## 3 Substrate Recognition by L/F-Transferase
### tRNA Recognition by L/F-Transferase  
While studies have demonstrated that L/F-transferase can utilize a number of different aminoacyl-tRNA substrates _in vitro_ such as tRNA\({}^{\text{Leu}}\), tRNA\({}^{\text{Phe}}\) and tRNA\({}^{\text{Met}}\)[26], _in vivo_ investigations indicate that tRNA\({}^{\text{Leu}}\) is the natural substrate [27]. The majority of recognition and specificity appears to arise from the recognition of RNA components of the aminoacyl-tRNAs as the inhibition of L/F-transferase by the aminoacyl-tRNA analogue puromycin (or another related compound) is minimal in comparison to the Km of aminoacyl-tRNA utilization by the enzyme [43]. In addition, a number of investigations have demonstrated the _in vitro_ utilization of tRNAs aminoacylated with a variety of unnatural amino acids of varying sizes [44; 45; 46; 47]. Investigations with mis-charging different tRNAs with different amino acids have revealed the major selectivity for the amino acid on an aminoacyl-tRNA substrate is the exclusion of C-beta branched amino acids [48]. This broad specificity for amino acid side chains is the basis for the proposed utilization of L/F-transferase for enzymatic labeling of polypeptides [45] for the rapid synthesis of reagents for applications such as positron emission tomography (PET) imaging [49].  
Recent investigations into the tRNA sequence specificity by L/F-transferase has confirmed the higher selectivity for tRNALeu but also preferential selectivity for a single tRNALeu isoacceptor (anticodon 5'-CAG-3), as has been previously suggested [48; 50; 51]. The step-wise mutagenesis of a poor tRNALeu substrate (with an 5'-GAG-3' anticodon) to that of the optimal tRNALeu substrate (with a 5'-CAG-3' anticodon) identified key recognition nucleotides for L/F-transferase [50]. Figure 2 shows the cloverleaf sequence and structure of _E. coli_ tRNALeu (CAG) and a 3D model structure using tRNAPhe (since there is no intact complex-free tRNALeu X-ray crystal structure solved to date, PDB ID: 1 EHZ). Key nucleotides recognized by L/F-transferase (red, squares), LeuRS (green, circles) and EF-Tu (blue, triangles) are highlighted. Of particular interest is the lack of overlap between key recognition nucleotides by L/F-transferase and the key recognition nucleotides by the translational machinery (leucyl-tRNA synthetase (LeuRS) or EF-Tu). Although L/F-transferase and EF-Tu both require the recognition of 3' aminoacyl-A76 (purple), they appear to be recognizing different faces of the acceptor stem/T-stem of the tRNA molecule. Meanwhile, LeuRS recognizes both the acceptor stem as well as the tertiary structure of the tRNA (D-/T-loop interactions). The lack of overlap suggests that there is an additional evolutionary constraint on this tRNALeu isoacceptor (5'-CAG-3) beyond that dictated by the translational machinery.  
Figure 2: Cloverleaf and 3D (sphere representation) model structure of tRNALeu (CAG) with key recognition nucleotides and their respective 3D (sphere-representation) surfaces recognized by L/F-transferase (red \(\circ\)), LeuRS (green \(\Circarrow\)), and EF-Tu (blue \(\Delta\)) highlighted. The 3’-aminoacyl adenosine (A76) is highlighted in purple to indicate that it is required to be recognized by both L/F-transferase and EF-Tu. The 3D model was generated using PDB ID 1 EHZ and PyMOL version 1.41, and nucleotide numbering is according to [52].