chm212 formal laboratory report due date: … formal laboratory report due date: tuesday, ......

CHM212 Formal Laboratory Report

Due date: Tuesday, Nov. 30, at the beginning of class.

So far, you have been writing detailed laboratory notebook entries that prepared you for your

experiment and documented what you did. For the formal laboratory report, you will write a report of

your experiment in a format consistent with a scientific publication. Read

G.M. Whitesides, Adv. Mater. (2004) 16, 1375

which is a valuable paper describing the process of writing a scientific paper. Please also read the paper

E. Carrilho et al., Anal. Chem. (2009) 81, 5990

to get a feel for how a scientific paper should look and sound. Keep in mind that each journal has its

own particular style. In this citation, Carrilho is the first author and the remaining authors are

represented by the Latin term “et al.” (as a Latin term in an English paper, it must be italicized). The full

journal title is Analytical Chemistry, and the paper was published on page 5990 of the 2009 volume 81 of

the journal. You must access this paper online through the URI library: Go to

http://www.uri.edu/library/, click on E-journals and fill in the search fields as prompted.

Throughout this course, the importance of peer-reviewed sources has been emphasized. Usually peer

review is kept confidential, but a number of journals, EMBO Journal among them, have started to pull

aside the curtains on peer review. Read through the attached peer review correspondence to get a feel

for how peer review is carried out. I have also attached the source paper, for those interested in having

context for the peer review correspondence.

Your formal laboratory report will be based on Experiment 4. You will follow the outline of the 2009

Analytical Chemistry paper referred to above. You must have the following sections in this order:

Title. This should encapsulate the point of your report.

Author list. Your name.

Affiliation list. Which department and university? Address, contact info.

Abstract. In one or two paragraphs, summarize the purpose of the paper and your conclusions. This

section should contain a motivation of why the work is important, the important data (here, Ka) and the

outstanding conclusions.

http://www.uri.edu/library/

Introduction. A short section outlining why you needed to do this work: what outstanding problem

does it solve, why does it matter to solve this problem, and to what is it relevant? Use your textbook as

a reference (be sure to cite it) to discuss why Ka is important and why you need to use the Gran plot

here. Introduce what the Gran plot is and define each of the terms. You don’t need to derive it, just

refer to the place in the textbook where it was derived. Put your references in as footnotes. You

should, however, really read through the Gran plot explanation in your textbook so that you understand

why you construct it in the way that you do. Assume that activity and ionic strength are well known

quantities to a trained chemist (because they should be)—you don’t need to give defining equations for

them.

Materials and Methods (more appropriate than Experimental Design for your report). Briefly outline

what you did. For Experiment 1, for example, you would write something like “Solution of ~0.1M

sodium hydroxide solution was standardized using ___g potassium hydrogen phthalate (KHP; Sigma

Aldrich Product #____, Lot #____) dried at 110°C for 1 hour. TheAliquots each contained ~___g KHP and

were titrated to a phenolphthalein endpoint. Laboratory temperature pressure were ___°C and ___kPA.

The buret was used as-supplied without further calibration”. You don’t have to write that you added

sodium hydroxide dropwise, or that you thoroughly mixed the solution in a volumetric flask before

diluting to the mark. You provide only sufficient relevant details so that a properly trained chemist

could duplicate your work. For the Gran plot, you don’t necessarily have to state what volume of titrant

you added in each step because your paper will include the titration curve that shows the volume steps.

However, if the volume added were especially critical, then you would certainly mention it.

Results and Discussion. Present your results and discuss their significance. You should include the

following figures:

1. Figure 1: the raw data as a titration curve. Figure 1a: a plot focused on the endpoint region.

Figure 1b: a smaller plot showing all of your titration data.

2. Figure 2: the Gran plot. Do all linear fitting and extrapolation using the built-in features in Excel

(or similar program).

Label all of your graphs (and all items on the graphs) clearly and include figure captions. I should almost

be able to understand your paper by looking at the figures and captions alone. No figures should be

larger than half a page.

In your text, you should mention the endpoints you calculated using the first and second derivative plots

used as an approximation to Ve and discuss any discrepancy with the value finally determined from the

Gran plot. Discuss the reason for any differences (you may alternatively have dealt with this in your

introduction when you discussed the Gran plot). You should mention the results of any calculations (eg.

activity calculations) you needed in order to extract meaning from the Gran plot. You do not, however,

have to present sample calculations of ionic strength or activity. You do, however, have to provide a

knowledgeable chemist enough information to calculate everything that you calculated. The titration

curve is a sufficient summary of your raw data (your lab notebook must have the raw values in case they

ask for it) and then you specify the reagent amounts so that the reader could calculate the activity

(specify the ionic radius). Clearly state any approximations that you have made, such as using an

average ionic strength (and at what volume you calculated it and what value it has). You need to do all

of this in as few words as possible.

Conclusions. State the outcome of your experiments. Ensure (in your head—don’t write it) that what

you learned is consistent with what you said you wanted to learn (title, abstract and introduction) and

what you said you learned (abstract). If it isn’t, then rewrite what you said you wanted to learn.

Calculate your % error compared to the accepted value, and also state and justify whether or not the

two values are equivalent. Show the final statistical measures, not example calculations. State any

reasons for the discrepancy, such as whether or not your approximations are reasonable (state, then

how you would do this more accurately). Your procedure will have mentioned whether or not you

calibrated your glassware—unless this is expected to be a major reason for the discrepancy (and then

you must show the calculation that shows that it is a major source), you should not mention it. Do not

mention something unknown like “It may have been due to a systematic error”. You should mention

only things that you can support with evidence (and then you must state that evidence).

This report should be no more than 3 pages, typed and single-spaced with standard margins and 10-

point font. Focus on writing concisely—if you can say everything in 2 pages, so much the better! All

plots must be legible and should be part of the flow of the text, as they are in the Anal. Chem. (2009)

paper you read. That is, figures should not be on separate pages, but be incorporated into the

document. Your text should explicitly refer to the figures, as in “The Ka calculated from the Gran plot

(Figure 2) is ___...”.

Your mark will be based on how clearly you explain your experiment and the results and how

professionally the report is prepared. Proper grammar and correct spelling will both be marked. You

should also conform to the style advice offered in the Whitesides paper, eg. “Describe experimental

results uniformly in the past tense.”

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The EMBO Journal Peer Review Process File - EMBO-2010-75316

© European Molecular Biology Organization 1

Manuscript EMBO-2010-75316 Optimization of speed and accuracy of decoding in translation Ingo Wohlgemuth, Corinna Pohl and Marina V. Rodnina Corresponding author: Marina V. Rodnina, Max Planck Institute for Biophysical Chemistry Review timeline: Submission date: 08 July 2010 Editorial Decision: 18 August 2010 Revision received: 20 August 2010 Accepted: 24 August 2010 Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.)

1st Editorial Decision 18 August 2010

Thank you for submitting your manuscript for consideration by The EMBO Journal. Let me first of all apologise for the delay in getting back to you with a decision. This was caused by the limited availability of suitable referees during the ongoing summer holiday period. Your manuscript has now finally been seen by three referees whose comments are shown below. As you will see referees 1 and 2 are positive about the manuscript and would support publication here after appropriate revision. Referee 3 is more critical and feels that the manuscript may be better suited to publication in a more specialised journal. On balance and given the clear positive vote by the majority of the referees I have come to the conclusion that we should be happy to consider a revised manuscript in which you need to address the issues raised by the referees in an adequate manner. I should remind you that it is EMBO Journal policy to allow a single round of revision only and that, therefore, acceptance of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Peer Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process initiative, please visit our website: http://www.nature.com/emboj/about/process.html Thank you for the opportunity to consider your work for publication. I look forward to your revision. Yours sincerely,

jrdwyer

Typewritten Text

Record of peer review.



Editor The EMBO Journal ------------------------------------------------ REFEREE COMMENTS Referee #1 (Remarks to the Author): This manuscript describes a re-analysis of some of the kinetic parameters of tRNA selection, attempting to reconcile discrepancies in the literature between the Rodnina and Ehrenberg groups. Overall the manuscript presents some very useful data that resolve most of the lingering controversies and should allow the field to move forward in a unified manner. In addition, the manuscript presents some interesting new data that directly demonstrate where competition by near-cognate tRNAs has an impact during tRNA selection (on GTPase activation, but not accommodation), thus resolving an important question in the field. The manuscript was clearly written though I have some recommendations to improve the clarity and to minimize certain details. Once the various issues are addressed, I support publication of this manuscript in EMBO. Specific points 1. I found some of the references in the introduction to be surprising choices and would recommend that the authors be certain of these selections (for example, citing Sergiev et al. for error frequency measurements and two single molecule citations, Blanchard and Marshall, in reference to "reading the A-site codon ..."). 2. Repeated references to the "extremely" (p 4, bottom) or "dramatically" (p 7, bottom) different Km measurements by Ehrenberg for the near-cognate tRNA seem overstated - the differences are about 10-fold, which might be better referred to as "substantially" or "quite". 3. There remains some question as to whether accommodation is generally rate limiting for PT or not for the whole set of aa-tRNAs (indeed the authors acknowledge that the situation might be different for Pro-tRNAPro on the top of p 7) - I would recommend that the authors make a softer claim at the top of p 4 where they quite generally claim that accommodation is rate limiting for PT 4. Figure 3 did not seem necessary - all values were adequately discussed in the text and the points were clearly made. As a bar graph, it seems somewhat confusing to have both measured and calculated values being compared - especially when one needs to read the legend carefully to figure things out. Another option might be to say Ecalc, rather than E1, for example. 5. Bottom p 8 - why is there only one value for P (= 0.03) when there should be a P value for both the different buffers. This was confusing. 6. The analysis of the discrepancy between the Ehrenberg and Rodnina groups is very clearly resolved and this is important information to be disseminated. That said, once it is made clear that the PEP and NTPs are chelating some of the Mg preferentially from the near cognate samples, the problem becomes immediately clear. I suspect that panels A-C of Figure 4 could be seen as overkill in establishing this point, since the most interesting information is found in the titration in Figure 4D. Also, it would be useful to the reader if the authors help them through the calculations that determine what will be the final concentration of Mg in the assays that the Ehrenberg group used, given the chelation (starting at 5 mM Mg, subtract 2 mM for the GTP and ATP, and then half of the remaining Mg when PEP is at the Kd of 10 mM). I found myself doing these calculations as I read, and I think it would be more straightforward to show these calculations in a clear way (perhaps a table??). 7. p 13, "If the ribosomes were designed in the way ..." should be changed to "If ribosomes function in the way proposed ..." - the term "design" has undesired connotations ("intelligent design") in this context. 8. p 14/15 - discussion of the relative contributions of thermodynamic and kinetic discrimination would be clearer with a few changes:



p14, sentence 4 - "Although excess near- and non-cognate ..." change to "Although excess near- and non-cognate ternary complexes reduce the rate of GTP hydrolysis in the cognate ternary complex, the overall faster rates of GTP hydrolysis relative to peptide bond formation buffer the inhibitory effects ..." p14, near middle - "This suggests ... is oversimplified and does not provide reliable predictions. ..." Could be softened to "This suggests that the predicted 7-fold decrease in the rate of dipeptide formation due to competition (Lovmar and Ehrenberg, 2006) will not be observed. Instead, in a model where there is competition between ternary complexes at the codon recognition step, but not after GTP hydrolysis, ..." Referee #2 (Remarks to the Author): The decoding process in translation involves a selection step during which aminoacyl-tRNA (aa-tRNA) is delivered by EF-Tu-GTP to the A-site of the bacterial ribosome and accepted or rejected, an irreversible GTP hydrolysis step after which EF-Tu-GDP is released, and a proof-reading step during which the acceptor stem of cognate aa-tRNA is either 'accommodated' in the ribosome's peptidyltransferase center or, in the case of near-cognate aa-tRNAs, preferentially released. There is a trade-off between the rate of this process and its accuracy, and it has generally been accepted that in order to permit rapid translation, the ribosome does not use its full inherent capacity for discrimination between cognate and near-cognate aa-tRNAs. However, Ehrenberg and colleagues recently reported a much lower mis-sense error rate in vitro than previously observed, coupled with an extremely high Km for near-cognate aa-tRNA, and a rate constant for peptidyl transfer that is not limited by the preceding accommodation step (Johanssen et al., 2008). Confirmation of these observations would necessitate significant revision of important aspects of current models for the translation process. Wohlgemuth et al. have accordingly undertaken thorough kinetic analyses to re-examine details of the speed and accuracy of the decoding process. They report that the rate constants and Km's for dipeptide formation at different temperatures and in different buffers are compatible with previous reports from various groups. However, in contrast to (Johanssen et al., 2008) but consistent with other reports (Pape et al., 1998, 1999), Wohlgemuth et al. report that the accommodation step for Phe-tRNA(Phe) is rate-limiting for peptide bond formation, and that the error frequency in vitro is 3 x 10-3 which is consistent with previous reports in vitro (e.g. Gromadski et al., 2006) and in vivo, but is four orders of magnitude greater than reported by Johanssen et al. These discrepancies are not due to differences in the activities of translational components, but the low error frequency reported by Johanssen et al. appears to be due to the low level of free Mg2+ in their 'polymix' buffer (due to uncompensated chelation of Mg2+ by NTPs and phospoenolpyruvate) which selectively impairs translation reactions involving near-cognate substrates. Wohlgemuth et al. also report that the efficiency of the proofreading stage is ensured by a much greater rate of accommodation for cognate than for near-cognate tRNAs, coupled with a rapid rate of rejection of the latter. The similarity in Km values for cognate and near-cognate ternary complexes described here and elsewhere (Gromadksi and Rodnina, 2004a) indicates that the ribosome does not utilize its inherent potential for substrate selection at the binding stage, in favor of ensuring speed of polypeptide synthesis. These data raise the question of how ribosomes escape inhibition in vivo by the presence of an excess of near-cognate ternary complexes. Wohlgemuth et al. report that the rate of GTP hydrolysis in cognate ternary complexes incubated with programmed ribosomes is significantly reduced by addition of an excess of competing noncognate complexes whereas the rate of subsequent peptide bond formation is unaffected. These results lead to the conclusion that the high rate of GTP hydrolysis in cognate complexes ensures rapid polypeptide synthesis (at the expense of fidelity) and also precludes a reduction in this rate by the presence of competing near-cognate ternary complexes. In summary, in this very clearly presented manuscript, Wohlgemuth et al. describe data that provide new insights into the decoding process and that support and extend the kinetic discrimination model



for selection of cognate aa-tRNAs by the ribosome that was previously proposed by Rodnina, Wintermeyer and colleagues. MINOR COMMENTS There are a few typographical and other errors that should be corrected (a) p4, line 26. "capable of increasing" instead of "capable to increase" (b) p7, lines 17-18. "Gromadski et al., 2004" is not present in the reference list (c) p9, line 12. The authors must at some point define 'PEP'. (d) p9, line 26. Do the authors mean "correspondingly" rather than "respectively"? Referee #3 (Remarks to the Author): The kinetics of the decoding step of translation is important for understanding how the ribosome can achieve optimal speed and accuracy. In principle the mechanism is well established. However, the Ehrenberg lab published recently some surprising data and reported a missense error rate that is about four orders of magnitude lower than previously established data. In the present paper Wohlgemuth and colleagues have carefully re-analysed the kinetics of peptid bond formation in two different buffer systems and have made considerable effort to reproduce the recent data from the Ehrenberg lab. Wohlgemuth and colleagues present convincing evidence that the unusually low missense error rate reported by the Ehrenberg lab was obtained due to an experimental mistake. Moreover, Wohlgemuth and colleagues show that an excess of non- and near-cognate ternary complexes reduces the rate of cognate GTPase but not of cognate peptide bond formation. The present paper is well written and the results are important as they clarify a recently introduced controversy. However, as the paper does not appear to present a sufficiently significant conceptual advance in our understanding of ribosomal decoding it may be better suited for a more specialised journal. 1st Revision - authors' response 20 August 2010

Referee #1 (Remarks to the Author): 1. I found some of the references in the introduction to be surprising choices and would recommend that the authors be certain of these selections (for example, citing Sergiev et al. for error frequency measurements and two single molecule citations, Blanchard and Marshall, in reference to "reading the A-site codon ..."). We checked the references and replaced Sergiev et al. by a recent review on misreading (Drummond and Wilke, 2009), where the type of missense errors observed in Sergiev et al. (i.e. errors frequencies measured in genetic screens) is included. However, Blanchard 2004 and Marshall 2008 is a correct reference for codon reading, which was identified as a distinct kinetic step in those single molecule studies. 2. Repeated references to the "extremely" (p 4, bottom) or "dramatically" (p 7, bottom) different Km measurements by Ehrenberg for the near-cognate tRNA seem overstated - the differences are about 10-fold, which might be better referred to as "substantially" or "quite". Done, p. 4. 3. There remains some question as to whether accommodation is generally rate limiting for PT or not for the whole set of aa-tRNAs (indeed the authors acknowledge that the situation might be different for Pro-tRNAPro on the top of p 7) - I would recommend that the authors make a softer claim at the top of p 4 where they quite generally claim that accommodation is rate limiting for PT



Done, p. 4 4. Figure 3 did not seem necessary - all values were adequately discussed in the text and the points were clearly made. As a bar graph, it seems somewhat confusing to have both measured and calculated values being compared - especially when one needs to read the legend carefully to figure things out. Another option might be to say Ecalc, rather than E1, for example. We would prefer to keep Figure 3, because visual information is usually more convincing than numbers, it is easier to compare data, and the standard deviation is indicated. Many people would not consider numbers in the text (without a Figure) as data. E1 was changed to Ecalc, as suggested. 5. Bottom p 8 - why is there only one value for P (= 0.03) when there should be a P value for both the different buffers. This was confusing. Clarification added (P is 0.03 in both buffers), p. 8. 6. The analysis of the discrepancy between the Ehrenberg and Rodnina groups is very clearly resolved and this is important information to be disseminated. That said, once it is made clear that the PEP and NTPs are chelating some of the Mg preferentially from the near cognate samples, the problem becomes immediately clear. I suspect that panels A-C of Figure 4 could be seen as overkill in establishing this point, since the most interesting information is found in the titration in Figure 4D. Fig. 4D provides the final evidence as to the differential effect of Mg2+ on cognate and near-cognate decoding; however, Figs. 4A-B are important to build up the case about the Mg2+-chelating properties of PEP. In A, we show that the results of Ehrenberg can be reproduced with the concentrations of NTPs and PEP used in that paper, but it is not clear yet which component is responsible for the effect. In B, we show that the effect is due to PEP and that the effect can be remedied by Mg2+ addition. Also the shape of the time courses showing differences in endpoints is crucial. We think it is important to keep Figures 4A, B, and D as is to provide convincing evidence for a critical reader. Figure 4C was meant to further support the Mg2+ effect, but it is not as essential as the other figures and – following the suggestion of the reviewer - we removed it and revised the text on p. 10 accordingly. Also, it would be useful to the reader if the authors help them through the calculations that determine what will be the final concentration of Mg in the assays that the Ehrenberg group used, given the chelation (starting at 5 mM Mg, subtract 2 mM for the GTP and ATP, and then half of the remaining Mg when PEP is at the Kd of 10 mM). I found myself doing these calculations as I read, and I think it would be more straightforward to show these calculations in a clear way (perhaps a table??). We added a sentence on p. 10 to explain the calculation of free Mg2+. A table with the values would be overdone, because there are only two conditions (with 1 mM ATP / 1 mM /GTP / 10 mM PEP or the double concentrations), and because the free Mg2+ concentration is only an estimation (this consideration is now also added on p. 10). The main point that we want to make is that high concentrations of PEP chelate Mg2+ and from a certain point on this affects decoding by near-cognate and cognate ternary complexes differently, which leads to apparently low error rates. 7. p 13, "If the ribosomes were designed in the way ..." should be changed to "If ribosomes function in the way proposed ..." - the term "design" has undesired connotations ("intelligent design") in this context. Done 8. p 14/15 - discussion of the relative contributions of thermodynamic and kinetic discrimination would be clearer with a few changes:



p14, sentence 4 - "Although excess near- and non-cognate ..." change to "Although excess near- and non-cognate ternary complexes reduce the rate of GTP hydrolysis in the cognate ternary complex, the overall faster rates of GTP hydrolysis relative to peptide bond formation buffer the inhibitory effects ..." p14, near middle - "This suggests ... is oversimplified and does not provide reliable predictions. ..." Could be softened to "This suggests that the predicted 7-fold decrease in the rate of dipeptide formation due to competition (Lovmar and Ehrenberg, 2006) will not be observed. Instead, in a model where there is competition between ternary complexes at the codon recognition step, but not after GTP hydrolysis, ..." Done, with thanks for valuable suggestions. Referee #2 (Remarks to the Author): MINOR COMMENTS There are a few typographical and other errors that should be corrected (a) p4, line 26. "capable of increasing" instead of "capable to increase" (b) p7, lines 17-18. "Gromadski et al., 2004" is not present in the reference list (c) p9, line 12. The authors must at some point define 'PEP'. (d) p9, line 26. Do the authors mean "correspondingly" rather than "respectively"? All errors corrected Referee #3 (Remarks to the Author): Referee 3 had no specific objections. As to the general criticism of limited conceptual advance, we would like to stress that the clarification of the "recently introduced controversy" is absolutely crucial, as it has far-reaching consequences for understanding bacterial fitness, modeling of gene expression by systems biology approaches, modeling response to environmental stress, cross-talk between translation and transcription, and understanding the quality control mechanisms of gene expression. Conceptually, we show for the first time that the GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation. This, we feel, confers sufficient experimental and conceptual importance to be published in EMBO J.

Optimization of speed and accuracy of decodingin translation

Ingo Wohlgemuth1, Corinna Pohl1

and Marina V Rodnina*

Department of Physical Biochemistry, Max Planck Institute forBiophysical Chemistry, Gottingen, Germany

The speed and accuracy of protein synthesis are fundamen-

tal parameters for understanding the fitness of living cells,

the quality control of translation, and the evolution of

ribosomes. In this study, we analyse the speed and accuracy

of the decoding step under conditions reproducing the high

speed of translation in vivo. We show that error frequency is

close to 10�3, consistent with the values measured in vivo.

Selectivity is predominantly due to the differences in kcat

values for cognate and near-cognate reactions, whereas the

intrinsic affinity differences are not used for tRNA discrimi-

nation. Thus, the ribosome seems to be optimized towards

high speed of translation at the cost of fidelity. Competition

with near- and non-cognate ternary complexes reduces the

rate of GTP hydrolysis in the cognate ternary complex, but

does not appreciably affect the rate-limiting tRNA accom-

modation step. The GTP hydrolysis step is crucial for the

optimization of both the speed and accuracy, which explains

the necessity for the trade-off between the two fundamental

parameters of translation.

The EMBO Journal (2010) 29, 3701–3709. doi:10.1038/

emboj.2010.229; Published online 14 September 2010

Subject Categories: proteins

Keywords: fidelity; rapid kinetics; ribosome; translation;

tRNA selection

Introduction

Speed and accuracy of translation determine cell growth and

the quality of newly synthesized proteins. The rate of protein

elongation in Escherichia coli was estimated to be 4–22 s�1

per codon at 371C (Bremer and Dennis, 1987; Sorensen and

Pedersen, 1991; Liang et al, 2000; Proshkin et al, 2010).

Estimations of error frequencies range between 10�5 and

10�3 depending on the type of measurement, concentrations

and nature of tRNAs that perform misreading, and the

mRNA context (Parker, 1989; Kramer and Farabaugh, 2007;

Drummond and Wilke, 2009). Aminoacyl-tRNAs (aa-tRNAs)

are delivered to the ribosome in a ternary complex with EF-Tu

and GTP. The selection of an aa-tRNA that is cognate to the

mRNA codon is accomplished by a kinetic discrimination

mechanism (Pape et al, 1999; Gromadski and Rodnina,

2004a), which operates in two stages: initial selection and

proofreading (Thompson and Stone, 1977; Ruusala et al,

1982). Initial selection begins with the codon-independent

initial binding of a ternary complex, EF-Tu .GTP .aa-tRNA, to

the ribosome (Figure 1A; Rodnina et al, 1996; Gromadski and

Rodnina, 2004a; Diaconu et al, 2005). Initial binding is

followed by sampling the A-site codon in the decoding centre

by the anticodon of the aa-tRNA (Blanchard et al, 2004;

Marshall et al, 2008). Correct codon–anticodon pairing re-

sults in conformational changes of the ribosome, aa-tRNA,

and EF-Tu (Rodnina et al, 1994; Ogle et al, 2001, 2002;

Rodnina and Wintermeyer, 2001; Cochella and Green, 2005;

Pan et al, 2008; Schmeing et al, 2009; Schuette et al, 2009;

Villa et al, 2009), which ultimately lead to GTP hydrolysis by

EF-Tu (Rodnina et al, 1995; Pape et al, 1999; Gromadski and

Rodnina, 2004a; Lee et al, 2007). If the codon–anticodon

duplex contains a mismatch, that is, the aa-tRNA is near-

cognate to the codon, the concerted rearrangements do not

occur, or are different (Ogle et al, 2002), and GTPase activa-

tion of EF-Tu is slow (Pape et al, 1999; Gromadski and

Rodnina, 2004a; Gromadski et al, 2006; Lee et al, 2007). In

addition, near-cognate ternary complexes dissociate rapidly

from the ribosome, whereas cognate ones are bound very

tightly (Thompson and Karim, 1982; Pape et al, 1999;

Gromadski and Rodnina, 2004a; Cochella and Green, 2005;

Daviter et al, 2006). Partitioning between GTPase activation

and ternary complex dissociation strongly favours acceptance

of cognate and rejection of near-cognate ternary complexes.

The hydrolysis of GTP irreversibly separates the initial selec-

tion stage from the proofreading stage. During the proof-

reading stage, the acceptor stem of aa-tRNA released from

EF-Tu .GDP moves into the ribosome and accommodates

at the peptidyl transferase centre. The accommodation of

cognate aa-tRNA is rapid and efficient; in contrast, the

accommodation of near-cognate tRNA is slow and results in

the preferential rejection of near-cognate aa-tRNA (Pape et al,

1999). Accommodation is followed by, and in some cases

may limit the rate of, irreversible peptide bond formation

(Pape et al, 1999; Bieling et al, 2006).

One major unresolved question concerns the maximum

capacity of the ribosome for tRNA discrimination. In the

codon-recognition complex, the Kd values for cognate and

near-cognate ternary complexes differ by about 1000-fold

(Thompson and Karim, 1982; Ogle et al, 2002; Gromadski

and Rodnina, 2004a; Daviter et al, 2006). However, this large

inherent DDG1 of binding is not fully used. This is because in

the cognate complex the following steps of GTPase activation

and GTP hydrolysis are rapid, precluding the equilibration of

the binding step and increasing the KM (Gromadski and

Rodnina, 2004a). Such design allows for rapid translation;

however, the maximum of intrinsically possible discrimina-

tion is not achieved, a phenomenon known as trade-off

between speed and accuracy (Thompson and Karim, 1982;

Lovmar and Ehrenberg, 2006). In fact, despite the largeReceived: 8 July 2010; accepted: 24 August 2010; published online:14 September 2010

*Corresponding author. Department of Physical Biochemistry, MaxPlanck Institute for Biophysical Chemistry, Am Fassberg 11, Gottingen,37077, Germany. Tel.: þ 49 0 551 201 2902; Fax: þ 49 0 551 2012905;E-mail: [email protected] authors contributed equally to this work

The EMBO Journal (2010) 29, 3701–3709 | & 2010 European Molecular Biology Organization | All Rights Reserved 0261-4189/10

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&2010 European Molecular Biology Organization The EMBO Journal VOL 29 | NO 21 | 2010

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Paper after peer review. The referees see an earlier draft of the paper and base their comments on that version.

differences in the affinities and in the rates of forward

reactions for cognate and near-cognate aa-tRNAs both at

initial selection and proofreading stages, the error frequency

obtained in vitro was close to 10�2–10�3 under a variety of

conditions (Pape et al, 1999; Gromadski and Rodnina, 2004a;

Cochella and Green, 2005; Daviter et al, 2006; Lee et al,

2007). Recently, Ehrenberg and colleagues reported a sub-

stantially lower missense error rate of 3�10�7, as calculated

from the kcat/KM values measured for Phe-tRNA on a cognate

UUU and a near-cognate CUU codon in ‘polymix’ buffer at

371C (Johansson et al, 2008). The KM value for the near-

cognate aa-tRNA was quite high at 420mM. These data may

have several important implications. First, the high KM value

would suggest that the ribosome is capable of increasing the

DDG1 of cognate and near-cognate codon–anticodon interac-

tions to an extent that has not been observed before. Second,

due to the high KM value for the near-cognate substrate, there

would be practically no competition with the cognate ones,

which, as a consequence, would decode their codons at

maximum speed. Third, very high accuracy would suggest

that there is essentially no trade-off between speed and

accuracy of decoding. Finally, the authors argued that,

under the conditions used by them, the accommodation

step was not rate-limiting for peptide bond formation

(Johansson et al, 2008). The conceptual importance of

these findings for understanding the decoding mechanism

has prompted us to re-analyse the kinetics of peptide bond

formation under conditions reproducing the high speed of

translation in vivo and to directly measure the error fre-

quency in polymix buffer at 371C. For comparison, we used

two buffer systems that were reported to mimic conditions of

rapid protein synthesis in the cell, the polymix buffer used by

Johansson et al (2008) and the HiFi buffer used by our group

(Gromadski and Rodnina, 2004a). Using rapid kinetics tech-

niques, we compared the rate constants of peptide bond

formation for cognate and near-cognate tRNAs, measured

error frequencies, and studied the effect of competition on the

speed of decoding by the cognate aa-tRNA.

Figure 1 Dipeptide formation on a cognate codon. (A) Schematic of the decoding mechanism. Kinetically resolved steps are indicated(Gromadski and Rodnina, 2004a). (B) Dipeptide (fMetPhe) formation in HiFi (open circles) or polymix (closed circles) buffer at 371C.Increasing amounts of ternary complex (TC¼EF-Tu .GTP .Phe-tRNAPhe) were added to initiation complex with a UUC codon at the A site.(C) Time courses of accommodation and dipeptide formation. Peptide bond formation is shown as consumption of fMet-tRNAfMet substrate(left y-axis). Accommodation was monitored by the fluorescence decrease after the binding of EF-Tu .GTP .Phe-tRNAPhe(Prf16/17) to theinitiation complex (right y-axis); a.u., arbitrary units.

Ribosome decodingI Wohlgemuth et al

The EMBO Journal VOL 29 | NO 21 | 2010 &2010 European Molecular Biology Organization3702

Results

Rate of dipeptide formation with cognate aa-tRNA

Time courses of fMetPhe formation were measured by

quench-flow (Materials and methods section), mixing ribo-

somal initiation complex containing fMet-tRNAfMet at the

P site and a UUC codon at the A site with excess of ternary

complex, EF-Tu .GTP .Phe-tRNAPhe. In contrast to our routine

protocol (Gromadski and Rodnina, 2004a), the ternary com-

plex was formed in situ and used without further purification,

as described previously (Johansson et al, 2008). Apparent

rate constants (kapp) for peptide bond formation were deter-

mined at increasing concentrations of ternary complex. The

concentration dependence of kapp was hyperbolic and at

saturation gave the rate constant of dipeptide formation,

kdip, of about 170 s�1 in polymix buffer at 371C (Figure 1B).

The titration yielded also KM¼ 4.8 mM (Figure 1B; Table I), in

agreement with the results of Johansson et al (2008), who

reported values of kdip¼ 130 s�1 and KM¼ 4.5 mM. In HiFi

buffer at 371C, the rate of dipeptide formation was higher,

200 s�1, and the KM value was approximately the same

(4 mM). Thus, the rate constant of peptide bond formation

at 371C in either buffer was compatible with the rate of

elongation in vivo. Analogous experiments in HiFi buffer at

201C, in which purified and non-purified ternary complexes

were compared or an excess of ribosome complex over ternary

complex was used, yielded kdip values between 12 (a value

close to 7 s�1 was obtained previously using purified compo-

nents and excess of initiation complex over ternary complex

(Gromadski and Rodnina, 2004a)) and 22 s�1 (excess unpur-

ified ternary complex (Table I)); the latter value reproduced

the value reported for polymix buffer at 201C, 26 s�1

(Johansson et al, 2008); thus, the two buffer systems yielded

comparable rates of peptide bond formation. The KM values

were consistently higher with unpurified ternary complexes,

suggesting that the effective concentration of EF-Tu .GTP .Phe-

tRNAPhe in the unpurified mixture was overestimated. It

should be noted that the rate of peptide bond formation is

strongly affected by Mg2þ and polyamines; therefore, compar-

isons with other buffer systems (e.g. without or with different

polyamines) have to be carried out with caution.

Next, we examined whether the accommodation step is in

fact not limiting the rate of peptide bond formation in

polymix at 371C, as suggested previously (Johansson et al,

2008). The time course of the accommodation step

was monitored by the fluorescence change in Phe-

tRNAPhe(Prf16/17); the label reports conformational changes

and movements of the tRNA through the ribosome but is

insensitive to peptide bond formation (Pape et al, 1998). The

resulting time course was biphasic, with the fast step

(fluorescence increase) reflecting the binding of the ternary

complex to the ribosome and all following steps up to GTP

hydrolysis (Figure 1C), whereas the slower phase (fluores-

cence decrease) reflects the accommodation of the aa-tRNA at

the A site (Pape et al, 1998; Gromadski and Rodnina, 2004a).

The rate of the second step at the present conditions was

40 s�1. The rate of peptide bond formation measured by

quench-flow in parallel was the same, 40 s�1, indicating

that, at least with Phe-tRNAPhe, accommodation is rate-limit-

ing for peptide bond formation not only in the buffers used

previously (Pape et al, 1998, 1999), but in polymix buffer at

371C (pH 7.5) as well. The rate-limiting step of peptidyl

transfer to other aa-tRNAs (e.g. prolyl-tRNA for which the

reaction seems to be slow (Pavlov et al, 2009)) remains to be

determined.

Missense error frequency

To estimate the error frequency of decoding, we used two

independent methods. First, we used the approach proposed

by Johansson et al (2008). An excess of ternary complex with

Phe-tRNAPhe was added to initiation complex displaying

the near-cognate CUC (Leu) codon at the A site; time courses

of fMetPhe formation were measured at increasing ternary

complex concentrations in polymix buffer at 371C

(Figure 2A). As the reactions were carried out at multiple-

turnover conditions, kapp values were determined by linear

fitting of the initial parts of the time courses. The concentra-

tion dependence of kapp was hyperbolic with kdip (near-

cognate)¼ 0.26 s�1 and KM¼ 1.9 mM (Figure 2B). From the

ratio between the kdip and KM values for near-cognate and

cognate codons, an error frequency of 3�10�3 was obtained.

This value was four orders of magnitude higher than that

reported by Ehrenberg and colleagues (Johansson et al,

2008), but in full agreement with our previous data measured

at 201C in HiFi buffer (Gromadski and Rodnina, 2004a;

Gromadski et al, 2006).

The KM value of 1.9 mM (Figure 2B), which is quite

different from that reported by Ehrenberg and colleagues

(near-cognate KM420mM), was further verified by the alter-

native approach by measuring dipeptide formation under

single-turnover conditions with an excess of initiation com-

plex over near-cognate ternary complex (Figure 2C). In the

absence of a competing cognate ternary complex, the initial

selection step is inefficient and near-cognate substrates are

rejected at the proofreading stage. Two parameters can be

extracted from the analysis of the time courses: The ampli-

tude of fMetPhe formation (fMetPhe/TC), which gives the

error frequency of proofreading alone, and the apparent rate

Table I Rates of cognate peptide bond formation

Buffer T (1C) Conditions kdip (s�1) KM (mM) kdip/KM (mM�1 s�1)

Polymix 37 Unpurified, TC4IC 174±4 4.8±0.2 36Polymixa 37 Unpurified, TC4IC 130±10 4.5±0.5 29HiFi 37 Unpurified, TC4IC 200±40 3.9±1.9 51Polymixa 20 Unpurified, TC4IC 26±1 2.0±0.3 13HiFi 20 Unpurified, TC4IC 22±4 1.4±0.5 16HiFi 20 Purified, TC4IC 14±4 0.5±0.4 28HiFi 20 Purified, IC4TC 12±1 0.3±0.1 40

IC, initiation complex; TC, ternary complex.aFrom Johansson et al (2008).


&2010 European Molecular Biology Organization The EMBO Journal VOL 29 | NO 21 | 2010 3703

constant (kapp), which at saturation is the sum of the

kdipþ krejection; the concentration dependence of either kapp

or of the amplitude yields an independent estimate of the KM

value for near-cognate binding. The time courses of dipeptide

formation were single-exponential, and the concentration

dependencies of both amplitude and kapp were hyperbolic,

yielding KM values of 1.5–2.0 mM (Figure 2D), consistent with

the KM value determined above under conditions of multiple

turnover. The yield of dipeptide at saturation, that is, the

error frequency of proofreading, was 0.055, in agreement

with our previous value, 0.06 (Gromadski and Rodnina,

2004a). An alternative calculation using the equation

kapp¼ kdipþ krejection, combined with the value kdip(near-

cognate)¼ 0.26 s�1, gives krejection¼ 7.1 s�1 and an error

frequency of proofreading of 0.035; in agreement, within

experimental error, with the value measured directly (also

see below).

As a second independent approach to determine the error

frequency, we directly measured the incorporation of Phe and

Leu on a CUC codon in a competition assay in which the

respective ternary complexes with [3H]Phe-tRNAPhe and

[14C]Leu-tRNALeu(CUC) were present in excess over ribo-

somes, both in polymix and HiFi buffer at 371C. For this

experiment, the two ternary complexes were purified by gel

filtration. Misincorporation was quantified by determining

the amounts of fMetLeu (cognate) and fMetPhe (near-cog-

nate) formed. The overall error frequency normalized for

equal concentrations for Phe-tRNAPhe and Leu-tRNALeu was

5.6�10�3 for polymix (Figure 3), in agreement with the value

of 3�10�3 determined above from the ratio of kdip/KM

values. The misincorporation error frequency in HiFi buffer

at 371C was slightly lower (4�10�3) and similar to that

measured in HiFi buffer at 201C (2�10�3; (Gromadski

and Rodnina, 2004a)). The relative contributions of initial

selection (I) and proofreading (P) stages were quite similar in

HiFi and polymix buffers (I¼ 0.12 and 0.22 in HiFi and

polymix, respectively; P is about 0.03 in both buffers).

Thus, the results obtained by the two different approaches

yield error frequencies in the range of 3–6�10�3.

Potential sources of apparent very high fidelity

The fact that very similar error frequencies were observed in

polymix and HiFi buffers excludes buffer composition as a

source of the marked fidelity differences observed in our

experiments and in those of Ehrenberg and colleagues.

Furthermore, rate constants of cognate dipeptide formation

reported by the two groups are very similar, arguing against

Figure 2 Dipeptide formation on a near-cognate codon. (A) Time courses of fMetPhe formation under multiple-turnover conditions in polymixbuffer. A fixed amount of initiation complex (CUC codon at the A site) was mixed with varying concentrations of EF-Tu .GTP .Phe-tRNAPhe: 10(closed circles), 5 (open circles), 2 (closed triangles), and 1 mM (open triangles). (B) Concentration dependence of dipeptide formation undermultiple-turnover conditions. (C) Time courses of dipeptide formation under single-turnover conditions measured with limiting concentrationof purified ternary complex and varying concentrations of initiation complex: 6 (closed circles), 3 (open circles), 1.2 (closed triangles), and0.6mM (open triangles). (D) Rate (closed circles, left y-axis) and amplitude (open circles, right y-axis) dependence of dipeptide formation undersingle-turnover conditions.

Figure 3 Error frequencies in HiFi and polymix buffers. Errorfrequencies were determined for polymix (black bars) and HiFi(white bars) buffers. The contribution of initial selection (I) wascalculated from the overall error frequency (E) and the measurederror frequency of proofreading (P). The calculated error frequency(Ecalc) was determined from the ratio of kcat/KM values determinedfor fMetPhe dipeptide formation on near-cognate and cognatecodons. The overall error frequency reported by Johansson et al(2008) is shown for comparison (J).



significant differences in the activities of ribosomes, EF-Tu, or

tRNAs. Differences due to the mRNA constructs were exam-

ined and excluded previously (Johansson et al, 2008).

Furthermore, the experimental setup (excess of initiation

complexes over ternary complexes or vice versa) does not

seem to have a strong effect. However, we note that

Johansson et al (2008) applied different experimental proto-

cols for the cognate and near-cognate decoding. In the

cognate case, ternary complex and initiation complex mix-

tures were prepared under the same conditions, that is, in the

presence of 1 mM ATP, 1 mM GTP, and 10 mM phosphoenol

pyruvate (PEP). However, when working with the near-

cognate complexes, Johansson et al (2008) used twice the

concentrations of ATP, GTP, and PEP (2 mM, 2 mM, and

20 mM, respectively) in the ternary complex mixture. When

we measured the concentration dependence of the initial

velocity of dipeptide formation on a near-cognate codon

using ATP, GTP, and PEP at these high concentrations, we

also observed very low kapp values that did not saturate even

at 15 mM ternary complex (Figure 4A, inset), indicating a very

high KM value similar to the data obtained by Ehrenberg and

colleagues. From the ratio of near-cognate and cognate kcat

and KM values obtained from these data, we could reproduce

the apparent very low error frequency of CUU decoding

reported by Johansson et al (2008) (Figure 4A).

Parameters such as pH, ionic strength, or free Mg2þ

concentration can be altered when reaction components are

added to the polymix buffer. In particular, the free Mg2þ

concentration decreases compared with the initial concentra-

tion (5 mM in polymix buffer) on the addition of 1 mM ATP,

1 mM GTP, and 10 mM PEP due to the chelation of Mg2þ . As

ATP and GTP each bind Mg2þ at a 1:1 ratio, Ehrenberg and

colleagues pointed out that the free Mg2þ concentration

decreased correspondingly on addition of NTPs (Pavlov

et al, 2009). However, also PEP binds Mg2þ ions with

millimolar affinity, with a Kd value of about 10 mM at

100 mM monovalent salt (Manchester, 1980). In fact, it was

shown that PEP shifts the Mg2þ optimum of poly(Phe)

synthesis by 0.5 mM Mg2þ for each additional 1 mM PEP,

suggesting a binding ratio of approximately 0.5 Mg2þ/PEP at

5–10 mM PEP (Manchester and Alford, 1979). To test the

effect of chelating Mg2þ ions, we mixed purified initiation

complex with a CUU codon at the A site with an excess of

near-cognate EF-Tu .GTP .Phe-tRNAPhe complex and mea-

sured time courses of product formation in polymix buffer

(Figure 4B, note the logarithmic y-axis). Under conditions of

multiple turnover, that is, excess of near-cognate ternary

complexes over ribosomes, all ribosomes should eventually

form a dipeptide with the near-cognate aa-tRNA, reaching the

end level close to one fMetPhe/IC. The addition of 1 mM ATP

and 1 mM GTP not compensated by additional Mg2þ results

in a much lower extent of near-cognate dipeptide formation;

the addition of 10 mM PEP had a comparable effect. The effect

of adding GTP, ATP, and PEP without compensating Mg2þ

was cumulative and virtually abolished near-cognate dipep-

tide formation. Notably, from the 5 mM Mg2þ added, 2 mM

Figure 4 Effect of the Mg2þ concentration on dipeptide formation on near-cognate codons. (A) Catalytic efficiency (kcat/KM) of dipeptideformation on CUC or CUU codons with (þMg2þ ) or without (�Mg2þ ) addition of Mg2þ to compensate for Mg2þ -chelating compounds (ATP,1 mM; GTP, 1 mM; PEP, 10 mM; black bars); the published value (Johansson et al, 2008) obtained under the latter conditions is also indicated(J, grey bar). Inset: Dipeptide formation (kapp) in the presence of uncompensated NTPs and PEP (CUU codon). (B) Effect of Mg2þ -bindingcompounds on dipeptide formation. Ternary complex with Phe-tRNAPhe was added to initiation complex (CUU at the A site) in polymix bufferwithout further additions (open circles) or after addition of 1 mM ATP/GTP each (closed triangles); 10 mM PEP (open triangles,); or 1 mM ATP/GTP each and 10 mM PEP (open squares). ATP, GTP, and PEP were added with the ternary complex without compensating by Mg2þ asdescribed previously (Johansson et al, 2008). As a control, the same amounts of non-compensated ATP, GTP, and PEP were added to thecognate reaction (UUC codon) either with the ternary complex (as in Johansson et al., 2008; closed circles) or with the initiation complex(closed squares). (C) Mg2þ dependence of fMetPhe dipeptide formation on cognate (UUC, closed circles) and near-cognate (CUU, open circles)codons. Amplitudes are plotted against the concentration of free Mg2þ . Inset: time courses of dipeptide formation on the near-cognate CUUcodon at increasing concentration of Mg2þ (mM): 0.5 (closed diamonds), 1.5 (open squares), 2 (closed squares), 2.5 (open triangles), 3 (closedtriangles), 3.5 (open circles) and 5 (closed circles).



Mg2þ were chelated by ATP and GTP, and addition of 10 mM

PEP can be expected to further reduce free Mg2þ concentra-

tion to close to 1.5 mM assuming a Kd value of 10 mM

(Manchester, 1980) or even lower assuming the binding

ratio 0.5 Mg2þ/PEP (Manchester and Alford, 1979). The

cognate reaction (UUC codon at the A site) was not affected

appreciably when the NTPs and PEP were added with the

ternary complex, but was strongly impaired by pre-incuba-

tion with the initiation complex, suggesting that initiation

complexes are particularly sensitive to very low concentra-

tions of Mg2þ . The effect can be reversed by compensating

NTPs and PEP with Mg2þ (Figure 4B, open circles and data

not shown), suggesting that the low efficiency of dipeptide

formation observed with near-cognate aa-tRNA can be attrib-

uted to the Mg2þ -chelating properties of NTPs and PEP.

The reduced end points of near-cognate reactions at the

artificially low concentration of free Mg2þ caused by adding

NTPs and PEP suggest inactivation of the reaction compo-

nents. To determine the lowest free Mg2þ concentrations that

can be tolerated without significant inactivation, reaction end

points were measured at decreasing Mg2þ concentrations at

low PEP concentrations (0.5 mM that is sufficient for the

conversion to GTP of the submicromolar amounts of GDP

formed in the reaction) and GTP (0.5 mM, compensated by

Mg2þ ). At Mg2þ concentrations above 3 mM, the end point

of dipeptide formation was similarly high for cognate and

near-cognate reactions (Figure 4C), as expected for a well-

behaved system. At lower Mg2þ concentrations, the end

points of the near-cognate reaction were decreased more

than those of the cognate one, and both reactions were

abolished at 1 mM free Mg2þ . These data demonstrate that

in the multiple-turnover assay substrates are partially inacti-

vated at free Mg2þ concentrations below 3 mM. Thus, the

apparent low error frequency reported by Johansson et al

(2008) at free Mg2þ concentration close to 1.5 mM seems to

be due to the preferential impairment of the reaction with

near-cognate substrates due to the chelation of Mg2þ in the

presence of a large excess of NTPs and PEP.

Effect of ternary complex competition on GTP

hydrolysis and peptide bond formation

Our present data confirmed that the KM values for near-

cognate ternary complexes can be comparable to those for

the cognate complexes (Gromadski and Rodnina, 2004a).

This raises the question of how the ribosome deals with the

competition between cognate and excess near- and non-

cognate ternary complexes prevailing in vivo. To address

this question, we measured the rates of GTP hydrolysis and

peptide bond formation for mixtures of cognate, near-cog-

nate, and non-cognate ternary complexes on ribosomes con-

taining f[3H]Met-tRNAfMet at the P site and a UUC (Phe)

codon at the A site. Separate ternary complexes were pre-

pared and purified by gel filtration: the cognate ternary

complex EF-Tu .GTP .Phe-tRNAPhe and the mixture of near-

and non-cognate ternary complexes prepared from total tRNA

by aminoacylation with a mixture of 19 non-radioactive

amino acids excluding phenylalanine (Materials and methods

section). The ternary complex mixture was prepared in such

a way that the concentration of cognate Phe-tRNAPhe ternary

complex was fixed and the concentrations of near-cognate

and cognate complexes were varied. The ternary complex

mixture was added to the initiation complex, and time

courses of GTP hydrolysis and peptide bond formation were

measured by quench-flow. The observed rate reflects GTP

hydrolysis in the cognate ternary complex, because GTP

hydrolysis in the near- and non-cognate ternary complexes

is much slower (4100-fold), even in the absence of the

cognate competitor (Gromadski and Rodnina, 2004a;

Gromadski et al, 2006), and is not significant in the time

window used in this study. The rate of GTP hydrolysis in the

cognate complex decreased more than 10-fold on addition of

a 30-fold excess of competing ternary complexes (Figure 5).

However, the rate of peptide bond formation with cognate aa-

tRNA was not affected by the addition of near- and non-

cognate competitors (Figure 5). Thus, the competition be-

tween cognate and near- or non-cognate ternary complexes

reduced the rate of GTP hydrolysis, but not that of subse-

quent peptide bond formation.

Discussion

Accuracy of translation

The overall accuracy of protein synthesis on the ribosome is

determined by three basic selection stages: the selection of

ternary complexes before GTP hydrolysis at the initial selec-

tion stage; preferential rejection of near-cognate aminoacyl-

tRNAs at the proofreading stage after GTP hydrolysis

(Thompson and Stone, 1977; Ruusala et al, 1982; Rodnina

and Wintermeyer, 2001) and preferential release of near-

cognate peptidyl-tRNAs by the termination factors (Zaher

and Green, 2009). The missense error frequency of transla-

tion in the cell depends on the combined efficacy of these

three stages and on the abundance of the aa-tRNA cognate to

the given codon relative to the near-cognate competitors. The

combined error frequency of initial selection and proofread-

ing in vitro was observed to be close to 10�3 at different

temperatures in both polymix and HiFi buffers (this study

and Gromadski and Rodnina, 2004a, 2004b; Gromadski et al,

2006). Consistently, fidelity measurements conducted in the

full range of published buffer systems with tRNA mixtures on

heteropolymeric mRNA suggested that in vitro protein synth-

esis proceeds with an error rate of 2�10�3–1�10�2 (Zaher

and Green, 2009). A single initial miscoding event is then

amplified by preferential incorporation of a wrong amino acid

Figure 5 Effect of competition on cognate reaction. Rates (kapp) ofGTP hydrolysis (closed circles) and dipeptide formation (closeddiamonds) were measured on addition of increasing concentrationsof competing near- and non-cognate ternary complexes. x-axis,molar excess of near- and non-cognate over cognate ternarycomplexes.



at the following codon, followed by premature chain termi-

nation, thereby reducing the observed error frequency of

synthesis of a full-length protein approximately 10-fold

(Zaher and Green, 2009). Thus, the overall error frequency

is expected to be around 10�4, which may be further modu-

lated by the tRNA concentrations in the cell. Assuming an

average protein length in E. coli of about 300 amino acids

(Netzer and Hartl, 1997), this means that most of the proteins

in the cell are synthesized correctly.

According to the present results, both rate and fidelity of

decoding measured in vitro are fully compatible with the

values observed in vivo and provide new insight into the

mechanism of aa-tRNA selection at the proofreading stage.

In polymix buffer at 371C, the rate of accommodation is 650-

fold higher with cognate (170 s�1) than with near-cognate

(0.26 s�1) aa-tRNA. Conversely, rejection of near-cognate aa-

tRNA during accommodation is rapid (7.1 s�1), resulting in

efficient proofreading, whereas cognate aa-tRNA does not

dissociate to any significant extent during accommodation.

Optimization of the speed of protein synthesis

A significant trade-off between speed and accuracy is a

consequence of the necessity for rapid protein synthesis in

the cell. If ribosomes functioned in the way proposed by

Johansson et al (2008), the trade-off between the speed and

accuracy would be negligible, because the KM value for the

near-cognate ternary complex would be much higher than

that for the cognate one, and thus the discrimination between

aa-tRNAs would use the (huge) DDG1 of binding, in addition

to potential differences in kcat values. Our present results

exclude this scenario and further support the kinetic discri-

mination mechanism derived from kinetic measurements

performed under a variety of conditions (Pape et al, 1999;

Gromadski and Rodnina, 2004a; Gromadski et al, 2006; Kothe

and Rodnina, 2007). Although kcat values for the peptidyl

transfer reaction are grossly different between cognate and

near-cognate aa-tRNAs, KM values are very similar, indicating

that the inherent affinity differences between cognate and

near-cognate complexes are not used for selection (see below

and Gromadski and Rodnina, 2004a). Thus, the potential for

very accurate substrate selection in translation is sacrificed in

favour of speed.

It has been argued that if the KM values for cognate aa-

tRNA were comparable to those for near-cognate and non-

cognate substrates, this would lead to slow cognate amino-

acid incorporation due to competition by the large excess of

incorrect ternary complexes (Bilgin et al, 1988; Lovmar and

Ehrenberg, 2006). The present data suggest why this does not

occur. Although excess near- and non-cognate ternary com-

plexes reduce the rate of GTP hydrolysis in the cognate

ternary complex, the overall faster rates of GTP hydrolysis

relative to peptide bond formation buffer the inhibitory effect

such that a 10-fold decrease in the rate of GTP hydrolysis, due

to a high excess of competing ternary complexes, is not

reflected in a decrease of the rate of cognate peptide bond

formation (Figure 5). This suggests that the predicted seven-

fold decrease in the rate of dipeptide formation due to

competition (Lovmar and Ehrenberg, 2006) will not be

observed. Indeed, a more realistic model in which there is

competition between ternary complexes at the codon

recognition step, but not after GTP hydrolysis, gives results

that are consistent with the experimental data obtained at

201C. Predictions for 371C are more challenging, because

many parameters, in particular the KM values for the

GTPase reaction, cannot be determined experimentally due

to the very high reaction rate for the cognate substrate.

Nevertheless, estimates can be made based on the KM values

for the peptidyl transfer reaction and the rate constants of

GTP hydrolysis and peptide bond formation reported in this

study and in the study by Johansson et al (2008). Assuming

conservatively that (i) the KM values for the GTPase reaction

are similar to or higher than that for peptide bond formation

and (ii) the KM values for cognate (2% of total aa-tRNA) are

roughly the same as for the mixture of near-cognate (about

10% of total aa-tRNA) and non-cognate ternary complexes,

the rate of peptide bond formation on a cognate codon in the

presence of a 30-fold excess of competing near- and non-

cognate ternary complexes is estimated to be close to 20 s�1.

This value is fully consistent with the in vivo rate of protein

synthesis. Thus, the in vitro data that lead to the kinetic

discrimination model of decoding are fully compatible with

the in vivo measurements of the rate and accuracy of protein

synthesis, and thus are likely to hold for the conditions

in vivo as well. The high speed of GTPase activation and

GTP hydrolysis in the cognate ternary complex causes a loss

in the fidelity of selection, but at the same time precludes that

the rate of cognate peptide bond formation is decreased by

competition with bulk ternary complexes. This suggests that

it is the rate of GTP hydrolysis, and not that of peptide bond

formation, which governs the evolution and the optimization

of speed and accuracy of translation, and explains why the

maximum accuracy intrinsic in the system is not achieved.

Materials and methods

Buffers and reagentsHiFi buffer: 50 mM Tris–HCl (pH 7.5), 70 mM NH4Cl, 30 mM KCl,3.5 mM MgCl2, 0.5 mM spermidine, 8 mM putrescine, and 2 mMDTT. Polymix buffer: 5 mM potassium phosphate (pH 7.5), 95 mMKCl, 5 mM NH4Cl, 5 mM magnesium acetate, 0.5 mM CaCl2, 1 mMspermidine, 8 mM putrescine, and 1 mM DTE. Buffer A: 50 mM Tris–HCl (pH 7.5), 70 mM NH4Cl, 30 mM KCl, and 7 mM MgCl2. Buffer B:20 mM HEPES–HCl (pH 7.5), 95 mM KCl, 5 mM NH4Cl, 5 mMmagnesium acetate, 0.5 mM CaCl2, 8 mM putrescine, 1 mM spermi-dine, and 1 mM DTE. Chemicals were from Roche MolecularBiochemicals, Sigma Aldrich, or Merck. Radioactive compoundswere from Hartmann Analytic. MNV mRNA (50-GGCAAGGAGGUAAAUAAUGNNNACGAUU-30, in which the coding sequence is initalics and the A-site codon (underlined) is NNN¼UUC, CUC, orCUU, coding for Phe or Leu, was purchased from Microsynth.

Ribosomes from E. coli MRE600, initiation factors, EF-Tu, andtRNAs were prepared as described previously (Gromadski andRodnina, 2004a). Initiation complexes were prepared by incubatingribosomes (1 mM) with a three-fold molar excess of mRNA,initiation factors 1, 2, and 3 (1.5mM each), f[3H]Met-tRNAfMet

(1.5 mM), and GTP (1 mM) in buffer A (for experiments in HiFibuffer) or B (for experiments in polymix buffer) for 45 min at 371C.The complexes were purified by centrifugation through 400mlsucrose cushions (1.1 M sucrose in buffer A or B, respectively) at260 000 g for 2 h. Pellets were dissolved in HiFi or polymix buffer toa final concentration of 12 mM, shock-frozen in liquid nitrogen, andstored at �801C. Ternary complexes, EF-Tu .GTP .Phe-tRNAPhe orEF-Tu .GTP . Leu-tRNALeu (specific for CUC codon) were preparedby incubating EF-Tu (30mM), GTP (1 mM), PEP (3 mM), pyruvatekinase (0.05 mg ml�1), purified Phe-tRNAPhe (14C- or 3H-labelled) or[14C]Leu-tRNALeu (10–20 mM) and EF-Ts (0.02mM) in polymix orHiFi buffer. Where indicated, ternary complexes were purified bygel filtration on two tandem Superdex 75 columns (Rodnina et al,1995) in polymix or HiFi buffer. Ternary complexes were diluted tothe final concentration immediately before the experiment. Where



indicated (e.g. in multiple-turnover experiments), additionalMg2þ was added to compensate for Mg2þ binding to GTP (1:1Mg2þ/GTP), ATP (1:1 Mg2þ/ATP), and PEP (0.3 Mg2þ/PEP).

Kinetic experimentsAll experiments were carried out at 371C, if not stated otherwise.The formation of cognate dipeptides was measured on rapidmixing of equal volumes (14ml) of initiation complexes (withUUC as second codon; 0.2mM) with excess ternary complex EF-Tu .GTP . [14C]Phe-tRNAPhe (1–10mM) in a quench-flow apparatus(RQF-3, KinTek Corporation; Figure 1B). Samples were quenchedwith 0.5 M KOH, hydrolyzed for 30 min at 371C, and neutralizedwith acetic acid. Amino acids and dipeptides were separated byHPLC on a Chromolith 100 RP 8 column (Merck) using a gradient of0–65% acetonitrile in 0.1% TFA. Radioactivity in the eluate wascounted after addition of Lumasafe Plus scintillation cocktail(Perkin Elmer). The time courses showed a delay of productformation followed by a single exponential phase and were fitted bynumerical integration (Scientist, MicroMath Scientific Software),using a model with two consecutive irreversible steps, in which thesecond step reflects dipeptide formation (kapp), and the delaycomprises all preceding reactions. The rate of dipeptide formationwas plotted versus ternary complex concentration and fitted to ahyperbolic function.

Product formation on a near-cognate codon in excess of ternarycomplex over ribosomes, that is, at multiple turnover conditions,was monitored by quench flow as above, using initiation complexes(CUC at the A site; 0.14mM) and excess ternary complex (1–10 mM)containing [14C]Phe-tRNAPhe. The kapp values were determinedfrom the initial part of the time courses (0.004–0.1 s) as describedpreviously (Johansson et al, 2008) and the concentration depen-dence of kapp values was evaluated by hyperbolic fitting (Figure 2Aand B). Time courses obtained at low Mg2þ concentration werefitted to straight lines and their slopes were plotted versus ternarycomplex concentration, yielding kcat/KM values (Figure 4A). Tomonitor the formation of near-cognate dipeptides by quench-flow atsingle-round conditions (Figure 2C and D), initiation complexes(0.6–6mM, CUC as second codon) were rapidly mixed with purifiedternary complexes (0.2mM) containing [3H]Phe-tRNAPhe. Timecourses were analysed by single exponential fitting. Rates and endpoints of dipeptide formation were plotted versus ternary complexconcentration and fitted to a hyperbolic function.

To examine the Mg2þ dependence of peptide bond formation(Figure 4B), initiation complexes (0.14 mM, UUC, CUU, or CUC assecond codon) were mixed with EF-Tu .GTP . [14C]Phe-tRNAPhe

(1mM) in polymix buffer at 371C. Where indicated, Mg2þ wasadded to compensate for added nucleoside triphosphates or PEP.The Mg2þ dependence of the amplitudes (Figure 4C) was examinedat the same conditions in HiFi buffer without ATP and in thepresence of GTP (0.5 mM compensated by 0.5 mM Mg2þ ) and PEP(0.5 mM) and pyruvate kinase (0.05 mg ml�1). Ternary complexeswere formed in HiFi buffer (3.5 mM Mg2þ , as described above) anddiluted to the Mg2þ concentration of the particular experimentsimmediately before the start of the reaction, taking into account theMg2þ added with ternary complex, initiation complex, and EF-Ts(0.02 mM). The amplitude determined at 64 min incubation wastaken to represent the final level of product formation.

To test the effect of excess of near- and non- cognate ternarycomplexes on the rate of GTP hydrolysis (Figure 5), two ternarycomplexes were prepared. The cognate ternary complex wasprepared by mixing EF-Tu (50mM), EF-Ts (0.02mM), [g-32P]GTP(3ml of undiluted commercial stock solution), [14C]Phe-tRNAPhe

(30 mM), PEP (3 mM), and pyruvate kinase (0.05 mg ml�1) in HiFibuffer at 371C. The mixture of near- and non-cognate ternarycomplexes was prepared from EF-Tu (180 mM), EF-Ts (0.1mM), totalaa-tRNA (120 mM) aminoacylated with a mixture of 19 amino acidsexcept Phe, GTP (1 mM), PEP (3 mM), and pyruvate kinase(0.05 mg ml�1). The two complexes were purified by gel filtration;mixed at indicated ratios and added to initiation complex (0.14 mM).Reactions were quenched with 40% formic acid, dried, anddissolved in 20ml H2O from which 4ml were used for TLC analysis(Polygram CEL300). To measure rates of peptide bond formation,ternary complexes were prepared in the same manner, except thatunlabelled GTP (1 mM) was used.

The overall error frequency was monitored by a competitionassay (Gromadski and Rodnina, 2004b) using purified ternarycomplexes (Figure 3). Initiation complexes (0.5mM; CUC codon atthe A site) were incubated with a mixture of cognate Leu (constantconcentration of 1 mM) and near-cognate Phe (1, 1.5, or 3 mM)ternary complexes for 10, 20, and 30 s. The reaction was quenchedand the reaction products were analysed as described above. Theratio of dipeptides fMetPhe/(fMetLeuþ fMetPhe), normalized toequal concentrations of ternary complexes, represents the observederror frequency (E). To measure proofreading (P), initiationcomplex (0.5mM, CUC codon at the A site) was incubated withnear-cognate ternary complex (Phe, 0.2 mM) for 10, 20, and 30 s.The reaction was stopped and products were analysed as describedabove. The amount of Phe in dipeptides represents the errorfrequency of proofreading (P). From the overall error frequency (E)and the error frequency of proofreading (P), the efficiency of initialselection (I) was calculated: (I)¼ (E)/(P).

Fluorescence stopped-flow experiments were performed on anApplied Photophysics stopped-flow apparatus, monitoring theproflavin fluorescence of EF-Tu .GTP .Phe-tRNAPhe(Prf16/17) (Papeet al, 1998), using an excitation wavelength of 463 nm. Theexperiments were performed by rapidly mixing equal volumes(60ml each) of ternary complex (0.2mM final concentration) andinitiation complex (2mM final concentration, UUC codon). The timecourses were evaluated by two-exponential fitting. The time courseof dipeptide formation, measured in parallel, was normalized byadjusting the start point to 1 and the end point to 0 and fitted bynumerical integration.

Acknowledgements

We thank Wolfgang Wintermeyer for critical reading the paper andCarmen Schillings, Simone Mobitz, and Sandra Kappler for experttechnical assistance. The study was supported by the DeutscheForschungsgemeinschaft.

Conflict of interest

The authors declare that they have no conflict of interest.

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