Heat Maps for Intramolecular Communication

Supplementary information to the paper:

Heat maps for intramolecular communication

in an RNP enzyme encoding glutamine

Annia Rodríguez-Hernández and John J. Perona

Summary of Supplementary Information:

Supplemental Figure S1

Supplemental Figure S2

Supplemental Figure S3

Supplemental Table S1

Supplementary Fig S1, related to Figure 1 and Figure 2. Cloverleaf representation of the E. coli tRNA2Gln isoacceptor used in these experiments. The modified nucleotides are indicated, although unmodified transcript RNA was used in this work (see Methods).

Supplementary Fig. S2, related to Figures 3, 5, 6, and 7. Single turnover kineticsfor WT GlnRS

a. Plots of Gln-tRNAGln product formation with time, under conditions of molar enzyme excess over tRNA, saturating levels of tRNA and ATP, and concentrations of glutamine varying from 0.01 – 10 mM. Each time course was fit to a single exponential function. b. Replot of the kobsvalues obtained from panel (a), as a function of the substrate glutamine concentration. Fitting the data to a hyperbolic binding isotherm yieldskobs,max andKd for glutamine. c. Example of the experimental data for a WT GlnRS timecourse performed at 0.05 mM glutamine (red curve in panel (a)). At right, Ap* designates AMP derived from A76 in substrate tRNA, after P1 nuclease digestion, while Gln-Ap* is derived from P1 nuclease digestion of the Gln-tRNAGln product.

Supplementary Fig. S3, related to Figure 7.Single turnover kineticsfor GlnRS R520A

a. Plots of Gln-tRNAGln product formation with time, under conditions of molar enzyme excess over tRNA, saturating levels of tRNA and ATP, and concentrations of glutamine varying from 0.01 – 10 mM. Each time course was fit to a single exponential function. b. Replot of the kobsvalues obtained from panel (a), as a function of the substrate glutamine concentration. Fitting the data to a hyperbolic binding isotherm yields kobs,max andKd for glutamine. c. Example of the experimental data for an R520A GlnRS timecourse performed at 0.5 mM glutamine (red curve in panel (a)). At right, Ap* designates AMP derived from A76 in substrate tRNA, after P1 nuclease digestion, while Gln-Ap* is derived from P1 nuclease digestion of the Gln-tRNAGln product.

Table S1, related to Figures 2, 3, 5, 6 and 7. Single Turnover Kinetics for WT GlnRS and Mutants

Enzyme / Kd, Gln (mM) / kobs(max) (sec-1) / kobs(max)/Kd (mM-1sec-1) / *Rel kobs(max)/Kd / ΔΔG, [Kd] (kcal/mol) / ΔΔG,[kobs(max)] (kcal/mol) / ΔΔG,[kobs(max)/Kd] (kcal/mol)
WT QRSH / 1.1 ± 0.04 / 28 ± 2 / 25.5 / 1.000 / 0.00 / 0.00 / 0.00
3’End GlnRS Mutants
T68A / 8.1±2.4 / 0.27±0.02 / 0.03 / 0.001 / 1.23 / 2.86 / 4.15
N69A / 6.8±2.4 / 1.7±0.2 / 0.25 / 0.010 / 1.12 / 1.73 / 2.85
I129A / 1.6±0.3 / 18.7±0.9 / 11.7 / 0.459 / 0.23 / 0.25 / 0.48
R192A / 31.2±1.4 / 3.3±0.3 / 0.11 / 0.004 / 2.06 / 1.32 / 3.35
K194A / 5.6±1.4 / 23±0.1 / 4.1 / 0.161 / 1.00 / 0.12 / 1.13
R133A / 17.1±1.5 / 9.2±0.3 / 0.5 / 0.020 / 1.69 / 0.69 / 2.42
L136A / 8.0±1.1 / 28±1.0 / 3.5 / 0.137 / 1.22 / 0.00 / 1.22
I183A / 1.4±0.07 / 10.7±0.15 / 7.6 / 0.298 / 0.15 / 0.59 / 0.75
Acceptor Stem GlnRS Mutants
Q234A / 0.9±0.3 / 12.1±1.0 / 13.4 / 0.525 / -0.12 / 0.52 / 0.40
D235A / 1.5±0.4 / 13.3±0.2 / 8.9 / 0.349 / 0.19 / 0.46 / 0.65
R237A / 2.5±0.5 / 19.0±3.0 / 7.6 / 0.298 / 0.51 / 0.24 / 0.75
R238A / 1.0±0.05 / 6.8±1.7 / 6.8 / 0.267 / -0.06 / 0.87 / 0.81
Inner Corner GlnRS Mutants
K317A / 3.4±0.3 / 10.9±0.25 / 3 / 0.118 / 0.70 / 0.58 / 1.32
D319A / 0.9±0.1 / 17.3±8.7 / 19.2 / 0.753 / -0.12 / 0.30 / 0.17
Q318A / 0.6±0.04 / 8.9±0.2 / 14.7 / 0.576 / -0.37 / 0.71 / 0.34
T316A / 0.52±0.08 / 4.2±0.4 / 8.1 / 0.318 / -0.46 / 1.17 / 0.71
E323A / 0.4±0.1 / 12.3±0.9 / 30.8 / 1.208 / -0.62 / 0.51 / -0.12
S329A / 1.2±0.2 / 28±4.0 / 23.3 / 0.914 / 0.05 / 0.00 / 0.06
N320A / 0.7±0.06 / 11.4±0.8 / 16.3 / 0.639 / -0.28 / 0.55 / 0.28
Anticodon Region GlnRS Mutants
N336A / 0.9±0.04 / 12.5±0.1 / 14.1 / 0.553 / -0.12 / 0.50 / 0.36
N370A / 0.8±0.2 / 12.7±1.9 / 16 / 0.627 / -0.20 / 0.49 / 0.29
R545A / 0.9±0.01 / 18.2±4.0 / 20.2 / 0.792 / -0.12 / 0.27 / 0.14
K401A / 0.5±0.01 / 9.1±1.9 / 18.2 / 0.714 / -0.49 / 0.69 / 0.21
R410A / 0.8±0.2 / 16.0±4.6 / 20 / 0.784 / -0.20 / 0.34 / 0.15
R412A / 1.1±0.1 / 7.9±0.4 / 7.2 / 0.282 / 0.00 / 0.78 / 0.78
Q517A / 0.72±0.02 / 11.6±0.8 / 16.1 / 0.631 / -0.26 / 0.54 / 0.28
T547A / 0.6±0.1 / 11.1±2.1 / 18.5 / 0.725 / -0.37 / 0.57 / 0.20
R520A / 2.2±0.1 / 5.1±0.1 / 2.3 / 0.090 / 0.43 / 1.05 / 1.48
R341A / 1.2±0.07 / 9.9±1.5 / 8.3 / 0.325 / 0.05 / 0.64 / 0.69
tRNAGln Mutant
U35C / 4.5±0.4 / 3.0±0.85 / 0.7 / 0.027 / 0.87 / 1.38 / 2.21