Fig 1.
Kinetic reaction scheme for class I and class II tRNA synthetases.
(A) Detailed map of the reaction pathway for monomeric class I AARS enzymes. The states of the enzyme Si are given in the legend on the right with abbreviations E = AARS enzyme, T = ATP, M = AMP, P = PPi, D = AA Adenylate, R = tRNA, and R* = AA-tRNA denoting the charged tRNA. For example, state S0 corresponds to the enzyme free of all substrates while S5 corresponds to the enzyme with bound amino acid adenylate. The two main catalytic events, activation of the amino acid to form amino acid adenylate plus pyrophosphate (activation), and transfer of the amino acid from the adenylate to the tRNA (transfer) are labelled. Red arrows denote reaction steps where a substrate associates to the enzyme. (B) The reaction pathway for class I dimer metRS and class II AARS enzymes follows the kinetic flip-flop mechanism reported in Guth et al. [17]. Here, the same kinetic steps in site 1 of the dimer occur as in the class I enzymes followed by a subsequent activation event in site 2 while charged aa-tRNA in site 1 remains bound. After activation in site 2, aa-tRNA is released from site 1. States with substrate bound in the first and second site are separated by a slash, e.g. state S15 with charged tRNA in the first site and AA is bound to the second catalytic site is denoted as ER*/A.
Fig 2.
Computer simulation of pyrophosphate exchange and single turnover kinetics.
(A) Reaction scheme used to simulate pyrophosphate exchange kinetics and, (B) reaction scheme used to simulate single turnover kinetics involving the transfer step. State notation follows that of Fig 1. (C) Example Woolf-Hanes plot and the resulting values of and kcat from a computer simulation of pyrophosphate exchange for E. coli cysRS. (D) Example plot from a computer simulation of single turnover kinetics for E. coli cysRS. Black squares represent data points from the computer simulation while the red curve gives the best fit exponential
to the data.
Table 1.
In vitro measurements of E. coli cysRS and kcat values. The experimental measurements for three separate kinetic assays are reported for the Class I cysRS aminoacyl-tRNA synthetase enzyme. PMID denotes the PubMed ID for the reference source of the data. Abbreviations are, PP = pyrophosphate exchange, AA = aminoacylation, ST = single turnover transfer, and SC = single turnover overall chemistry. Units for kcat are in s−1 and
are in
M.
Fig 3.
Kinetic model of class I cysRS tRNA charging.
(A) Range of kcat and Km values determined by in vitro pyrophosphate and aminoacylation assays (black dots) are compared with the values of the kinetic model (green triangles). (B) Pre-steady state kinetics simulation with starting concentrations of M, [ATP] = 5 mM and
M and
M show an initial burst of cys-tRNA charging followed by a slower steady state recapitulating what was observed experimentally in Ref. [9]. (C) Single turnover simulation of the transfer rate (ktran) and (D) of the overall chemistry step (kchem) show excellent agreement with experimentally observed values in Ref. [9].
Table 2.
In vitro measurements of E. coli hisRS and kcat values. The experimental measurements for three separate kinetic assays are reported for the Class II hisRS aminoacyl-tRNA synthetase enzyme. PMID denotes the PubMed ID for the reference source of the data. Abbreviations are, PP = pyrophosphate exchange, AA = aminoacylation, ST = single turnover transfer, and SC = single turnover overall chemistry. Units for kcat are in s−1 and
are in
M. nd/ns = not determined/stated.
Fig 4.
Kinetic model of class II hisRS tRNA charging.
(A) Range of kcat and Km values determined by in vitro pyrophosphate and aminoacylation assays (black dots) are compared with the values of the kinetic model (green triangles). (B) Pre-steady state kinetics simulation with starting concentrations of M, [ATP] = 5 mM and
M and
M show no burst of his-tRNA charging as observed experimentally in [17]. (C) Single turnover simulation of the transfer rate (ktran) and (D) of the overall chemistry step (kchem) show excellent agreement with experimentally observed values in Ref. [17].
Fig 5.
Average tRNA turnover rates in vivo for CysRS and HisRS.
(A) Violin plot of the average tRNA turnover rates for (A) cysRS and (B) hisRS computed using the 12 proteomics measurements. Red line corresponds to the consensus turnover rate used for computing average enzyme numbers for use in translation simulations.
Fig 6.
Comparison between in vivo activity verses in vitro kcat measurements in class I and II aminoacyl tRNA synthetases.
Black lines with error bars indicate the range of in vivo activity for each AARS enzyme determined from proteomics data, while black triangles give the turnover rate used to calculate the number or enzymes for computational simulations. Red lines with error bars correspond to the range of experimental in vitro kcat measurements. Red triangles represent the optimized kcat values which are able to support maximum ribosome elongation rate that would be possible following a nutrient up-shift. Data for (A) class I tRNA synthetases and (B) class II tRNA synthetases.
Fig 7.
Experimental in vitro Km measurements in class I and class II aminoacyl-tRNA synthetases.
Violin plots of the experimental in vitro Km measurements are given as black lines. Green triangles represent the optimized Km values used in translation simulations. (A) values for tRNA as substrate in the aminoacylation reaction for class I enzymes and (B) class II enzymes. (C)
values for amino acid as substrate in the aminoacylation reaction for class I enzymes and (D) class II enzymes.
Fig 8.
Computational Simulations of tRNA Charging and Translation in vivo.
(A) The kinetics of tRNAcys charging by cysRS during translation. (B) The kinetics of tRNAhis charging by hisRS during translation. (C) The movement of tRNA during the translation cycle. Uncharged tRNAs (black) are charged by AARS enzymes (blue) and released as aa-tRNA (red). The aa-tRNA then binds to EfTu:GTP to form ternary complex (green) which is recruited to the ribosome. Ribosomes (purple) incorporate the amino acid into the peptide chain releasing EfTu:GDP and uncharged tRNAs. (D) Partitioning of the total amount of tRNA in the cell. Percentage of total tRNA as free uncharged tRNAs (black), free charged tRNAs (red), free tRNAs in ternary complex (green), and tRNA bound to either ribosomes (purple) or AARS enzymes (blue) is shown as a pie chart at a growth rate of (
min).
Fig 9.
Theoretical and Experimental Estimates of tRNA Charging Fractions in Minimal Media.
The total fraction of tRNAs charged in exponentially growing E. coli cells. Black lines with error bars indicate the range of experimental measurement from several different experiments [39–42], while green triangles give the results of the stochastic translational model with aminoacyl tRNA synthetase kinetics following the reaction scheme in Fig 1 at a growth rate of h−1.