Kinetics of initiating polypeptide elongation in an IRES-dependent system
- University of Pennsylvania, United States
Figures
Figure 1 with 1 supplement
Structure of CrPV-IRES bound to the 80S ribosome superposed on A, P, and E tRNA binding sites.
The position of the first codon is indicated. Adapted from Fernandez et al. (2014).
Figure 1—figure supplement 1
In vitro translation of firefly luciferase with WT and mutated F-IRES mRNA.
https://doi.org/10.7554/eLife.13429.004
Figure 2
Proposed scheme for initial tetrapeptide synthesis on CrPV IRES-programmed ribosomes.
This simplified scheme neglects the several substeps, including GTP hydrolysis, Pi release, and elongation factor release, that accompany both productive binding of ternary complex to the ribosome …
Figure 3 with 2 supplements
Rates of initial Phe-tRNAPhe binding measured by fluorescence anisotropy or Phe-tRNAPhe cosedimentation.
Fluorescence anisotropy changes were monitored after rapid mixing of Phe-tRNAPhe (Prf) ternary complex (0.1 µM final concentration, containing 1 mM GTP)with 80S·FVKM-IRES complex (0.1 µM final …
Figure 3—figure supplement 1
Corrected IRES-dependent time courses for initial Phe-tRNAPhe binding as measured by fluorescence anisotropy.
Experiments were carried out as described in Figure 3, but in the presence or absence of added IRES. Fluorescence anisotropy changes were monitored after rapid mixing of Phe-tRNAPhe(Prf) ternary …
Figure 3—figure supplement 2
Corrected IRES-dependent time courses for initial Phe-tRNAPhe binding as measured by Phe-tRNAPhe cosedimentation.
Phe-TC (1.6 µM final concentration) was rapidly mixed with either 80S·FVKM-IRES complex (0.8 µM final concentration) or just 80S (0.8 µM final concentration). These experiments were carried out …
Figure 4 with 2 supplements
Kinetics of peptide synthesis and Met-tRNAMet cosedimenting with ribosomes.
Reaction mixtures were quenched at various times after mixing. Peptide synthesis aliquots were quenched with 0.8 M KOH, and the released [35S]-containing peptide was resolved and quantified by TLE …
Figure 4—figure supplement 1
Time courses for formation of PheMet dipeptide and PheLysMet tripeptide as determined by TLE.
(A) Dipeptide synthesis: 80S·FM-IRES complex with Phe-tRNAPhe in the P site was mixed with [35S]-Met-TC. (B) Tripeptide synthesis: 80S·FKM-IRES complex with Phe-Lys-tRNALys (Δ) in the P site was …
Figure 4—figure supplement 2
Added 30S carrier does not significantly change the amount of FVKM-tRNAMet co-sedimenting with 80S ribosomes in the presence and absence of FVKM-IRES.
https://doi.org/10.7554/eLife.13429.012
Figure 5 with 1 supplement
Tetrapeptide translocation (Step 12) is faster than tripeptide translocation (Step 9).
(A) Puromycin reaction with PheValLys-tRNALys bound either at the A site (D) or at the P-site (O) of the 80S·FVKM-IRES complex or being translocated from the A site to the P site (□). (B) Puromycin …
Figure 5—figure supplement 1
Octapeptide synthesis: 80S·FKVRQWLM-IRES complex with FKVRQWLM-tRNAMet in the P-site was prepared using the standard procedure (see Complex Preparations in Materials and methods) and incubating the 80S-IRES complex with the eight relevant TCs (including [35S]-Met-TC) for 40 min.
The resulting labeled octapeptide, released by base hydrolysis, was analyzed by TLE. Migration positions of [35S]-Met and [35S]-labeled FKVRQWLM (*) are indicated.
Tables
Table 1
Apparent rate constants for Steps 1 and 2.
| Apparent rate constants (s-1) | -eEF2 | +eEF2 |
|---|---|---|
| k1 | 0.0071 ± 0.0033 | 0.0033 ± 0.0001 |
| k-1 | 0.15 ± 0.04 | 0.0034 ± 0.0001 |
| k2 ([Phe-TC] = 0.1 µM) | 0.11 ± 0.04 | 0.0256 ± 0.0002 |
Table 2
t1/2 values*.
| Step (s) | t1/2 (s) |
|---|---|
| 1† 1 (+eEF2)† | 230 ± 5 237 ± 5 |
| 2‡ 2 (+eEF2)‡ | 15 ± 9 30 ± 5 |
| 3 | 210 ± 10 |
| 4 + 5 | 8 ± 2 |
| 4-8 | 98 ± 15 |
| 6 = (4-8) – (4+5) – (7+8)§ | 84 ± 16 |
| 7 | 3 ± 1 |
| 8 = (7+8) – 7§ | 4 ± 2 |
| 7 + 8 | 6 ± 2 |
| 7-11 | 128 ± 26 |
| 9 = (7-11) – (7+8) – (10+11)§ | 110 ± 30 |
| 10 | 2 ± 1 |
| 11 = (10 + 11) – 10§ | 7 ± 3 |
| 10 + 11 | 9 ± 2 |
| 12 | <10 |
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* Error ranges shown are based on the variances of fits to single or double exponentials of the results presented in Figure 4, unless otherwise noted.
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† Calculated as 0.69 (k-1 + k2)/k1k2 (see Table 1).
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‡ Calculated as 0.69 (k-1 + k2)/k22 (see Table 1).
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§ Error ranges for these steps, which are not observed directly, are based on the error ranges of the directly observed steps.
Additional files
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Supplementary file 1
Initial coding sequences of variants used in this work.
- https://doi.org/10.7554/eLife.13429.016
