Active site titration to determine the concentration of HisRS. The disappearance of ATP is fit to an equation that is based on a first order exponential decay followed by a linear phase. The concentration of active enzyme is determined from the burst amplitude. The two plots shown represent two different concentrations of histidyl-tRNA synthetase, with the top plot representing a reaction performed using 2 μM HisRS, and the lower plot representing HisRS at 4 μM. Note that the steady state rate of ATP consumption in both cases is the same: the key difference is in the amplitude of the exponential.

Valve system on the Kintek RQF-3, indicating the arrangement of the sample loops relative to the drive syringes and the central mixer. Figure courtesy of the Kin-Tek Corporation (http://www.kintek-corp.com/)

Stopped-flow fluorescence emission spectra for the formation and pyrophosphorylation of the TyrRS•Tyr~AMP complex. (A) reaction trace for the formation of the TyrRS•Tyr~AMP complex, as determined by monitoring the decrease in the fluorescence emission above 320 nm. Bacillus stearothermophilus tyrosyl-tRNA synthetase was preincubated in the presence of tyrosine and subsequently mixed with MgATP. (B), conversion of TyrRS•Tyr~AMP + pyrophosphate to TyrRS + Tyr + ATP, determined by monitoring the increase in the fluorescence emission above 320 nm. Data acquisition for both curves was split, with 500 data points measured during the initial 20% of each reaction trace and 500 data points measured during the remainder of the reaction trace. Residual values are shown beneath each reaction trace.

Reaction mechanism for the aminoacylation of tRNA. The reaction mechanism for the amino acid activation and the aminoacyl transfer steps of the tRNA aminoacylation reaction are shown. In this mechanism, the amino acid (AA) and ATP are assumed to bind to the aminoacy-tRNA synthetase (E) in random order. “•” and “~” represent noncovalent and covalent bonds, respectively. Rate and dissociation constants are shown next to the steps to which they correspond.

Quantifying the interaction between K230 and T234 in the transition state of Bacillus stearothermophilus tyrosyl-tRNA synthetase. The double mutant free energy cycle shown is used to determine the standard free energy of interaction (ΔΔG^o_int) for the interaction between the K230 and T234 side chains in the E•[Tyr~ATP]^‡ transition state of Bacillus stearothermophilus tyrosyl-tRNA synthetase. Standard free energies for each variant are calculated using equation 23. Standard free energies corresponding to the addition of each side chain (ΔG_a through ΔG_d) are calculated by subtracting the standard free energies of each variant. For example, to calculate ΔG_a, the standard free energy of the K230A/T234A variant is subtracted from that of the K230/T234A variant (ΔG^o_a = ΔG^o_K230/T234A − ΔG^o_K233A/T234A). The standard free energy of interaction is calculated by subtracting ΔG^o_a from ΔG^o_d (ΔΔG^o_int = ΔG^o_d − ΔG^o_a). Since free energy is a state function, the standard free energy of interaction can also be calculated by subtracting ΔG^o_b from ΔG^o_c (ΔΔG^o_int = ΔG^o_c − ΔG^o_b). Analogous double mutant free energy cycles can be used to calculate the free energy of interaction of the K230 and T234 side chains for each step along the reaction pathway. Rate and kinetic constants used to calculate the standard free energies of each variant are taken from [83].

Schematic of synthetic template transcription. Overlapping synthetic nucleotides are used as a template for Klenow fragment extension by annealing and extension cycles. The template strand contains 2′-O-methyl modifications on the two terminal 5′ residues (inset). The double-stranded fragment then acts as a template for T7 RNA polymerase. The DNA modifications are thought to cause the polymerase to dissociate from the template before adding nontemplated residues, thereby increasing product homogeneity [30]. From Sherlin et al., 2001 [31].

Example of a single-turnover noncognate glutamylation reaction by E. coli GlnRS. The reaction was conducted in a rapid chemical-quench instrument using 71 μM GlnRS, 33 μM [³²P]-labeled tRNA^Gln, 2 M glutamate, and 10 mM ATP. The quenched time points of the reaction were analyzed according to Wolfson-Uhlenbeck method [56]. The ratio of Glu~AMP and AMP gives the fraction aminoacylated at each timepoint. k_obs is determined by single-exponential fit of the data.

Representative single turnover and multiple turnover rapid chemical quench kinetic experiments performed in the HisRS and CysRS systems. (A) single turnover aminoacylation kinetics of wild type HisRS:wild type tRNA cognate interaction (blue circles), and the interaction of wild type HisRS with G34U tRNA^His (red triangles). The inset provides a close-up of the first 100 milliseconds of both reactions, illustrating the faster rate of wild type relative to the tRNA^His variant. (B) pre-steady kinetics of HisRS AMP and His-tRNA^His formation, for the identity-compromised C73U tRNA^His variant. Filled blue circles, AMP formation by wild type HisRS in the absence of tRNA. The progress curve is fit to a double exponential followed by a linear phase. Green triangles, AMP formation by wild type HisRS in the presence of C73U tRNA^His. The plot is fit to a single exponential followed by a linear phase. Red squares, His-tRNA^His formation by the C73U tRNA^His variant tRNA. This curve is fit to a linear equation. The difference in slope between the linear portions of the plots for AMP formation and His-tRNA^His formation by the C73U tRNA^His variant represents the stoichiometry of ATP consumption relative to aminoacylated tRNA product formation. The data to derive plots A and B were reported in [66]. (C) time course of synthesis of Cys-tRNA^Cys under single turnover (enzyme concentration in excess) conditions. The concentration of tRNA^Cys was held fixed at 0.5 μM, while that for CysRS was varied between 1 and 20 μM. (D) Re-plot of amplitudes from plot C against the concentration of Cys-tRNA^Cys by hyperbolic fit to derive the K_d for tRNA^Cys. Plots C and D are adapted from Zhang et al.[63].

Comparison of single and double exponential fits to stopped flow fluorescence data. The reaction trace shown records the formation of His-tRNA^His by rapid mixing of HisRS·His~AMP with tRNA^His, employing MDCC-labeled HisRS. Fits to both a single and double exponential are shown on the plot; and the resulting residuals are plotted below. The wave-like pattern of the residuals relative to zero for the single exponential is indicative of a poorer fit.

Standard free energy profile for the wild type and Q195A variants of Bacillus stearothermophilus tyrosyl-tRNA synthetase. Standard free energy values (ΔG^o) are shown for each intermediate and transition state along the tRNA aminoacylation pathway for the wild type and Q195A variants of Bacillus stearothermophilus tyrosyl-tRNA synthetase. Standard free energies were calculated from rate and dissociation constants as described in the text. Values for the rate and dissociation constants are taken from [85] and [86].