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The Role of Initiator tRNAi met in Fidelity

of Initiation of Protein SynthesisHadieh Monajemi a , Nasr Y. M. Omar b , Mohamad Noh Daud b ,Sharifuddin Mohd. Zain b & Wan Ahmad Tajuddin Wan Abdullah a ca Department of Physics, Faculty of Science, University of Malaya,Kuala Lumpur, Malaysiab Department of Chemistry, Faculty of Science, University of Malaya,Kuala Lumpur, Malaysiac Faculty of Computer Science and Information Technology,University of Malaya, Kuala Lumpur, Malaysia

Available online: 08 Sep 2011

To cite this article: Hadieh Monajemi, Nasr Y. M. Omar, Mohamad Noh Daud, Sharifuddin Mohd. Zain& Wan Ahmad Tajuddin Wan Abdullah (2011): The Role of Initiator tRNAi

met in Fidelity of Initiation ofProtein Synthesis, Nucleosides, Nucleotides and Nucleic Acids, 30:9, 726-739

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Nucleosides, Nucleotides and Nucleic Acids, 30:726–739, 2011Copyright C© Taylor and Francis Group, LLCISSN: 1525-7770 print / 1532-2335 onlineDOI: 10.1080/15257770.2011.605780

THE ROLE OF INITIATOR tRNAimet IN FIDELITY

OF INITIATION OF PROTEIN SYNTHESIS

Hadieh Monajemi,1 Nasr Y. M. Omar,2 Mohamad Noh Daud,2

Sharifuddin Mohd. Zain,2 and Wan Ahmad Tajuddin Wan Abdullah1,3

1Department of Physics, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia2Department of Chemistry, Faculty of Science, University of Malaya,Kuala Lumpur, Malaysia3Faculty of Computer Science and Information Technology, University of Malaya,Kuala Lumpur, Malaysia

� The proper arrangement of amino acids in a protein determines its proper function, which isvital for the cellular metabolism. This indicates that the process of peptide bond formation requireshigh fidelity. One of the most important processes for this fidelity is kinetic proofreading. As bio-chemical experiments suggest that kinetic proofreading plays a major role in ensuring the fidelity ofprotein synthesis, it is not certain whether or not a misacylated tRNA would be corrected by kineticproofreading during the peptide bond formation. Using 2-layered ONIOM (QM/MM) computa-tional calculations, we studied the behavior of misacylated tRNAs and compared the results withthese for cognate aminoacyl-tRNAs during the process of peptide bond formation to investigate theeffect of nonnative amino acids on tRNAs. The difference between the behavior of initiator tRNAi

met

compared to the one for the elongator tRNAs indicates that only the initiator tRNAimet specifies the

amino acid side chain.

Keywords ONIOM; peptide bond formation; tRNA; ribosome

INTRODUCTION

Proteinsynthesis proceeds by transfer of the growing polypeptide chainfrom the peptidyl-tRNA bound to the ribosomal P-site to the incomingaminoacyl-tRNA in the adjacent A-site. After the translocation of the nascentpeptidyl-tRNA from A-site to the P-site and the misacylated tRNA from theP-site to the E-site of the ribosome by the action of elongation factor G, thenext codon in the empty A-site becomes exposed to the new aminoacyl-tRNA

Received 1 May 2011; accepted 12 July 2011.The authors would like to acknowledge the financial support provided by the Institute of Research

Management and Consultancy of the University of Malaya.Address correspondence to Hadieh Monajemi, Department of Physics, Faculty of Science, University

of Malaya, 50603 Kuala Lumpur, Malaysia. E-mail: h.monajemi@hotmail.com

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Role of Initiator tRNAimet in Fidelity of Initiation of Protein Synthesis 727

(aa-tRNA) ternary complex.[1] The tRNA binding sites are located at the in-terface between the ribosomal subunits, with the decoding center (DC) onthe small subunit[2] and the peptidyl transfers center (PTC) on the largesubunit.[3] Synthetic polynucleotide containing AUG and/or GUG codonsas well as natural mRNA have been used extensively in order to elucidate themechanism of initiation of protein synthesis.[4] The initial codon–anticodoninteraction occurs directly without the presence of the large subunit. Ex-actly how the correct tRNA binds to the mRNA without the presence of theribosome is still unclear.[2]

Fidelity in protein synthesis is contributed by several mechanisms, i.e.,fidelity in mRNA transcription, tRNA aminoacylation, and codon–anticodoninteraction. The cognate codon–anticodon interaction is followed by the ac-commodation of the aa-tRNA in the A-site of the ribosome due to the inducedfit mechanism.[5–7] Compared to the cognate anticodon, the rate of GTP hy-drolysis and accommodation is significantly lower for the near/noncognateanticodons, which results in their rejection from the ribosome.[5] This pro-cess, i.e., the kinetic proofreading, plays a major role in the fidelity of proteinsynthesis. However, it is not clear whether or not a misacylated tRNA influ-ences this conformational change and whether or not it is affected by kineticproofreading process during peptide bond formation.[7,8]

The conformational change of the tRNA in the PTC is also indepen-dent of the codon–anticodon interaction since no fundamental differencesare observed between the PTCs of the 70S and 50S structures during inter-action with the full aa-tRNA and the small oligonucleotide, respectively.[4]

It is worth mentioning that this is not in conflict with the idea behind ki-netic proofreading since the kinetic proofreading is based on the kineticdifferences between the accommodation process of cognate and non/near-cognate codon–anticodon interaction rather than the structural differences.Moreover, the tRNA stabilization in the A-site of the ribosome after the EF-Tu dissociation is believed to be partly affected by the nature of the aminoacid it is attached to. Though, whether this arises from the ribosomal affinityto the amino acid side chain itself or the correct acylated tRNA remains adilemma, since the interaction of the amino acid side chain with ribosomalbases is not clearly observed.[4] Thus, we assume that this stability is inde-pendent of the amino acid’s interaction with ribosomal bases or ribosomalproteins. This assumption is based on what has been reported regardingnonsequence-specific ribosomal interaction with aa-tRNA.

In addition, the fact that the ribosome might specify the amino acid sidechain in the A-site is not applicable for initiator tRNAi

met since it initiallybinds to the small subunit and the mRNA with the absence of large subunit.Furthermore, the initiator tRNAi

met initially interacts with the P-site of thelarge subunit rather than the A-site. Thus, the fidelity of protein synthesisfor a misacylated tRNA might be due to the different function of the tRNAstructure itself. To investigate these assumptions, we analyzed the structure

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728 H. Monajemi et al.

of six different tRNAs attached to both cognateand noncognate amino acidsusing computer simulation. Considering the structural differences betweenthe initiator tRNAi

met and elongator tRNAmet, we used both structures inour calculations. The energetic differences between these structures indicatethat only the initiator tRNAi

met behaves in a different manner when attachedto a noncognate amino acid.

It is somehow impossible to use quantum mechanics methods to studythe system due to the large size of the tRNA molecule. In order to dealwith the large molecules with reasonable cost of calculation and accurate re-sults, one can use hybrid methods, which will allow the combination of twoor more computational techniques in one calculation.[9–12] Most of thesemethods can only combine quantum mechanics (QM) and molecular me-chanics (MM), called in general QM/MM methods. There are a few hybridmethods which can combine QM with QM as well as MM methods. One ofthe most famed methods from this region is known as ONIOM (Our ownN-layered integrated molecular orbital and molecular mechanics) method,which may contain two or more different methods.[9] This method dividesthe system into several onion-like layers and uses a proper computationalchemistry method on each layer based on its size and role in the chemi-cal reaction.[13] One example is using quantum methods to study the bondbreaking/forming process in the active site of an enzyme,[14,15] while therest of the molecule is studied using the MM method. In this study, theamino acid attached to the adenosine in the aminoacyl-arm of the tRNA ishandled with a QM method while the rest of the tRNA is handled with theMM method.

COMPUTATIONAL METHODS

In this study, we used the X-ray crystal structures of six different tRNAs(i.e., tRNAi

met, tRNAmet, tRNAarg, tRNAglu, tRNAgln, and tRNAphe), [16–21]

which are available in the Protein Data Bank (ID codes: 1YFG, 2CSX, 1F7V,1G59, 1QRS, and 1EVV, respectively). These structures are used as the start-ing geometries of all calculations. All computations were carried out usingthe Gaussian 09 program. Molecular visualization was facilitated with the useof Gauss view.[22]

Using the full fragment of the tRNA molecule in our calculations resultsin a large molecular size (near 2500 atoms). In order to have accurate yetcomputationally feasible calculations, we employed the ONIOM method,[9]

which is used for large systems since it enables one to divide the system intotwo (high and low) or three (high, medium, and low) different layers thatcan be treated with different levels of theory. A 2-layer ONIOM(QM/MM)calculation is used in this study where the amino acid arm of the tRNAis treated with the MP2 (second-order Moller–Plesset perturbation theory)

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Role of Initiator tRNAimet in Fidelity of Initiation of Protein Synthesis 729

FIGURE 1 ONIOM partitioning model: QM region is the active site of aa-tRNA molecule in whichpeptide bond formation occurs and MM region is the rest of the tRNA molecule.

method and the rest of the tRNA is treated with the UFF (Universal ForceField) molecular mechanics method (Figure 1).

We used the link atom model for covalent interaction between the tworegions and the resulted dangling bond from the partitioning is capped withthe hydrogen atom. The electrostatic interaction between the MM and QMregions is handled with electronic embedding (EE) where the partial chargesfrom the MM region are included in the QM Hamiltonian. This brings moreaccuracy to the calculations compared to the mechanical embedding (ME),which is used in Gaussian by default.

The energy expression for the ONIOM(QM/MM) method is:

E ONIOM = E Model,High + E Real,Low − E Model,Low (1)

where EModel,High is the energy of the QM region with a high level of calcula-tion, EReal,Low is the energy of the MM region with a low level of calculation,and EModel,Low is the energy of the QM region with the low level of calcula-tion (similar to the one used in EReal,Low). In a 2-layer ONIOM calculationfor large molecules, the classical molecular mechanics method is normallyused for the larger portion of the system and ab-initio calculations for thesite of interest. Defining a proper layer is important in ONIOM calculations.The general rule, which is common in all ONIOM calculations, is to use thehigh level of theory for the chemically active region (i.e., the model system),

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730 H. Monajemi et al.

while the low level of theory is used for the whole molecule (i.e., the realsystem).

Additional to this general rule, there are three important rules to definea proper layer (partitioning) in ONIOM calculations:

1. Bond breaking/formation must take place in the model system.2. The boundaries between regions should not fall on double/triple chem-

ical bonds. Likewise, all atoms in a ring must remain in the same region.3. In a chemical reaction, all of the parameters in the MM region must be

the same in both reactants and products. In this case, it is safer to extendthe model system at least three bonds away from where the reaction istaking place.

In order to have accurate results, it is necessary to follow all these rules.Furthermore, the S-value (substituent-value) test can also validate eitherthe aptness of the ONIOM (QM:MM) partitioning or the accuracy of theONIOM approximation for a specific molecule and/or reaction. These twopurposes are satisfied with two different methods. For the latter, Gaussianprovides the keyword ONIOM = S-value. This purpose is sometimes nec-essary for verifying the accuracy of the ONIOM approximation for somemolecules. However, the S-value test for the latter requires a high-level cal-culation on the real system which makes it computationally expensive and isnot advised to be used for biomolecules (Equation 2)

�SONIOM−approx. = {E high(Real)−E high(Model)} (2)

−{E low(Real)−E low(Model)}.

The former, on the other hand, requires four MM calculations (Equation3). It verifies whether or not the partitioning is appropriate for a specificstructure and the chemical reaction.

�SMM−partition = {E MM(react, Real)−E MM(react, Model)} (3)

−{E MM(pro, Real)−E MM(pro, Model)}.

Since this study is not focusing on the chemical reaction itself, there isno necessity for carrying out the S-value test. Therefore, we followed the rulenumber 2 for extending the QM region in the aminoacyl-tRNA molecule.

Using different substitutions in a molecule, which can act as a “field” todistort the system’s charge distribution, enables us to study electronic effectson the system. We have applied this distortion to the aa-tRNA molecules bysubstituting the hydrogen atom of the α-carbon (Cα9) of the amino acidmoiety with halogen atoms. Each of the six different tRNAs were attached tofive different amino acids (we used the same methionine structure for both

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Role of Initiator tRNAimet in Fidelity of Initiation of Protein Synthesis 731

FIGURE 2 Atomic substitution models: (A) Four halogen atomic substitutions. (B) Single and doublemethyl, ethyl, and phenyl substitutions.

tRNAimet and tRNAmet), one cognate and four noncognate to that specific

tRNA. This results in 30 different aminoacyl-tRNAs of which 6 are cognateand 24 are noncognate structures. Different substitutions on the aminoacid moiety apply different electronic conditions to the molecule. The firstsubstitution is the halogen atom substitution where the hydrogen of theCα9 is substituted with four different halogen atoms, giving us a total of 150substituted and nonsubstituted aa-tRNA structures (Figure 2A). The overallcomparison is based on the increase of electronegativity from hydrogen tofluorine (H < I < Br < Cl < F).

The second type of substitution concerns the electron donat-ing/withdrawing groups. Methyl (electron donating), ethyl (electron do-nating), and phenyl (electron withdrawing) substitutions were used for thispurpose. In this substitution, the three mentioned groups substitute the hy-drogen atom attached to the amino nitrogen (N8) (similar to the structureof N-alkylamino acids in[23]). In order to apply the distortion in the chargedistribution of the molecule, the three substituents are initially bonded tothe N8, replacing only one of the hydrogen atoms and then another group(phenyl, ethyl, or methyl) is added to the system, replacing the second hy-drogen atom attached to N8 (Figure 2B). The normal condition where bothhydrogen atoms are attached to N8 in the amino group of the amino acidmoiety is then compared with these six substitutions. These calculations arecarried out for the six different tRNA molecules where each one is attachedto five different amino acids resulting in an overall number of 210 substitutedand nonsubstituted aa-tRNA structures.

We performed single point energy (SPE) calculations using ONIOM(MP2/6-31G (d,p):UFF) with no geometry optimization on the full fragmentof the tRNA molecule. On the other hand, the geometry of the amino acid

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732 H. Monajemi et al.

molecule is optimized at the MP2 level before it is attached to the tRNAmolecule. The geometry of this attachment is based on the geometry of thepreviously optimized aminoacyl-tRNA with molecular mechanics methods.

RESULTS AND DISCUSSION

The aim of this study is to investigate the structural differences betweencognate and noncognate aminoacyl-tRNAs. Knowing the difference betweenthe initiation and elongation processes in terms of fidelity made us involvethe initiator tRNAi

met in the comparison. We used electronegativity to distortthe charge distribution of the active site of the system where ester bonddissociation and peptide bond formation occur.

Effects of High Electronegativity on aa-tRNA Molecules

The influence of halogen substitutions on cognate and noncognateamino acids attached to tRNA reveals useful information on the behaviorof these molecules under different conditions. The high electronegativityof these atoms affects the charge distribution of the atoms around the esterbond, which is of our interest. Therefore, in order to reduce computa-tional time, we have measured the charge distribution of a short fragmentof aminoacyl-tRNA (i.e., alanyl-ribose) with these atomic substitutions in or-der to have a better understanding of the effect of electronegativity on thesurrounding atoms. Figure 3 shows the unsubstituted and four halogenicsubstitutions for the alanine amino acid attached to the ribose of Adenosine(excluding the purine base). The first structure is the unsubstituted ala-nine. We then apply these substitutions on the full fragment of cognate andnoncognate aminoacyl-tRNAs to observe the behavior of these moleculesunder different substitutions.

As shown in Figure 3, there is a polar ester bond between the oxygenof the ribose sugar (O11) and the carbon of the amino acid moiety (C10).This is due to the high electronegativity of the O11 (i.e., 3.4) compared tothe carboxylic carbon (C10). However, halogenic substitutions cause a morepolar ester bond in this molecule with the exception of fluorine substitution(F25) (which results in the least polar C10–O11 ester bond).

Table 1 illustrates the charge values on Cα9, C10, and O11 for differenthalogenic substitutions (F, Cl, Br, and I) as well as the one without substitu-tion (H).

As observed in this table, the least polar ester bond (the bond betweenC10 and O11) in the molecule is related to fluorine substitution (F25). Onthe other hand, the ester bond polarities related to Cl25 and Br25 are higherthan that of F25. Iodine substitution results in the most polar ester bond inthe system compared to the others. In this case, the relatively high negativecharge on Cα9 results from the high nuclear charge of I25, which acts in

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Role of Initiator tRNAimet in Fidelity of Initiation of Protein Synthesis 733

FIGURE 3 The atomic charges in different halogen atomic substitutions for alanine attached to theribose sugar.

an electropositive way to Cα9. The higher electropositivity results in a lowerionization energy, which is also observed from the calculated first ionizationenergies of the alanine–ribose structure for all five conditions based onKoopmans’ theorem. As observed in Table 2, the lowest ionization energy isobserved for the iodine substitution and the highest is observed for fluorineand hydrogen (unsubstituted).

TABLE 1 Atomic charges in the molecule for different substitutions

Substituents Cα9 C10 O11

H25 (0.210) −0.06 0.821 −0.631I25 (0.271) −0.401 1.021 −0.700Br25 (−0.087) −0.026 0.868 −0.616Cl25 (−0.004) −0.065 0.906 −0.618F25 (−0.395) 0.542 0.784 −0.623

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734 H. Monajemi et al.

TABLE 2 The first ionization energy of the alanine–ribose sugar molecule for fourhalogen substituents

Substituents Electronegativity Ionization energy (eV)

H 2.2 0.339I 2.66 0.243Br 2.96 0.306Cl 3.16 0.329F 3.98 0.339

To observe the behavior of cognate and noncognate aminoacyl-tRNAsunder different charge distribution and ester bond polarity, we have per-formed the same halogenic substitutions on the amino acid attached tothe full fragment of tRNA. The H25 is substituted with each of the fourhalogen atoms (i.e., F, Cl, Br, and I) after optimization of the amino acidattached to the adenosine in order to keep the structure the same for allsubstitutions.

Since the movement of aminoacyl-tRNAs in the ribosome is via the in-teractions between tRNA and ribosomal bases (mainly purine–pyrimidinehydrogen bonds), the overall energy of the molecule is not constant andvaries based on the location (A-site or P-site) and on the state (hybrid ornormal) of the molecule in the ribosome. Thus, the important issue understudy is the relative energy variation, which is related to the behavior of thecognate and noncognate aminoacyl-tRNAs under different distortion fieldswithin the system. The relative energy variation with respect to increase inelectronegativity for different aminoacyl-tRNA molecules is shown in Figures4A–4F. The second vertical axis on the right-hand side of Figure 4A is forthe relative energy of the cognate met-tRNAi

met, which is too large in scaleto show in the same scale with the noncognate ones. This is most severe foriodine substituents on the cognate met-tRNAi

met. As seen in Table 2, thelowest ionization energy on the molecule is caused by the iodine substituentwhich results in a relatively “unstable” structure. Moreover, the highly polarester bond caused by iodine substitution makes the structure more likelyto undergo chemical reactions. However, the highly polar ester bond onlyaffects met-tRNAi

met (Figure 4A). Other aminoacyl-tRNAs show similar be-havior for different substitutions. Furthermore, the met-tRNAi

met moleculeis more stable (lower energy) compared to the misacylated tRNAi

met struc-tures as well as to cognate and noncognate elongator aminoacyl-tRNAs underunsubstituted condition.

Since the ester bond is highly labile,[24] the stability of the met-tRNAimet

under normal unsubstituted condition prevents the cognate methioninefrom being hydrolyzed. However, this stability is not observed for the cognateelongator aminoacyl-tRNAs. This indicates that there is a difference betweenthe structure of initiator and elongator tRNAs and the way they functionunder different conditions.

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Role of Initiator tRNAimet in Fidelity of Initiation of Protein Synthesis 735

FIGURE 4 Relative energy variation for cognate and noncognate aa-tRNAs with halogen substitutionson their amino acid moiety: (A) The energy variation for the initiator tRNAi

met attached to the fivedifferent amino acids. (B–F) The energy variation for the other five elongator tRNA structures attachedto the same amino acids.

Effect of Electron Donating/Withdrawing Groups on aa-tRNA

Based on the effect of methyl groups on the rate of peptide bond forma-tion[23] and to further investigate the behavior of the cognate and noncog-nate aminoacyl-tRNAs under different substitutions, alkyl and phenyl groupswere introduced in the amino side of the amino acid moiety.

In this section, we study the effects of substitution on the charge dis-tribution of the cognate and noncognate aminoacyl-tRNAs by substitutingelectron donating groups (EDG) and electron withdrawing groups (EWG)on the hydrogen atoms attached to the amino nitrogen of the amino acidmoiety. Methyl, ethyl, and phenyl groups were used as substituents on both

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736 H. Monajemi et al.

FIGURE 5 Relative energy variation for five elongator cognate and noncognate aa-tRNAs (i.e., aa-tRNAmet, aa-tRNAarg, aa-tRNAgln, and aa-tRNAglu (the four inset graphs) and aa-tRNAphe (the maingraph)) with electron donating/withdrawing substitutions on the amino nitrogen.

hydrogen atoms of the amino nitrogen. Based on the atom these groups areattached to (i.e., N8), each of these groups acts as either EDG or EWG. Thatis, the methyl and ethyl groups have an electron donating inductive effect(+I) on the amino acid moiety whereas the phenyl group has an electronwithdrawing mesomeric effect (−M). All seven systems are calculated for thesix different tRNAs attached to their cognate amino acids and four differentnoncognates (Figures 5 and 6). The strength of electron donation is deter-mined by the increase in the N8 negative charge of the amino acid moiety.The electron-withdrawing substitutions result in smaller negative chargeson N8. The N8 negative charge is near zero in the presence of di-phenylsubstituent.

In Figure 5, we compare the relative energy variation of cognate andnoncognate aminoacyl-tRNAs. Overall, similar behavior is observed wheneach tRNA is attached to cognate and noncognate amino acids (especiallyin tRNAgln). As mentioned in Section 3.1, our goal in this study is to observethe relative energy variations of cognate and noncognate aa-tRNAs underdifferent conditions and as observed in Figure 5, they are almost similar forelongator tRNAs. On the other hand, we found the initiator tRNAi

met behav-ior to be different when attached to noncognate amino acids (Figure 6).

In contrast to elongator tRNAs, met-tRNAimet is more stable under nor-

mal condition with no substitution, which is imperative to protect the methio-nine from being hydrolyzed when met-tRNAi

met is attached to the complex ofmRNA-smaller ribosomal subunit before attachment to the larger ribosomalsubunit. During the elongation process, this role is played by the universally

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Role of Initiator tRNAimet in Fidelity of Initiation of Protein Synthesis 737

FIGURE 6 Relative energy variation for the cognate and noncognate aa-tRNAimet with electron donat-

ing/withdrawing substitutions on the amino nitrogen.

conserved rRNA base U2585, which protects the peptidyl tRNA’s ester bondfrom being hydrolyzed when the A-site is empty[4] (after translocation), andthe initiator met-tRNAi

met lacks this protection when it is not yet bound tothe larger ribosomal subunit. Such behavior is similar to that for the elec-tronegative atomic substitutions discussed in Section 3.1. The unsubstitutedamino acid structure is most stable for cognate met-tRNAi

met compared tothe other structures (Figure 4).

The arginine amino acid, on the other hand, with a basic side chainand positive charge, is expected to attract electrons from the active site(i.e., the carboxyl group attached to the ribose) and results in a more stablemolecule. Moreover, the presence of EDG in the amino side of arginineresults in a positive charge at the other end of the arginine moiety (i.e., thecarboxyl group). However, this amino acid behaves in a different way whenattached to the tRNAi

met. Arg-tRNAimet is relatively unstable in the presence

of EDG and under a normal unsubstituted condition. This would ultimatelylead to the hydrolysis of the structure and dissociation of the noncognateamino acid from the initiator tRNAi

met. The situation is almost the samefor gln-tRNAi

met (excluding the steric effect of di-phenyl substitution) andphe-tRNAi

met. It is in contrast with the elongator tRNAmet, where its attach-ment to the noncognate amino acids results in highly stable structure inthe presence of EDG as well as EWG. These different behaviors of tRNAi

met

for different amino acids compared to the elongator tRNAs indicate theimportance of the amino acid side chain identity for the initiator tRNAi

met.

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738 H. Monajemi et al.

CONCLUSION

Based on previous biochemical studies on the structure and functionof the ribosome, it is believed that the type of amino acid side chain af-fects the stability of the tRNA binding at the A-site of the ribosome afterthe EF-Tu dissociation.[4] However, it is not clear whether the amino acidside chain interacts with the ribosomal bases at the A-site. In this study, weobserved the specificity of initiator tRNAi

met toward its cognate amino acidside chain (i.e., methionine). This specificity does not exist in elongator tR-NAs. Knowing that the advantage of elongator tRNAs over tRNAi

met is theirinteraction with the ribosomal A-site, the results in this study demonstratethe importance of the larger ribosome in stabilizing the correct acylatedtRNA at the A-site. Furthermore, the initiator tRNAi

met binds initially to theP-site of the larger ribosome, the site that is less likely to bind to the aminoacid side chain compared to the A-site.[4] Therefore, the difference betweeninitiator tRNAi

met and other elongator tRNAs is in the latter’s tendency tobe stabilized at the ribosomal A-site, and in the former’s less likeliness to bestabilized because of its initial attachment to the P-site. This indicates thelack of substrate specification at the P-site, which agrees well with existingbiochemical data.[4]

REFERENCES

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