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

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<h2> Translational System </h2>
 
<h2> Translational System </h2>
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<h4> Overview</h4>
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<h4> Transational Mechanism</h4>
 
 
 
 
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<h2> Translational Mechanism and amber-codon</h2>
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<h2> Amber-codon</h2>
 
 
 
 
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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.
 
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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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 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. <br>
 +
 
  
 
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 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 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></i>, 2006).
 
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></i>, 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 (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).
 
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></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).
 +
<br>
 
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 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></i>, 1999). In contrast, 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 (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).
 
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 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></i>, 1999). In contrast, 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 (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).
 +
<br>
 
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).
 
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).
 
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.
 
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.

Revision as of 11:56, 28 August 2017

Translational System

Transational Mechanism

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 (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 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 (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 i n 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 is mixed out of antiparallel β-sheed folds, flanked by α-helices, 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 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 et 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 (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 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 (Liu et al., 1999). In contrast, 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 (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.