Polypeptide Substrates Research Articles

Origins of Catalytic Specificity in Bacterial Oligosaccharyltransferase

Modification of polypeptides through asparagine (N)-linked glycosylation is involved in a broad range of biological functions. N-linked glycosylation is catalyzed by oligosaccharyltransferase (OST), an endoplasmic reticulum membrane protein, which promotes the formation of an N-glycosidic linkage between the acceptor asparagine and an oligosaccharide donor. Broad polypeptide substrate specificity is characteristic of N-linked glycosylation, resulting from a short consensus sequence: N-X-S/T (where X is any amino acid except P). However, experimental evidence has shown that the N-glycosylation efficiency is affected by the sequence within this consensus motif.We investigated the catalytic specificity of Campylobacter lari OST using molecular dynamics simulations. In OST, a large external loop (EL5) pins the substrate in the binding pocket located between the transmembrane and periplasmic domains while two acidic catalytic residues, E319 and D56 in C. lari, are in position to form hydrogen bonds with the acceptor asparagine and prime it for nucleophilic attack on the oligosaccharide donor. To explore how different substrates affect the efficiency of N-linked glycosylation, four substrates of differing N-glycosylation efficiencies were examined: the optimal consensus sequence (NAT) and three sub-optimal variants (NAS, NFS, and NWS). Our simulations of OST in complex with the optimal substrate (NAT) show a conformational change of EL5 and of the periplasmic domain is necessary to promote optimal hydrogen bond formation between the acceptor asparagine and E319/D56 by restricting side chain motion in the catalytic pocket. In addition, we found that binding of the other three substrates (NAS, NFS, and NWS) affects the structure and dynamics of OST. These changes lower the probability of forming hydrogen bonds with the acceptor asparagine, essential for catalysis, and accelerate substrate release for NFS, providing two mechanisms for modulating the glycosylation efficiency of OST for various consensus sequence substrates.

The unique functional role of the C-HS hydrogen bond in the substrate specificity and enzyme catalysis of type 1 methionine aminopeptidase.

It is intriguing how nature attains recognition specificity between molecular interfaces where there is no apparent scope for classical hydrogen bonding or polar interactions. Methionine aminopeptidase (MetAP) is one such enzyme where this fascinating conundrum is at play. In this study, we demonstrate that a unique C-HS hydrogen bond exists between the enzyme methionine aminopeptidase (MetAP) and its N-terminal-methionine polypeptide substrate, which allows specific interaction between apparent apolar interfaces, imposing a strict substrate recognition specificity and efficient catalysis, a feature replicated in Type I MetAPs across all kingdoms of life. We evidence this evolutionarily conserved C-HS hydrogen bond through enzyme assays on wild-type and mutant MetAP proteins from Mycobacterium tuberculosis that show a drastic difference in catalytic efficiency. The X-ray crystallographic structure of the methionine bound protein revealed a conserved water bridge and short contacts involving the Met side-chain, a feature also observed in MetAPs from other organisms. Thermal shift assays showed a remarkable 3.3 °C increase in melting temperature for methionine bound protein compared to its norleucine homolog, where C-HS interaction is absent. The presence of C-HS hydrogen bonding was also corroborated by nuclear magnetic resonance spectroscopy through a change in chemical shift. Computational chemistry studies revealed the unique role of the electrostatic environment in facilitating the C-HS interaction. The significance of this atypical hydrogen bond is underscored by the fact that the function of MetAP is essential for any living cell.

Perspectives and Insights into the Competition for Aminoacyl-tRNAs between the Translational Machinery and for tRNA Dependent Non-Ribosomal Peptide Bond Formation.

Aminoacyl-tRNA protein transferases catalyze the transfer of amino acids from aminoacyl-tRNAs to polypeptide substrates. Different forms of these enzymes are found in the different kingdoms of life and have been identified to be central to a wide variety of cellular processes. L/F-transferase is the sole member of this class of enzyme found in Escherichia coli and catalyzes the transfer of leucine to the N-termini of proteins which result in the targeted degradation of the modified protein. Recent investigations on the tRNA specificity of L/F-transferase have revealed the unique recognition nucleotides for a preferred Leu-tRNALeu isoacceptor substrate. In addition to discussing this tRNA selectivity by L/F-transferase, we present and discuss a hypothesis and its implications regarding the apparent competition for this aminoacyl-tRNA between L/F-transferase and the translational machinery. Our discussion reveals a hypothetical involvement of the bacterial stringent response that occurs upon amino acid limitation as a potential cellular event that may reduce this competition and provide the opportunity for L/F-transferase to readily increase its access to the pool of aminoacylated tRNA substrates.

GroEL2 of Mycobacterium tuberculosis Reveals the Importance of Structural Pliability in Chaperonin Function

Intracellular protein folding is mediated by molecular chaperones, the best studied among which are the chaperonins GroEL and GroES. Conformational changes and allosteric transitions between different metastable states are hallmarks of the chaperonin mechanism. These conformational transitions between three structural domains of GroEL are anchored at two hinges. Although hinges are known to be critical for mediating the communication between different domains of GroEL, the relative importance of hinges on GroEL oligomeric assembly, ATPase activity, conformational changes, and functional activity is not fully characterized. We have exploited the inability of Mycobacterium tuberculosis GroEL2 to functionally complement an Escherichia coli groEL mutant to address the importance of hinge residues in the GroEL mechanism. Various chimeras of M. tuberculosis GroEL2 and E. coli GroEL allowed us to understand the role of hinges and dissect the consequences of oligomerization and substrate binding capability on conformational transitions. The present study explains the concomitant conformational changes observed with GroEL hinge variants and is best supported by the normal mode analysis. Conformational changes and allosteric transitions are hallmarks of the chaperonin mechanism. We have exploited the inability of M. tuberculosis GroEL2 to functionally complement a strain of E. coli in which groEL expression is repressed to address the importance of hinges. The significance of conservation at the hinge regions stands out as a prominent feature of the GroEL mechanism in binding to GroES and substrate polypeptides. The hinge residues play a significant role in the chaperonin activity in vivo and in vitro.

ArnT proteins that catalyze the glycosylation of lipopolysaccharide share common features with bacterial N-oligosaccharyltransferases.

ArnT is a glycosyltransferase that catalyzes the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to the lipid A moiety of the lipopolysaccharide. This is a critical modification enabling bacteria to resist killing by antimicrobial peptides. ArnT is an integral inner membrane protein consisting of 13 predicted transmembrane helices and a large periplasmic C-terminal domain. We report here the identification of a functional motif with a canonical consensus sequence DEXRYAX(5)MX(3)GXWX(9)YFEKPX(4)W spanning the first periplasmic loop, which is highly conserved in all ArnT proteins examined. Site-directed mutagenesis demonstrated the contribution of this motif in ArnT function, suggesting that these proteins have a common mechanism. We also demonstrate that the Burkholderia cenocepacia and Salmonella enterica serovar Typhimurium ArnT C-terminal domain is required for polymyxin B resistance in vivo. Deletion of the C-terminal domain in B. cenocepacia ArnT resulted in a protein with significantly reduced in vitro binding to a lipid A fluorescent substrate and unable to catalyze lipid A modification with l-Ara4N. An in silico predicted structural model of ArnT strongly resembled the tertiary structure of Campylobacter lari PglB, a bacterial oligosaccharyltransferase involved in protein N-glycosylation. Therefore, distantly related oligosaccharyltransferases from ArnT and PglB families operating on lipid and polypeptide substrates, respectively, share unexpected structural similarity that could not be predicted from direct amino acid sequence comparisons. We propose that lipid A and protein glycosylation enzymes share a conserved catalytic mechanism despite their evolutionary divergence.

Insights into the molecular mechanism of allostery in Hsp70s.

Hsp70s chaperone an amazing number and variety of cellular protein folding processes. Key to their versatility is the recognition of a short degenerate sequence motif, present in practically all polypeptides, and a bidirectional allosteric intramolecular regulation mechanism linking their N-terminal nucleotide binding domain (NBD) and their C-terminal polypeptide substrate binding domain (SBD). Through this interdomain communication ATP binding to the NBD and ATP hydrolysis control the affinity of the SBD for polypeptide substrates and substrate binding to the SBD triggers ATP hydrolysis. Genetic screens for defective variants of Hsp70s and systematic analysis of available structures of the isolated domains revealed some residues involved in allosteric control. Recent elucidation of the crystal structure of the Hsp70 homolog DnaK in the ATP bound open conformation as well as numerous NMR and mutagenesis studies bring us closer to an understanding of the communication between NBD and SBD. In this review we will discuss our current view of the allosteric control mechanism of Hsp70 chaperones.

Characterization of transporter activities in fresh isolated primary hepatocytes of different species by using fluorescent substrates

Prediction of human pharmacokinetic profiles of drug candidates is an essential step toward first-in-human studies. However, it remains difficult to quantitatively predict hepatic clearance, particularly when hepatic uptake is mediated by transporter(s). Using 15 organic anion transporting polypeptide (OATP) substrate drugs, we tested 3 in vitro–in vivo extrapolation (IVIVE) approaches to predict overall hepatic intrinsic clearance in vivo (CLint,all,vivo). IVIVE approaches involved metabolic intrinsic clearance in human liver microsomes (CLint,met) with or without hepatocyte-to-buffer maximum unbound concentration ratio (Kp,uu,max) correction and uptake intrinsic clearance at 37°C (PSinf,37°C) in human hepatocyte suspensions. Kp,uu,max and PSinf,37°C values were determined in 2 hepatocyte batches, and all tested compounds showed temperature-dependent uptake, consistent with the fact of transporter-mediated uptake. CLint,met substantially underestimated CLint,all,vivo. By multiplying CLint,met by Kp,uu,max values, the prediction performance was much improved; however, in vitro–in vivo correlation was poor. Among the IVIVE approaches, PSinf,37°C showed the most robust correlation with CLint,all,vivo, and one of the hepatocyte batches could predict CLint,all,vivo with a minimal empirical scaling factor. These results suggested IVIVE with hepatic uptake clearance determined in hepatocyte suspensions as one of the most practical approaches for predicting CLint,all,vivo of OATP substrate drugs.

Conserved Omp85 lid-lock structure and substrate recognition in FhaC.

Omp85 proteins mediate translocation of polypeptide substrates across and into cellular membranes. They share a common architecture comprising substrate-interacting POTRA domains, a C-terminal 16-stranded β-barrel pore and two signature motifs located on the inner barrel wall and at the tip of the extended L6 loop. The observation of two distinct conformations of the L6 loop in the available Omp85 structures previously suggested a functional role of conformational changes in L6 in the Omp85 mechanism. Here we present a 2.5 Å resolution structure of a variant of the Omp85 secretion protein FhaC, in which the two signature motifs interact tightly and form the conserved ‘lid lock'. Reanalysis of previous structural data shows that L6 adopts the same, conserved resting state position in all available Omp85 structures. The FhaC variant structure further reveals a competitive mechanism for the regulation of substrate binding mediated by the linker to the N-terminal plug helix H1.

Conformational Changes of the Clamp of the Protein Translocation ATPase SecA

Post-translational protein translocation across the bacterial plasma membrane is mediated by the interplay of the SecA ATPase and the protein-conducting SecY channel. SecA consists of several domains, including two nucleotide-binding domains (NBD1 and NBD2), a polypeptide cross-linking domain (PPXD), a helical scaffold domain (HSD), and a helical wing domain (HWD). PPXD, HSD, and NBD2 form a clamp that positions the polypeptide substrate above the channel so that it can be pushed into the channel by a two-helix finger of the HSD. How the substrate is accommodated in the clamp during translocation is unclear. Here, we report a crystal structure of Thermotoga maritima SecA at 1.9Å resolution. Structural analysis and free-energy calculations indicate that the new structure represents an intermediate state during the transition of the clamp from an open to a closed conformation. Molecular dynamics simulations show that closure of the clamp occurs in two phases, an initial movement of PPXD, HSD, and HWD as a unit, followed by a movement of PPXD alone toward NBD2. Simulations in the presence of a polypeptide chain show that the substrate associates with the back of the clamp by dynamic hydrogen bonding and that the clamp is laterally closed by a conserved loop of the PPXD. Mutational disruption of clamp opening or closure abolishes protein translocation. These results suggest how conformational changes of SecA allow substrate binding and movement during protein translocation.

Hydrolytic inhibition of α-chymotrypsin by 2,8,14,20-tetrakis(d-leucyl-d-valinamido)resorc[4]arenecarboxylic acid: a spectroscopic NMR and computational combined approach

The stereochemical features of 2,8,14,20-tetrakis(D-leucyl-D-valinamido)resorc[4]arenecarboxylic acid and the N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-4-nitroanilide polypeptide substrate were investigated by nuclear magnetic resonance spectroscopy. Proton selective relaxation parameters gave the basis for the inhibitory activity of resorcin[4]arene in the hydrolysis of the polypeptide substrate by α-chymotrypsin. Results showed that an interaction between the resorcin[4]arene and α-chymotrypsin does occur, and involves the hydrophobic moiety of the macrocycle. This interaction is further reinforced by polar groups located on the side chains of the resorcin[4]arene, whereas the macrocycle-polypeptide substrate interaction is negligible. Conformational analysis and interaction studies carried out by molecular modeling are in good agreement with the NMR data, thus providing an additional support to the rationalization of the inhibitory potential of resorcin[4]arenes on the α-chymotrypsin activity.

Free-Energy Landscapes of the Translocation of a Substrate in Four Proteasome - Activator Complexes Analyzed using Molecular Dynamics Simulations

Proteasome is involved in the degradation of proteins. Proteasome activators bind to the proteasome core particle (CP) and facilitate opening a gate of the CP, where Tyr8 and Asp9 in the N-termini tails of the CP form the ordered open gate. In a double mutant (Tyr8Gly/Asp9Gly), the N-termini tails are disordered and the stabilized open-gate conformation cannot be formed. To understand how proteasome is responsible for the efficient proteolysis of the substrate, four different molecular dynamics (MD) simulations were carried out: ordered- and disordered-gate models of the CP complexed with an ATP-independent PA26 and ordered- and disordered-gate models of the CP complexed with an ATP-dependent PAN-like activator. The free-energies of the translocation of a poly-peptide substrate moving through the gate were estimated using an MD simulation program called SCUBA developed by the Japan Atomic Energy Agency (JAEA). In the ordered-gate models, the substrate in the activator was more stable than that in the CP. The conformational entropy of the N-termini tails of the CP was larger when the substrate was in the activator than in the CP. In the disordered-gate models, the substrate in the activator was more destabilized than in the ordered-gate models. The mutated N-termini tails became randomized and their increased conformational entropy could no longer increase further even when the substrate was in the activator, meaning the randomized N-termini tails had lost the ability to stabilize the substrate in the activator. Thus, it was concluded that the dynamics of the N-termini tails entropically play a key role in the translocation of the substrate.

Toward Prospective Prediction of Pharmacokinetics in OATP1B1 Genetic Variant Populations.

Physiologically based pharmacokinetic (PBPK) models are increasingly being used to provide human pharmacokinetic (PK) predictions for organic anion-transporting polypeptide (OATP) substrates based on in vitro assay data. As a natural extension in the application of these models, in this study, we incorporated in vitro information of three major OATP1B1 genetic variants into a previously reported PBPK model to predict the impact of OATP1B1 polymorphisms on human PK. Using pravastatin and rosuvastatin as examples, we showed that the predicted plasma concentration–time profiles in groups carrying different OATP1B1 genetic variants reasonably matched the clinical observations from multiple studies. This modeling and simulation approach may aid decision making in early pharmaceutical research and development as well as patient-specific dose adjustment in clinical practice.

Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation

Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were driven by hydrophobic and electrostatic complementarity between amino acids in the peptide-binding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we used in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification.

Examination of polypeptide substrate specificity for Escherichia coli ClpB.

Escherichia coli ClpB is a molecular chaperone that belongs to the Clp/Hsp100 family of AAA+ proteins. ClpB is able to form a hexameric ring structure to catalyze protein disaggregation with the assistance of the DnaK chaperone system. Our knowledge of the mechanism of how ClpB recognizes its substrates is still limited. In this study, we have quantitatively investigated ClpB binding to a number of unstructured polypeptides using steady-state anisotropy titrations. To precisely determine the binding affinity for the interaction between ClpB hexamers and polypeptide substrates the titration data were subjected to global non-linear least squares analysis incorporating the dynamic equilibrium of ClpB assembly. Our results show that ClpB hexamers bind tightly to unstructured polypeptides with binding affinities in the range of ∼3-16 nM. ClpB exhibits a modest preference of binding to Peptide B1 with a binding affinity of (1.7 ± 0.2) nM. Interestingly, we found that ClpB binds to an unstructured polypeptide substrate of 40 and 50 amino acids containing the SsrA sequence at the C-terminus with an affinity of (12 ± 3) nM and (4 ± 2) nM, respectively. Whereas, ClpB binds the 11-amino acid SsrA sequence with an affinity of (140 ± 20) nM, which is significantly weaker than other polypeptide substrates that we tested here. We hypothesize that ClpB, like ClpA, requires substrates with a minimum length for optimal binding. Finally, we present evidence showing that multiple ClpB hexamers are involved in binding to polypeptides ≥152 amino acids.

Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å(3) pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism.

Physiologically based pharmacokinetic prediction of telmisartan in human.

A previously developed physiologically based pharmacokinetic model for hepatic transporter substrates was extended to an organic anion transporting polypeptide substrate, telmisartan. Predictions used in vitro data from sandwich culture human hepatocyte and human liver microsome assays. We have developed a novel method to calibrate partition coefficients (Kps) between nonliver tissues and plasma on the basis of published human positron emission tomography (PET) data to decrease the uncertainty in tissue distribution introduced by in silico-predicted Kps. With in vitro data-predicted hepatic clearances, published empirical scaling factors, and PET-calibrated Kps, the model could accurately recapitulate telmisartan pharmacokinetic (PK) behavior before 2.5 hours. Reasonable predictions also depend on having a model structure that can adequately describe the drug disposition pathways. We showed that the elimination phase (2.5-12 hours) of telmisartan PK could be more accurately recapitulated when enterohepatic recirculation of parent compound derived from intestinal deconjugation of glucuronide metabolite was incorporated into the model. This study demonstrated the usefulness of the previously proposed physiologically based modeling approach for purely predictive intravenous PK simulation and identified additional biologic processes that can be important in prediction.

Synergism between a foldase and an unfoldase: reciprocal dependence between the thioredoxin-like activity of DnaJ and the polypeptide-unfolding activity of DnaK.

The role of bacterial Hsp40, DnaJ, is to co-chaperone the binding of misfolded or alternatively folded proteins to bacterial Hsp70, DnaK, which is an ATP-fuelled unfolding chaperone. In addition to its DnaK targeting activity, DnaJ has a weak thiol-reductase activity. In between the substrate-binding domain and the J-domain anchor to DnaK, DnaJ has a unique domain with four conserved CXXC motives that bind two Zn2+ and partly contribute to polypeptide binding. Here, we deleted in DnaJ this Zn-binding domain, which is characteristic to type I but not of type II or III J-proteins. This caused a loss of the thiol-reductase activity and strongly reduced the ability of DnaJ to mediate the ATP- and DnaK-dependent unfolding/refolding of mildly oxidized misfolded polypeptides, an inhibition that was alleviated in the presence of thioredoxin or DTT. We suggest that in addition to their general ability to target misfolded polypeptide substrates to the Hsp70/Hsp110 chaperone machinery, Type I J-proteins carry an ancillary protein dithiol-isomerase function that can synergize the unfolding action of the chaperone, in the particular case of substrates that are further stabilized by non-native disulfide bonds. Whereas the unfoldase can remain ineffective without the transient untying of disulfide bonds by the foldase, the foldase can remain ineffective without the transient ATP-fuelled unfolding of wrong local structures by the unfoldase.

Adult human liver contains intermediate-type proteasomes with different enzymatic properties

Background. The 20S proteasome is the proteolytic core of the major intracellular protein degradative system, the ubiquitin-proteasome system. Since little is known about proteasomes of human liver, we have investigated the proteasome spectrum in adult human liver.Material and methods. 20S proteasomes were chromatographically purified from adult human liver and from HuH7 cells. They were divided into subpopulations and subtypes and characterized with regard to their proteolytic activities using short fluorogenic oligo- and long poly-peptide substrates. Their subunit composition was studied by immunoblotting.Results. Proteasomes from adult human liver tissue can be separated into three subpopulations (I, II, III), each of which is composed of several subtypes, which total to a spectrum of 14 different subtypes. Two minor subtypes contain only the immuno-subunits β1i and β5i but not their standard counterparts; all others are intermediate subtypes containing β1 and β5 standard- and β1i and β5i immuno-subunits in various compositions. With regard to the proteolytic activities we observed that a decreasing content of subunit β1i in the subtypes goes along with a decreasing ratio of chymotrypsin-like/caspase-like activity, whereas the degradation rate of a 30 mer polypeptide substrate increased with decreasing β1i content. By comparison, 20S proteasomes from HuH7 cells do not contain immuno-subunits but are pure standard proteasomes, which can be separated into three subtypes.Conclusion. These findings suggest that adult human liver contains a spectrum of 14 different 20S proteasome subtypes with different enzymatic properties reflecting most probably an adaptive response of liver cell functions to challenging factors during lifetime.

Chance, Destiny, and the Inner Workings of ClpXP

AAA+ proteases are responsible for protein degradation in all branches of life. Using single-molecule and ensemble assays, Cordova etal. investigate how the bacterial protease ClpXP steps through a substrate's polypeptide chain and construct a quantitative kinetic model that recapitulates the interplay between stochastic and deterministic behaviors of ClpXP.

The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase.

Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3' aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA(Leu) (anticodon 5'-CAG-3') isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA(Leu) (CAG)-a G₃:C₇₀ base pair and a set of 4 nt (C₇₂, A₄:U₆₉, C₆₈)-that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.