Difference between revisions of "Team:Bielefeld-CeBiTec/Project/translational system/translation mechanism"

 
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Translational Mechanism
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<h2>Short Summary</h2>
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The incorporation of non-canonical amino acids (ncAAs) is only possible if the translational mechanisms are adapted. One possible way to incorporate the ncAAs is the repurposing of codons like the amber stop or rarely used leucine codons. Another way would be the incorporation of an unnatural base into the DNA to get new codons. For both, an orthogonal tRNA/aminoacyl-synthetase is necessary, which could charge the ncAA to the tRNA.
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<h2> Library and Selection </h2>
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<h2> Adaption of the Translational Mechanism to Incorporate Non-canonical Amino Acids </h2>
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The incorporation of a non-canonical amino acid (ncAA) requires a <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/translational_system/library_and_selection#">tRNA/aminoacyl-synthetase (tRNA/aaRS)</a> pair which is able to accept and bind the ncAA (to charge the tRNA with the ncAA). The tRNA/aaRS pair should be very specific in the incorporation of the ncAA. Therefore a library of the mutated <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/translational_system/library_and_selection"> orthogonal tRNA/aaRS </a> is generated and undergoes numerous rounds of positive and negative selection for the adaption of the codon recognition and the amino acid binding. The selection results in a synthetase which can be expressed efficiently in E.coli and is able to reliable incorporate an unnatural amino acid.
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The genetic code, consisting of the four bases adenine, guanine, thymidine, and cytosine, provides the framework for the synthesis of peptides and proteins, and thus builds the foundation of all living organisms. Three bases of DNA in protein coding sequences determine an amino acid, and multiple amino acids in turn form a polypeptide. Every possible base triplet codes for an amino acid or a translation stop, such that there are no unoccupied triplets. In order to incorporate  a non-canonical amino acid into a polypeptide, it is necessary that a certain base triplet is dedicated to encoding for this amino acid. As there are no unoccupied triplets available, an existing codon needs to be reassigned. The amber stop codon (UAG/TAG) has been a prominent candidate, as it is one of the three codons signaling translation stop and occurs relatively rarely (roughly 8% of all stop codons in <i>Escherichia coli</i> K-12 genome. An amber suppressor system, consisting of a mutated tRNA and its corresponding tRNA synthetase, is then used to replace the stop-function of the codon with the incorporation of the novel amino acid. More specifically, the tRNA is adapted to suppress the stop-function. In synthetic biology, this is commonly achieved by exchanging a nucleotide at the anticodon part, in a manner that the amber stop codon is read as a codon for an amino acid.  Such systems are common in research (Liu <i>et. al.</i> 2010, Anaelle <i>et. al.</i> 2015, Santoro <i>et. al.</i> 2002). Especially the studies by Liu and Schultz (2010) served as a guideline for our own ncAA incorporation system, as they provide us with comprehensive information on the suppressor system.
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By the incorporation of the <a href=!https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/unnatural_base_pair/unnatural_base_pairs">unnatural base pairs (UBP)</a>, the translational system enables the recognition of the new bases and the interaction of a matching tRNA/synthetase pair. This is the starting point for the engineering of an orthogonal tRNA/synthetase pair for the translational incorporation of  certain <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox">non-canonical amino acids (ncAA)</a>.
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In the process of protein biosynthesis, the DNA is first transcribed into mRNA and in the next step into amino acid polypeptides.
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Therefore, the ribosome binds on a certain position for the mRNA and supports the binding of the interaction for the mRNA and the matching amino acyl tRNA.
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The ribosome contains three binding sites. The tRNA first binds in the A site and the peptidyl tRNA is bound in the P-site. The polypeptide is then transferred from the peptidyl tRNA in the P-site to the aminoacyl-tRNA in the A-site. In the process of translocation, the ribosome moves on to the next codon on the mRNA and the tRNA is aminoacylated by the tRNA synthetase (aaRS). The tRNA leaves the complex of mRNA and ribosome through the E-site.
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Thus, the polypeptide chain is extended in numerous of these processes and the polypeptide is formed.
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This complex mechanism had to be adapted due to the use of UBPs.
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<h2> Tyrosine and Pyrosyl-Lysine tRNA/aminoacyl-synthetase </h2>
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<h2> tRNA/Aminoacyl-Synthetase  </h2>
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<h4> Tyrosine tRNA/aminoacyl-synthetase </h4>
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There are several amino acids which have been incorporated into peptides and proteins in <i> Escherichia coli </i>. Among others, it was possible to incorporate ncAA with heavy atoms, keto and alkaline side chains, photo crosslinking and so on (Zhang<i>  et al.</i>2005).
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Synthetases form a family of enzymes which attach amino acids to the tRNA in a two-step reaction. Therefore, each tRNA synthetase (aaRS) aminoacylates all the tRNAs in an isoaccepting group, representing a particular amino acid. ‘Isoaccepting’ implies that each amino acid can be transported to the ribosome by more than one tRNA.
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For the binding of the ncAA, an adaption of the synthetase is necessary. This is done by certain mutations of several regions of the synthetase, leading to a conformational change of the binding pocket and resulting in the binding of a new amino acid.  
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The tRNA recognition is based on particular nucleotides, labelled as identity set. This identity set is often concentrated in the acceptor stem and anticodon loop region of the tRNA. However, the anticodon sequence of the tRNA is not necessary for a specific tRNA synthetase recognition due to identity determinants which vary in their importance. <br>
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The tyrosine synthetase is the first orthogonal <i> E. coli </i> tRNA/aaRS pair generated from archaea and the best known so far (Wang<i>  et al.</i>2001). This tyrosine synthetase (TyrRS) has a small anticodon loop binding domain (Steer<i>  et al.</i>1999), so the change of the recognition in order to change the anticodon loop of its cognate tRNA to CUA is not as difficult as with a larger anticodon loop binding domain (Steer<i>  et al.</i>1999). The binding side is located deep inside a small pocket of the TyrRS (Tian<i>  et al.</i>2004). This leads to assume, that no large conformational changes are necessary for the incorporation of the ncAA. Rather very small conformational changes result in a larger or smaller binding pocket and an altered hydrogen-binding interaction with the ligand (Zhang<i>  et al.</i>2005).
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The TyrRS binding pocket is highly hydrophilic, preventing the binding of phenylalanine, whose structure is similar to the one of tyrosine (Goldgur<i>  et al.</i>1997). Changing the characteristic of the binding pocket into a hydrophobic kind, could favor the binding of phenylalanine derivates (Goldgur<i>  et al.</i>1997). In the middle of the negative charged binding pocket (Zhang<i>  et al.</i>2005), there is an aspartic acid on position 158. The entrance of the binding pocket is positioned between two glutamic acids at positions 36 and 172. Upon tRNA binding, the aspartic amino acid Asp158 forms two hydrogen bonds with the glycine at position 34 (Zhang<i>  et al.</i>2005).
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The first step of the two-step reaction is the formation of an aminoacylated intermediate by the reaction of the amino acid and ATP. This is possible through the Rossmann fold, a domain consiting of a six-stranded parallel β-sheet with connecting helices. The Rossmann fold acts analog to the active site of hydrogenases and is responsible for binding ATP, the amino acid, and the 3’-terminus of the tRNA.
 
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The second step is the nucleophilic attack of the carbonyl group of the anhydride from either the 2’-OH or the 3’-OH group on the 3’-terminus of the tRNA. This leads to the delivery of the amino acid to the tRNA.
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The synthetase can either recognize directly a base specific functional group or the enzyme  binds directly to the nonspecific portions of the tRNA. Both readouts function within the context of mutual induced fit, so conformational changes in both interaction partners occur while forming a catalytic complex.
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<p class="figure subtitle"><b>Figure 1: Tyrosine-binding site in apo <i> M. jannaschii TyrRS</i> </b><br> The electrostatic distribution around the tyrosine-binding site is shown. Positive potential is blue (10 mV), neutral potential (0 mV) is white, and negative potential (−10 mV) is red (Zhang<i>  et al.</i>2005).</p>
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The recognition requires determinants in the tRNA which are often located in the acceptor stem and the anticodon loop, but the identity signal can be located in the tertiary core region as well (Krebs<i>  et al.</i></i>, 2014).
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aaRS can be categorized into two classes, based on mutually exclusive sets of sequence motifs and properties of structural domains. The synthetases of these two classes are functionally different. While the class I synthetases aminoacylate tRNAs at the 2’-OH position, the class II synthetases aminoacylate tRNAs at the 3’-OH position. Nevertheless, the position of the initial aminoacylation is related to the binding orientation of the tRNA to the enzyme.
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The class I synthetases are monomeric synthetases and feature all structurally similar active site Rossmann fold domains in the region of the N-terminus. Beside this region, there are no significant structural or sequence similarities among the class I enzymes.
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There is an acceptor binding site inserted into the Rossmann-fold domain at a common location which binds the single stranded terminal end of the tRNA, while its C-terminal domain binds in the minor groove of the L-shaped tRNA and the anticodon arm. That is the point for the discrimination among the different tRNAs. The binding requires the formation of a hairpin structure of the single stranded 3’-terminus with the amino acid and the ATP in the active site (Chang<i>  et al.</i></i>, 2010, Krebs<i>  et al.</i></i>, 2014).
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<p class="figure subtitle"><b>Figure 1: Crystal structure of a class I tRNA/aminoacyl synthetase. </b><br> The tRNA is shown in red and the protein in blue (Krebs<i>  et al.</i></i>, 2014).</p>
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When the ligand tyrosine is bound, hydrogen bonds to the Tyr32, Asp158, Glu36, Gln173, Tyr151 and Gln155 are formed, resulting in subtle movements of the side chains within the tyrosine-binding pocket (Zhang<i>  et al.</i>2005). Beside the binding pocket, also other domains are affected by the binding of the tyrosine. For example, the loop 73-83, positioned at the entrance of the binding pocket, which due to the conformational changes then provides a hydrophobic lid over the binding pocket. This is assumed to have the effect of separating the activated tyrosine from water during the catalytic reaction (Zhang<i>  et al.</i>2005).
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The class II synthetases build a quaternary structure, which in general consists of dimers. They own a structurally conserved active side domain, located towards the C-terminal end of the synthetase. This domain of comprises antiparallel β-sheed folds, flanked by α-helices, which is dissimilar to the Rossmann-fold (see class I synthetase) (Wang<i>  et al.</i></i>, 2000). In this domain, there are three conserved short sequences. One of these sequences is important for the multimerization and the other two are essential for the enzymatic activity. The class II synthetases bind to the major groove side of the tRNA acceptor stem, whereby unlike the class I synthetases no hairpin structure of the single stranded 3’-terminus into the active site is required (Chang<i>  et al.</i></i>, 2010, Krebs<i>  et al.</i></i>, 2014).
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<h4> Pyrosyl-Lysine tRNA/aminoacyl-synthetase </h>
 
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<h2> Generating the Library </h2>
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<h2> An Orthogonal tRNA/Aminoacyl-Synthetase Pair </h2>
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Being able to pass as many as possible mutated synthetases through the cycle of the two selections for the adaption of the specific ncAA, a large library of different synthetase versions has to be created. The large library of random variants of sequences can be generated by side specific mutagenesis using randomized primers with the NNK scheme to avoid the incorporation of stop codons. The randomized N codes for the bases A,C,G,T and the randomized K codes for the bases G,T. This leads to the use of 32 codons, so every codon except the three stop codons and the one start codon is encoded through the NNK scheme (Yuval<i>  et al.</i>2011). By comparison to other randomized schemes, the NNK has a relatively low ratio between the most common and rarest coded amino acid (3/32 vs. 1/32) (Yuval<i>  et al.</i>2011).
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The incorporation of non-canonical amino acids (ncAA) requires a tRNA/synthetase (tRNA/aaRS) pair which can accept and bind the ncAA. The tRNA/aaRS pair should be highly specific for the incorporation of the ncAA and most importantly must not cross-react with endogenous amino acids (Ryu <i>  et al.</i></i>, 2006).
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By the randomization of more than one position, numerous more sequence variants occur. If one position is randomized, 130 variants of the sequences are needed to attain a 0.99 probability of discovering the best variant (Yuval<i>  et al.</i>2011). At the same time, for the randomization of three positions, there are statistically 102,478 variants needed to discover the best variant. In this context, the probability of full codon coverage changes from 0.82 &#37 for one randomizes position to 3.25 • 10<sup>-36</sup> &#37 for three randomized position (Yuval<i>  et al.</i>2011). Regarding, that not the full coverage is essential, but rather the discovering of the best variant, the NNK is a solid method for the generation of a synthetase library for the selection of the best ncAA binding site.
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Therefore, orthogonal tRNA/aaRS pairs are applied, which do not cross-react with the endogenous components of the translational system. The cognate tRNA is not recognized by the endogenous synthetase and the cognate synthetase does not incorporate the endogenous amino acids due to differences in tRNA identity elements, especially in the acceptor stem (Kwok<i>  et al.</i></i>, 1980). At the same time, the orthogonal tRNA/synthetase pair has a good translational function (Wang<i>  et al.</i></i>, 2001, Chang<i>  et al.</i></i>, 2010).
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Since the tRNA recognition by the aaRS can be domain or species specific (Kwok<i>  et al.</i></i>, 1980),  a heterologous aaRS/tRNA pair from a different organism is used. A possible source for an orthogonal tRNA/synthetase pair for the application  in bacterial cells are eukaryotes. However, the adaption of these tRNA/aaRS pair which aminoacylates in <i>E. coli</i> is very difficult (Liu<i>  et al.</i></i>, 1999). Evolved synthetases from archaea can be expressed efficiently in <i>E. coli</i> (Wang<i>  et al.</i></i>, 2001, Wang<i>  et al.</i></i>, 2000) and at the same time are more similar to eukaryotic organisms than to the prokaryotic host (Kwok<i>  et al.</i></i>, 1980, Zhang<i>  et al.</i></i>, 2005). The reason are significant differences in the acceptor stem and anticodon binding recognition domain between prokaryotic and archaea tRNA/aaRS (Wang<i>  et al.</i></i>, 2000).
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One possible orthogonal tRNA/aaRS pair is the tyrosine tRNA/aaRS pair of <i>Methanococcus jannaschii</i>, where the first base pair of the acceptor stem CG as a tRNA<sup>Tyr</sup>identity element differs from those of <i>E. coli</i> tRNA<sup>Tyr</sup> containing a GC (Wang<i>  et al.</i></i>, 2001).
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The orthogonal tRNA/aaRS pair is incorporated as a response of the amber stop codon, which does not incorporate any of the 20 canonical amino acids. We use the amber stop codon (UAG), because this codon is rare in <i>E.coli</i> and incorporation of random amino acids has been shown before.
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<h2> Selection </h2>
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For the translational incorporation of ncAAs, a corresponding tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair is essential. The tRNA/aaRS pair must recognize and deliver the novel amino acid in response to a unique codon that does not encode any of the 20 canonical amino acids. This leads to a modification of the tRNA for the recognition of the amber-codon and to a modification of the synthetase for the specific binding of a new amino acid.
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<b>Zhang Y, Wang L, Schultz PG, Wilson IA. </b>(2005). Crystal structures of apo wild-type <i>M. jannaschii tyrosyl-tRNA synthetase (TyrRS)</i> and an engineered TyrRS specific for O-methyl-L-tyrosine. <b>Protein Sci 14(5): 1340–1349</b>
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To ensure specificity and orthogonality, a tRNA/aaRS pair from a distinct microbial organism is used to generate tRNA and aaRS libraries which are used to evolve a suitable tRNA/aaRS pair in iterative negative and positive selection steps (Wang<i>   et al.</i>2006). Therefore the tRNA and the aaRS each have to be separately mutated and selected.
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<b>Wang L, Xie J, Schultz PG. </b> (2006). Expanding the Genetic Code. <b>Annu. Rev. Biophys. Biomol. Struct. 2006;35:225–49. </b>
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<b>Liu CC, Schultz PG.</b> (2010). Adding New Chemistries to the Genetic Code. Annu. Rev. Biochem. 2010;79:413–44.</b>
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<b>Kwok Y, Wong JT. </b> (1980). Evolutionary relationship between Halobacterium cutirubrum and eukaryotes determined by use of aminoacyl-tRNA synthetases as phylogenetic probes. Canadian Journal of Biochemistry. <b>58(3): 213-218</b>
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<b>Krebs J.</b>(2014) Lewin's GENES XI
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<b>Nov Y. </b> 2011. When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis,<b> Applied and Environmental Microbiology 0099-2240/12/12.00 </b>
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<b>Anaëlle, Lerche , Spicer, Davis. </b>(2015), Designing logical codon reassignment–expanding the chemistry in biology. <b> Chemical Science 6.1 (2015): 50-69</b>
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<b>Stephen W. Santoro, Wang, Herberich,  King, Schultz. </b> (2002) An efficient system for the evolution of aminoacyl-tRNA synthetase specificity." <b> Nature biotechnology 20.10 </b>
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<h4> Modification of the aaRS </h4>
 
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In the negative selection steps, the synthetase (aaRS) library is tested for orthogonality of the exogenous tRNAs towards the endogenous translation system. The expression of a barnase gene is prevented by an amber stop codon in its coding region. If the exogenous synthetase binds an endogenous amino acid, the amber stop codon preventing the expression of barnase is suppressed, which leads to cells death. Therefore, only cells with orthogonal or non-functional synthetases survive (Wang<i>  et al.</i>2006, Liu<i>  et al.</i>2010).
 
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The orthogonal aaRS are then subjected to positive selection, in which the tRNA/aaRS pairs are tested for their functionality. The expression of an antibiotic resistance gene is prevented by an amber stop codon. Only if the exogenous aaRS incorporates any amino acid, the amber stop codon is suppressed and the cell attains antibiotic resistance. Resulting in the death of cells who do not own a functional aaRS (Wang<i>  et al.</i>2006, Liu<i>  et al.</i>2010).
 
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<p class="figure subtitle"><b>Figure 2: Adaption of the aminoacyl-synthetase.</b><br> Positive and negative selection for the specificity of the orthogonal synthetase for the ncAA in <i> E. coli</i>. </p>
 
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Modification of the tRNA:
 
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Similar to the adaption by selection of the aaRS is the selection of a proper variant of the tRNA. Regarding the unnatural base pairs (UBP), the recognition of the unnatural bases through the tRNA for the incorporation of the non-canonical amino acids requires significant modifications of the tRNA. The recognition of the amber stop codon has already been achieved in other laboratories (Wang<i>  et al.</i>2006, Liu<i>  et al.</i>2010). Therefore, a selection cycle of negative and positive selections similar to the one of the aaRS, mentioned above, was performed. The main difference is the exchange of the tRNA by the aaRS on the plasmids used for the selection (Wang<i>  et al.</i>2006, Liu<i>  et al.</i>2010). The outcome is a tRNA, only able to recognize the amber codon and at the same time recognizes its orthogonal synthetase.
 
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The combination of the tRNA and aaRS modification results in an orthogonal tRNA/aaRS pair capable of specifically incorporating a certain ncAA if the amber codon is present.
 
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Latest revision as of 23:07, 1 November 2017

Translational Mechanism

Short Summary

The incorporation of non-canonical amino acids (ncAAs) is only possible if the translational mechanisms are adapted. One possible way to incorporate the ncAAs is the repurposing of codons like the amber stop or rarely used leucine codons. Another way would be the incorporation of an unnatural base into the DNA to get new codons. For both, an orthogonal tRNA/aminoacyl-synthetase is necessary, which could charge the ncAA to the tRNA.

Adaption of the Translational Mechanism to Incorporate Non-canonical Amino Acids

The genetic code, consisting of the four bases adenine, guanine, thymidine, and cytosine, provides the framework for the synthesis of peptides and proteins, and thus builds the foundation of all living organisms. Three bases of DNA in protein coding sequences determine an amino acid, and multiple amino acids in turn form a polypeptide. Every possible base triplet codes for an amino acid or a translation stop, such that there are no unoccupied triplets. In order to incorporate a non-canonical amino acid into a polypeptide, it is necessary that a certain base triplet is dedicated to encoding for this amino acid. As there are no unoccupied triplets available, an existing codon needs to be reassigned. The amber stop codon (UAG/TAG) has been a prominent candidate, as it is one of the three codons signaling translation stop and occurs relatively rarely (roughly 8% of all stop codons in Escherichia coli K-12 genome. An amber suppressor system, consisting of a mutated tRNA and its corresponding tRNA synthetase, is then used to replace the stop-function of the codon with the incorporation of the novel amino acid. More specifically, the tRNA is adapted to suppress the stop-function. In synthetic biology, this is commonly achieved by exchanging a nucleotide at the anticodon part, in a manner that the amber stop codon is read as a codon for an amino acid. Such systems are common in research (Liu et. al. 2010, Anaelle et. al. 2015, Santoro et. al. 2002). Especially the studies by Liu and Schultz (2010) served as a guideline for our own ncAA incorporation system, as they provide us with comprehensive information on the suppressor system.

By the incorporation of the unnatural base pairs (UBP), the translational system enables the recognition of the new bases and the interaction of a matching tRNA/synthetase pair. This is the starting point for the engineering of an orthogonal tRNA/synthetase pair for the translational incorporation of certain non-canonical amino acids (ncAA). In the process of protein biosynthesis, the DNA is first transcribed into mRNA and in the next step into amino acid polypeptides. Therefore, the ribosome binds on a certain position for the mRNA and supports the binding of the interaction for the mRNA and the matching amino acyl tRNA. The ribosome contains three binding sites. The tRNA first binds in the A site and the peptidyl tRNA is bound in the P-site. The polypeptide is then transferred from the peptidyl tRNA in the P-site to the aminoacyl-tRNA in the A-site. In the process of translocation, the ribosome moves on to the next codon on the mRNA and the tRNA is aminoacylated by the tRNA synthetase (aaRS). The tRNA leaves the complex of mRNA and ribosome through the E-site. Thus, the polypeptide chain is extended in numerous of these processes and the polypeptide is formed. This complex mechanism had to be adapted due to the use of UBPs.

tRNA/Aminoacyl-Synthetase

Synthetases form a family of enzymes which attach amino acids to the tRNA in a two-step reaction. Therefore, each tRNA synthetase (aaRS) aminoacylates all the tRNAs in an isoaccepting group, representing a particular amino acid. ‘Isoaccepting’ implies that each amino acid can be transported to the ribosome by more than one tRNA. The tRNA recognition is based on particular nucleotides, labelled as identity set. This identity set is often concentrated in the acceptor stem and anticodon loop region of the tRNA. However, the anticodon sequence of the tRNA is not necessary for a specific tRNA synthetase recognition due to identity determinants which vary in their importance.
The first step of the two-step reaction is the formation of an aminoacylated intermediate by the reaction of the amino acid and ATP. This is possible through the Rossmann fold, a domain consiting of a six-stranded parallel β-sheet with connecting helices. The Rossmann fold acts analog to the active site of hydrogenases and is responsible for binding ATP, the amino acid, and the 3’-terminus of the tRNA.
The second step is the nucleophilic attack of the carbonyl group of the anhydride from either the 2’-OH or the 3’-OH group on the 3’-terminus of the tRNA. This leads to the delivery of the amino acid to the tRNA. The synthetase can either recognize directly a base specific functional group or the enzyme binds directly to the nonspecific portions of the tRNA. Both readouts function within the context of mutual induced fit, so conformational changes in both interaction partners occur while forming a catalytic complex. The recognition requires determinants in the tRNA which are often located in the acceptor stem and the anticodon loop, but the identity signal can be located in the tertiary core region as well (Krebs et al., 2014). aaRS can be categorized into two classes, based on mutually exclusive sets of sequence motifs and properties of structural domains. The synthetases of these two classes are functionally different. While the class I synthetases aminoacylate tRNAs at the 2’-OH position, the class II synthetases aminoacylate tRNAs at the 3’-OH position. Nevertheless, the position of the initial aminoacylation is related to the binding orientation of the tRNA to the enzyme.
The class I synthetases are monomeric synthetases and feature all structurally similar active site Rossmann fold domains in the region of the N-terminus. Beside this region, there are no significant structural or sequence similarities among the class I enzymes. There is an acceptor binding site inserted into the Rossmann-fold domain at a common location which binds the single stranded terminal end of the tRNA, while its C-terminal domain binds in the minor groove of the L-shaped tRNA and the anticodon arm. That is the point for the discrimination among the different tRNAs. The binding requires the formation of a hairpin structure of the single stranded 3’-terminus with the amino acid and the ATP in the active site (Chang et al., 2010, Krebs et al., 2014).

Figure 1: Crystal structure of a class I tRNA/aminoacyl synthetase.
The tRNA is shown in red and the protein in blue (Krebs et al., 2014).

The class II synthetases build a quaternary structure, which in general consists of dimers. They own a structurally conserved active side domain, located towards the C-terminal end of the synthetase. This domain of comprises antiparallel β-sheed folds, flanked by α-helices, which is dissimilar to the Rossmann-fold (see class I synthetase) (Wang et al., 2000). In this domain, there are three conserved short sequences. One of these sequences is important for the multimerization and the other two are essential for the enzymatic activity. The class II synthetases bind to the major groove side of the tRNA acceptor stem, whereby unlike the class I synthetases no hairpin structure of the single stranded 3’-terminus into the active site is required (Chang et al., 2010, Krebs et al., 2014).

An Orthogonal tRNA/Aminoacyl-Synthetase Pair

The incorporation of non-canonical amino acids (ncAA) requires a tRNA/synthetase (tRNA/aaRS) pair which can accept and bind the ncAA. The tRNA/aaRS pair should be highly specific for the incorporation of the ncAA and most importantly must not cross-react with endogenous amino acids (Ryu et al., 2006). Therefore, orthogonal tRNA/aaRS pairs are applied, which do not cross-react with the endogenous components of the translational system. The cognate tRNA is not recognized by the endogenous synthetase and the cognate synthetase does not incorporate the endogenous amino acids due to differences in tRNA identity elements, especially in the acceptor stem (Kwok et al., 1980). At the same time, the orthogonal tRNA/synthetase pair has a good translational function (Wang et al., 2001, Chang et al., 2010).
Since the tRNA recognition by the aaRS can be domain or species specific (Kwok et al., 1980), a heterologous aaRS/tRNA pair from a different organism is used. A possible source for an orthogonal tRNA/synthetase pair for the application in bacterial cells are eukaryotes. However, the adaption of these tRNA/aaRS pair which aminoacylates in E. coli is very difficult (Liu et al., 1999). Evolved synthetases from archaea can be expressed efficiently in E. coli (Wang et al., 2001, Wang et al., 2000) and at the same time are more similar to eukaryotic organisms than to the prokaryotic host (Kwok et al., 1980, Zhang et al., 2005). The reason are significant differences in the acceptor stem and anticodon binding recognition domain between prokaryotic and archaea tRNA/aaRS (Wang et al., 2000).
One possible orthogonal tRNA/aaRS pair is the tyrosine tRNA/aaRS pair of Methanococcus jannaschii, where the first base pair of the acceptor stem CG as a tRNATyridentity element differs from those of E. coli tRNATyr containing a GC (Wang et al., 2001). The orthogonal tRNA/aaRS pair is incorporated as a response of the amber stop codon, which does not incorporate any of the 20 canonical amino acids. We use the amber stop codon (UAG), because this codon is rare in E.coli and incorporation of random amino acids has been shown before.

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