Figure 1.
Schematics for the polypeptide chain elongation cycle.
(A) The ribosome is divided into four phases: the A-sites vacated POST phase RO ready to receive a cognate aminoacyl-tRNA (phase “0”), the PRE phase bearing a growing peptidyl-tRNA in the A site (phase “1”), the PRE ribosomal complex
in transition of translocation (phase “2”), and the POST ribosomal complex ROEGDP (phase “3”). Phases are interconnected by four reactions representing the EF1α-initiated peptide elongation (k0), the combined factor binding and GTP hydrolysis (k1), the EF2-mediated tRNA translocation (k2), and EF2•GDP release (k3). Reactions associated with ADPR•EF2 were depicted in gray, and the corresponding reaction rate constants distinguished by an asterisk superscript. (B) Model variations in the PRE ribosomal binding step and subsequent ADPR•EF2 turnover. The reversible factor binding and subsequent GTP hydrolysis are combined together as
= ka kGTP/(kd+kGTP). Three minor model variations are:
= 0 (model V1),
≠ 0 and the dissociated factor is
(model V2), and
≠ 0 and the dissociated factor is
(model V3).
Figure 2.
Statistical reasoning in constructing the temporary stall probability.
(A) Cartoon illustration of arranging 3 stagnant ribosomes (k = 3) within an mRNA ORF containing 7 ribosomes (m = 7). Number of arrangement variations is given at the right column according to the position of the leading stagnant ribosome in the series, j. The formula is summarized as (m–j)!/{(k–1)![(m–j)–(k–1)]!}. (B) Plot of the actual translocating population F versus the translocation-active fraction f. (C) The stall function U(f) [ = F(f)/f] replotted in the normalized form within a physiological range of f
Table 1.
Model parameters used in simulations.
Figure 3.
Effects of partial impairment in the ADPR•EF2-associated processes on the polypeptide elongation activities.
The constant toxin dose x is set equal to 0.16 nM (equivalent to kcat = 0.0045 s−1). Insets display the cumulative incorporations of amino acids over time.
Table 2.
Seven extreme modes (A to G) of ADPR•EF2 inhibition.
Figure 4.
Transient ribosomal phase distributions and the decays of protein synthesis simulated by model V1 under the action of a constant toxin dose.
Simulations were conducted using [T] = 0.16 nM (or kcat = 0.0045 s−1) and the parameter values listed in Table 1 for the six inhibition modes defined in Table 2, with the mode G omitted. The normalized rates of the elongation cycle ([RO], blue lines) and of the inactivation of EF2 ([EGTP]+[EGDP], black lines) are drawn in semi-log scale. Nearby numerical values denote the apparent first-order inhibition slopes. A sharp transition of the inhibition slope is marked by a colored vertical line resulting from either a zero (red) or induction of the dead-end translocation block (green). Insets display superposition of the two normalized rate expressions Ωck2
+ Ω
and k0[RO].
Figure 5.
14C-Phe incorporation activities restored by additions of native EF2.
(A) After thorough intoxication of a cell-free system made up of a constant [R]t and varying [EF2]t, [T] was reset to zero and a specific amount of native EF2 equal to the system [EF2]t and bolus 14C-Phe-tRNA in excess (6 mM) were added. Cumulative 14C-Phe incorporation for the next 40 min was calculated. Control dot (•) was obtained from a toxin-free system containing the same [R]t and the [EF2]t specified by the abscissa without the later EF2 addition. (B) Simulation procedures similar to (A), except that the system [EF2]t was fixed to 0.6 µM. Namely, the added amount of native EF2 is independent of the original system [EF2]t. The control 14C-Phe incorporation (•) obtained under no toxins and no EF2 addition represents the pre-intoxication level of the system.
Figure 6.
Protection exerted by empty ribosomes during ADP-ribosylation of native EF2.
(A) The modeled reaction scheme, including the irreversible ADP-ribosylation of free EF2 (kcat) and two mutually exclusive reversible bindings of EF2 and ADPR•EF2 to the 80S empty ribosome. Simulations use the following parameters: k1 = 96 s−1 µM−1 and k−1 = 1 s−1 for native EF2, ( = k−1) for ADPR•EF2, kcat = 0.0072 s−1, [EF2]t = 0.6 µM, and [R]t between zero and 1 µM. (B-C) Percentages of ADP-ribosylation, represented as ([E*]+[R•E*])/[EF2]t, simulated by an unaltered
( = k1) and a completely inhibited
( = 0), respectively. (D) Plot of the instant extent of ADP-ribosylation at time zero extrapolated from the pseudo-linear curves in (C) versus the system [R]t.
Figure 7.
Delayed onset of protein synthesis inhibition by excessive EF2 could be produced only by modes inhibiting ADPR•EF2-ribosome interactions.
(A) Comparisons of the transient declines in the protein synthesis rate and the intact EF2 concentration as modeled by the inhibition modes A ( = 0) and mode D (
= 0) of model V3 (
= 0.3 s−1) in a control system ([R]t = 0.5 µM and [EF2]t = 0.6 µM). Insets show the same profiles in semi-log-linear scale. (B) A tenfold increase in [EF2]t lengthens the latency for the onset of protein synthesis decline in the mode D, but accelerates the inhibition of protein synthesis more than EF2 inactivation in the mode A.
Figure 8.
Optimal ADPR•EF2 parameters that produce simulation results consistent with Nygård and Nilsson [17].
In accordance to the experimental settings in [17], the final populations for the intact and the ADPR•EF2-modified
were obtained from a cell-free system made up of [EF2]t = 1.1 µM (82 pmole) and [R]t = 1.0 µM (75 pmole). This system was intoxicated by 1.2 µg toxins in 100 µL solution (equivalent to kcat = 5.3 s−1) for 30 min. A higher premature turnover rate constant
( = 3 s−1) was used in the simulations of model V2 and V3. The red contour lines mark the levels for intact
at 8% and ADPR-modified
at 4%–5%.
Figure 9.
Double reciprocal plots of the elongation velocity v versus native EF2 in the absence and presence of a fixed amount of ADPR•EF2.
A mixture of ADPR•EF2 (0.6 µM) and native EF2 in various concentrations was added into a cell-free poly(U)-translating system containing a constant [R]t = 0.5 µM. The resulting stead-state elongation velocity v was recorded and plotted reciprocally versus the concentration of native EF2. Note that the elongation velocity v under the inhibition mode A of model V1 is zero and hence could not be displayed in the reciprocal form.