeptide synthesis, not frameshifting Given that the biggest losses in processivity were due to the penultimate dC and that this is part of the standard chemoenzymatic method used for decades for incorporating unnatural AAs in translation, the remainder of this study investigated the mechanism of these losses. The mRNA mMA5KT used in the assays of the five consecutive incorporations contained five GCA alanine codons in a row. As triplet repeats were found in several cases to be slippery sequences that allowed frameshifting (25, 26), the processivity loss when incorporating five Ala-tRNA AlaB _dC substrates in a row ugc might be the result of frameshifting. We used radioactively-labeled Ser-tRNASer (reading the -1 frame AGC codon) and Gln-tRNAGln (reading the +1 frame CAG codon) to probe for potential frameshifting events in fMA4 synthesis with

Ala-tRNA AlaB _dC (see Supplementary Methods). However, neither Ser nor Gln ugc incorporation was observed in the experiments (Supplementary Figure 3), indicating no frameshifting occurred in the reaction. The other most likely reason for the processivity loss was peptidyl-tRNA drop-off during hexapeptide synthesis. We thus added peptidyl-tRNA hydrolase (PTH) in reactions with 5 Ala incorporations as PTH catalyzes deacylation of peptidyltRNAs free from the ribosome but not those bound to the ribosome (22). The


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synthesis was left for 5 sec to reach the saturation level before quenching with formic acid. After centrifugation of the quenched reaction mixture, the supernatant contained all the [3H]peptides hydrolyzed by PTH from the dropped-off peptidyl-tRNAs, whereas ribosome bound peptidyl-tRNAs were pelleted. The pelleted portion of [3H]peptides were then hydrolyzed off the tRNA by KOH. HPLC analyses revealed considerable intermediate peptides in the supernatant when synthesizing the hexapeptide

with

Ala-tRNA AlaB _dC ugc

(Figure

4c,d),

but

not

with

Ala-tRNA AlaB _enzymatic (Figure 4a,b). This indicated that the penultimate dC ugc facilitated drop off of the intermediate peptidyl-tRNAs from the ribosome. This confirms a long-standing prediction of increased drop off for unnatural substrates (1) and is consistent with the slower incorporation steps measured here as they would allow more time for drop off. Thus ribosomal drop off of intermediates, not frameshifting or stalling (27, 28), is responsible for the loss of processivities in five consecutive incorporations in the Ala-tRNA AlaB _dC case. ugc In conclusion, we enabled measurement of the rates of ribosomal synthesis of every intermediate in hexapeptide synthesis from unnatural AA-tRNA substrates. This revealed that the most detrimental change was not the anticodon swap, but rather the penultimate 2’ H group which increased peptidyl-tRNA drop off. This problem was circumvented in 2 ways to enable efficient ribosomal unnatural polymer syntheses.



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Methods Materials and experimental methods are described in the Supporting Information.

Supporting Information Supporting Information contains materials and methods, supplementary figures and data. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements We are very grateful to R. Fowler for technical assistance, R. Green for a PTH clone, and M. Ehrenberg and A. Borg for advice. This work was supported by grants from the Swedish Research Council (project grants and Linnaeus Uppsala RNA Research Centre to A.C.F.).

Declaration ACF owns patent US6977150 licensed to Ra Pharmaceuticals, Inc., owns shares in the company and is a member of its scientific advisory board.


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Figure 1. Kinetic assay of fMA5 synthesis. (a) HPLC separation of each intermediate in fMA5 synthesis. (b) Representative plots of normalized time-evolution of each intermediate in the kinetic Ala assay of five consecutive incorporations using native Ala-tRNA ugc _modified .

Figure 2. Kinetics of five consecutive incorporations using different AA-tRNAs. (a) Constructions of artificial AA-tRNAs. Synthetic tRNAs are based on native tRNAAla (sequence in black, anticodon redAlaB boxed) and lack modifications (orange). tRNA ugc has the wild-type UGC anticodon (red box).

tRNA AlaB tRNA AlaB ggu has GGU anticodon (purple) and guu has GUU anticodon (pink). Natural and unnatural AAs (black box) are charged on synthetic tRNAs. When the standard chemoenzymatic method is used, the tRNA has a penultimate dC (green). Rates (b) and processivities (c) at each incorporation step in the five consecutive incorporations using each of the AA-tRNA substrate are shown in bar figures and the yields of full-length products are plotted (d). Error bars represent standard deviations of the data points calculated as weighted averages from at least two independent experiments.

Figure 3. Yields of full-length products in five consecutive incorporations using different AA-tRNAs lacking dC. Data from Figure 2d are in color, with additional data in black. tBulk indicates E. coli total tRNA was added to the reaction as a control; rC_chemo indicates the substrates were synthesized from our N-NVOC-AA-pCpA method; rC_ribozyme indicates the substrates were synthesized via the flexizyme ribozyme acylation method. Error bars represent standard deviations of the data points calculated as weighted averages from at least two independent experiments.

Figure 4. Intermediate peptidyl-tRNAs drop-off along fMA5 hexapeptide synthesis. PTH was added to AlaB the reaction mixtures for fMA5 synthesis from [3H]fMet-tRNAfMet and from Ala-tRNA ugc _dC or

Ala-tRNA AlaB _enzymatic . The dropped-off fraction of intermediates was separated from the ribosome ugc bound fraction and both were analyzed on HPLC. Drop-off fraction (a) and ribosome bound fraction



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AlaB (b) for assay with Ala-tRNA ugc _dC . Drop-off fraction (c) and ribosome bound fraction (d) for assay

AlaB with Ala-tRNA ugc _enzymatic . Representative results are shown for duplicate experiments.


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Figure 1 43x14mm (300 x 300 DPI)

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Kinetics of Ribosome-Catalyzed Polymerization Using Artificial

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