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

Abstract

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.

Keywords: L/F transferase; N-end rule; aminoacyl-tRNA protein transferase; isoacceptors; quantitative mass spectrometry; tRNA recognition.

FIGURE 1.

Cloverleaf structures of E. coli tRNA isoacceptors for leucine, phenylalanine, and methionine. The two tRNA^Leu isoacceptor species of importance in this study are highlighted in the gray box. Two representative elongator methionyl-tRNA species are selected for this study with their respective gene names.

FIGURE 2.

A preference of leucyl-tRNA (CAG) isoacceptor by L/F transferase. (A) Graphical analysis of product formation over time for tRNA^Leu (CAG) (♦), tRNA^Phe (GAA) (◊), and tRNA^Leu (GAG) (Δ) when using an initial tRNA substrate concentration of 1.25 μM. Errors represented are standard deviation of triplicate measurements of a single independent experiment. Initial rate of product formation is calculated from the slope of the linear tangent line (gray) drawn to the curve. (B) A graphical display of initial rate of product formation vs. tRNA concentration for isoacceptors tRNA^Leu (CAG) (♦), tRNA^Leu (UAG) (□), tRNA^Leu (CAA) (▪), tRNA^Leu (UAA) (▴), tRNA^Phe (GAA) (◊), tRNA^Leu (GAG) (Δ), tRNA^Met (CAU) ileX (•), and tRNA^Met (CAU) metT (○). Errors represented are the standard deviation of three independent experiments. (C) A bar graph presenting the maximal percent aminoacylation for natural isoacceptors after 7 min of aminoacylation. Errors represented are the standard deviation of three independent experiments.

FIGURE 3.

Acceptor stem hybrids identify two independent sequence elements for optimal substrate utilization. (A) Cloverleaf structures of tRNA hybrid constructs 1–11. (B) A graphical display of initial rate of product formation vs. tRNA concentration for tRNA^Leu (CAG) (♦), tRNA^Leu (GAG) (Δ), constructs 1 (▴), 2 (▾), 3 (∇), 4 (○), and 5 (▪). Constructs 1–4 display low activity similar to GAG, while construct 5 displays a mid-range activity. Errors represented are the standard deviation of three independent experiments. (C) A graphical display of initial rate of product formation vs. tRNA concentration for tRNA^Leu (CAG) (♦), tRNA^Leu (GAG) (Δ), constructs 6 (□), 7 (♀), 8 (×), 9 (♂), 10 (☆), and 11 (•). Constructs 6–10 display mid-range activity (overlapping), while construct 11 displays high activity. Errors represented are the standard deviation of three independent experiments. (D) A bar graph presenting the maximal percent aminoacylation of all hybrid constructs after 7 min of aminoacylation. Errors represented are the standard deviation of three independent experiments.

FIGURE 4.

No significant recognition contribution by the D-stem and T-stem of the tRNA body. (A) Cloverleaf structures of tRNA hybrid constructs 12–15. (B) A graphical display of initial rate of product formation vs. tRNA concentration for tRNA^Leu (CAG) (♦), tRNA^Leu (GAG) (Δ), constructs 12 (), 13 (⋄), 14 (⊗), and 15 (⊠). Errors represented are the standard deviation of three independent experiments.

FIGURE 5.

Reverse hybrids validate the identified two independent sequence elements for optimal substrate utilization. (A) Cloverleaf structures of tRNA hybrid construct 16–18. (B) A graphical display of the initial rate of product formation vs. tRNA concentration for tRNA^Leu (CAG) (♦), tRNA^Leu (GAG) (Δ), constructs 16 (□), 17 (▪), and 18 (•). Errors represented are the standard deviation of three independent experiments.

FIGURE 6.

A proposed docking model of aminoacyl-tRNA binding to L/F transferase. Our model suggests that, in addition to the 3′ aminoacyl adenosine recognition and electrostatic interaction, the positive cluster (R76, R80, K83, and R84) of L/F transferase may play a role in the specific recognition of the acceptor stem of an aminoacyl-tRNA. To generate the model, the structure of L/F transferase-rA-Phe complex (shown as electrostatic surface, PDB ID: 2Z3K) (Watanabe et al. 2007) was superimposed on the FemX-peptidyl-RNA complex (PDB ID: 4II9) (Fonvielle et al. 2013) via the conserved core of the GNAT domain. The combined 3′ CCA ends (the cytosines 74 and 75 of the peptidyl-RNA and adenosine of rA-Phe) were then used as references to dock the yeast tRNA^Phe (shown as ribbon, PDB ID: 1EHZ) (Shi and Moore 2000). The model was generated using PyMOL (version 1.41), and electrostatic potentials were calculated by APBS (version 1.8).

FIGURE 7.

A summary of Leu-tRNA^Leu (CAG) recognition nucleotides by L/F transferase (□), LeuRS (○), and EF-Tu (Δ). Solid squares represent the major determinant G₃:C₇₀ base pair; meanwhile, dashed line squares represent the set of 4 nt (C₇₂, A₄:U₆₉, C₆₈) for L/F transferase aa-tRNA recognition. Data for LeuRS recognition were from Asahara et al. (1993a,b, 1998) and Larkin et al. (2002), and data for EF-Tu recognition were from Schrader et al. (2009, 2011) and Schrader and Uhlenbeck (2011). Nucleotides numbering is according to Sprinzl et al. (1998).