Difference between revisions of "Team:Bielefeld-CeBiTec/Project/translational system/library and selection"

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<h2> Library and Selection </h2>
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<h2> Translational System </h2>
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The incorporation of a non-canonical amino acid (ncAA) requires a tRNA/aminoacyl-synthetase (tRNA/aaRS) 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 orthogonal tRNA/aaRS 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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<h2> Translational Mechanism and amber-codon</h2>
 
 
 
 
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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 et al., 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 identity determinants which vary in their importance.  
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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 et al., 2001). This tyrosine synthetase (TyrRS) has a small anticodon loop binding domain (Steer et al., 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 et al., 1999). The binding side is located deep inside a small pocket of the TyrRS (Tian et al., 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 et al., 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 et al., 1997). Changing the characteristic of the binding pocket into a hydrophobic kind, could favor the binding of phenylalanine derivates (Goldgur et al., 1997). In the middle of the negative charged binding pocket (Zhang et al., 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 et al., 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. This domain describes a six-stranded parallel β-sheet with connecting helices. The Rossmann fold acts homologous to the active site domains 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 directly binds nonspecific portions of the tRNA. Both readouts function within the context of mutual induced fit, so conformational changes in both tRNA and synthetase 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 et al., 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 in the tertiary core region as well (Krebs<i>et al</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 of A76, 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-terminal. 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>, 2010, Krebs<i>et al</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 i n blue (Krebs<i>et al</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 et al., 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 et al., 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 is mixed out of antiparallel β-sheed folds, flanked by α-helices, dissimilar to the Rossmann-fold (see class I synthetase) (Wang<i>et al</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>, 2010, Krebs<i>et al</i>, 2014).
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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 et al., 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 et al., 2011).
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The incorporation of a non-canonical amino acid (ncAA) requires a tRNA/synthetase (tRNA/aaRS) pair which can accept and bind the ncAA. The tRNA/aaRS pair should be very specific in the incorporation of the ncAA and most importantly does not cross-react with endogenous amino acids (Ryu<i>et al</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 et al., 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 % for one randomizes position to 3.25 • 10<sup>-36</sup> % for three randomized position (Yuval et al., 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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This is achieved by the use of an orthogonal tRNA/aaRS pair, which does 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>, 1980). At the same time, the orthogonal tRNA/synthetase pair has a good translational function (Wang<i>et al</i>, 2001, Chang<i>et al</i>, 2010).
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Since the tRNA recognition by the aaRS can be domain or species specific (Kwok<i>et al</i>, 1980) a heterologous aaRS/tRNA pair from a different organism is used. A possible source for an orthogonal tRNA/synthetase pair to be applied in bacterial cells can be eukaryotic. However, the adaption of these tRNA/aaRS pair which aminoacylates in <i>Escherichia coli</i> is very difficult (Liu<i>et al</i>, 1999). In contrast, evolved synthetases from archaea can be expressed efficiently in <i>E. coli</i> (Wang<i>et al</i>, 2001, Wang<i>et al</i>, 2000) and at the same time are more similar to eukaryotic organisms than to the prokaryotic (Kwok<i>et al</i>, 1980, Zhang<i>et al</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>, 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>, 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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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 et al., 2006). Therefore the tRNA and the aaRS each have to be separately mutated and selected.
 
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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 et al., 2006, Liu et al., 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 et al., 2006, Liu et al., 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 et al., 2006, Liu et al., 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 et al., 2006, Liu et al., 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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Revision as of 02:54, 27 August 2017

Translational System

Overview

Translational Mechanism and amber-codon

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 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. This domain describes a six-stranded parallel β-sheet with connecting helices. The Rossmann fold acts homologous to the active site domains 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 directly binds nonspecific portions of the tRNA. Both readouts function within the context of mutual induced fit, so conformational changes in both tRNA and synthetase 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 in the tertiary core region as well (Krebset 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 of A76, 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-terminal. 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 (Changet al, 2010, Krebset al, 2014).

Figure 1: Crystal structure of a class I tRNA/aminoacyl synthetase.
The tRNA is shown in red and the protein i n blue (Krebset 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 is mixed out of antiparallel β-sheed folds, flanked by α-helices, dissimilar to the Rossmann-fold (see class I synthetase) (Wanget 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 (Changet al, 2010, Krebset al, 2014).

An orthogonal tRNA/aminoacyl-synthetase pair

The incorporation of a non-canonical amino acid (ncAA) requires a tRNA/synthetase (tRNA/aaRS) pair which can accept and bind the ncAA. The tRNA/aaRS pair should be very specific in the incorporation of the ncAA and most importantly does not cross-react with endogenous amino acids (Ryuet al, 2006). This is achieved by the use of an orthogonal tRNA/aaRS pair, which does 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 (Kwoket al, 1980). At the same time, the orthogonal tRNA/synthetase pair has a good translational function (Wanget al, 2001, Changet al, 2010). Since the tRNA recognition by the aaRS can be domain or species specific (Kwoket al, 1980) a heterologous aaRS/tRNA pair from a different organism is used. A possible source for an orthogonal tRNA/synthetase pair to be applied in bacterial cells can be eukaryotic. However, the adaption of these tRNA/aaRS pair which aminoacylates in Escherichia coli is very difficult (Liuet al, 1999). In contrast, evolved synthetases from archaea can be expressed efficiently in E. coli (Wanget al, 2001, Wanget al, 2000) and at the same time are more similar to eukaryotic organisms than to the prokaryotic (Kwoket al, 1980, Zhanget al, 2005). The reason are significant differences in the acceptor stem and anticodon binding recognition domain between prokaryotic and archaea tRNA/aaRS (Wanget 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 (Wanget 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.