Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 1997 Sep 15;11(18):2396–2413. doi: 10.1101/gad.11.18.2396

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 1997, Cold Spring Harbor Laboratory Press

PMC Copyright notice

Ternary complex formation by purified wild-type and mutant eIF-2 complexes. (A) Purified wild-type eIF-2 (□) and mutant eIF-2γ^N135K (⋄) were each assayed for the ability to promote GTP-dependent binding to the [³H] methionine charged initiator-tRNA [³H]Met–tRNA_i^Met, 60,000 cpm/pmole, 0.075 μm) as a function of protein concentration (μg). Identical reactions without GTP were performed as a control for nonspecific GTP-independent binding activity. The number of picomoles of [³H]Met–tRNA_i^Met bound in the absence of GTP was subtracted from the number of pimoles of [³H]Met–tRNA_i^Met] bound in the presence of GTP to determine the GTP-dependent-binding activity, for each respective eIF-2 complex. (B) Wild-type eIF-2 (□), the mutant eIF-2γ^N135K complex (⋄), and the mutant eIF-2β^S264Y complex (○) were each assayed for their ability to dissociate from the [³H]Met–tRNA_i^Met in the presence of the nonhydrolyzable GTP analog, GppNp. For the wild-type eIF-2 and eIF-2β^S264Y complexes, ternary complex formed after 5 min of incubation with GppNp and [³H]Met–tRNA_i^Met (0.075 μm) was competed with various concentrations of unlabeled, charged initiator-tRNA for an additional 5 min and the level of labeled ternary complex determined by the filter-binding assay. The same reaction conditions were used to assay the eIF-2γ^N135K complex with the exception that a 10-min incubation time was used in the initial step to enhance the level of labeled ternary complex. Ternary complex without addition of unlabeled charged initiator-tRNA was stable at 37°C for up to 15 min (data not shown). Identical reactions without GppNp were performed as a control for nonspecific binding of the labeled tRNA and subtracted from the respective assays as background. The amount of eIF-2 preparation in each reaction was adjusted to compensate for similar initial levels of ternary complex formation in the presence of GppNp. Total protein in each reaction was 1.25 μg for the wild-type eIF-2 and 5 μg for each of the mutant complexes. (C) The eIF-2γ^N135K complex (5 μg of total protein) was assayed for its ability to bind [³H]Met–tRNA_i^Met (60,000 cpm/pmole) in the presence of GppNp as compared with to the wild-type eIF-2 (5 μg of total protein). (Lanes 1–3) The amount of ternary complex formed by wild-type eIF-2 in the presence of GTP (25 μm), and 25 μm, and 1 mm GppNp, respectively, in a 5-min reaction. (Lanes 4–6) The amount of ternary complex formed by mutant eIF-2γ^N135K complex in the presence of GTP (25 μm); and 25 μm, and 1 mm GppNp, respectively, in a 10-min reaction. The 10-min time point used for the mutant complex analysis was to maximize the amount of initiator-tRNA binding. However, only a 10% increase in binding is observed by using a 10-min incubation vs. a 5-min incubation period. (D) Same as C except using purified mutant eIF-2β^S264Y complex (5 μg of total protein) compared with wild-type eIF-2 complex (2.5 μg of total protein). The different levels of total protein added to each reaction adjusts for the lower yields of eIF-2 in the former preparation as determined by Western blot analysis using antibodies directed against the α subunit of eIF-2. (Lanes 1,2) The amount of ternary complex formed by wild-type eIF-2 in the presence of GTP (25 μm); and 25 μm GppNp, respectively, in a 5-min reaction. (Lanes 3–5) The amount of ternary complex formed by mutant eIF-2β^S264Y complex in the presence of GTP (25 μm), and 25 μm, and 1 mm GppNp, respectively, in a 5-min reaction. (E) Same as C except using purified mutant eIF-2β^L254P complex (5 μg of total protein) compared with wild-type eIF-2 complex (0.17 μg of total protein). The different levels of total protein added to each reaction adjusts for the lower yields of eIF-2 in the former preparation as determined by Western blot analysis using antibodies directed against the α subunit of eIF-2. (Lanes 1,2) The amount of ternary complex formed by wild-type eIF-2 in the presence of GTP (25 μm), and 25 μm GppNp, respectively in a 5-min reaction. (Lanes 3,4) The amount of ternary complex formed by mutant eIF-2β^L254P complex in the presence of GTP (25 μm); and 25 μm GppNp, respectively, in a 5-min reaction. The data in panels B–E represent the average of two independent experiments with a standard deviation <15%.

Ternary complex formation by purified wild-type and mutant eIF-2 complexes. (A) Purified wild-type eIF-2 (□) and mutant eIF-2γ^N135K (⋄) were each assayed for the ability to promote GTP-dependent binding to the [³H] methionine charged initiator-tRNA [³H]Met–tRNA_i^Met, 60,000 cpm/pmole, 0.075 μm) as a function of protein concentration (μg). Identical reactions without GTP were performed as a control for nonspecific GTP-independent binding activity. The number of picomoles of [³H]Met–tRNA_i^Met bound in the absence of GTP was subtracted from the number of pimoles of [³H]Met–tRNA_i^Met] bound in the presence of GTP to determine the GTP-dependent-binding activity, for each respective eIF-2 complex. (B) Wild-type eIF-2 (□), the mutant eIF-2γ^N135K complex (⋄), and the mutant eIF-2β^S264Y complex (○) were each assayed for their ability to dissociate from the [³H]Met–tRNA_i^Met in the presence of the nonhydrolyzable GTP analog, GppNp. For the wild-type eIF-2 and eIF-2β^S264Y complexes, ternary complex formed after 5 min of incubation with GppNp and [³H]Met–tRNA_i^Met (0.075 μm) was competed with various concentrations of unlabeled, charged initiator-tRNA for an additional 5 min and the level of labeled ternary complex determined by the filter-binding assay. The same reaction conditions were used to assay the eIF-2γ^N135K complex with the exception that a 10-min incubation time was used in the initial step to enhance the level of labeled ternary complex. Ternary complex without addition of unlabeled charged initiator-tRNA was stable at 37°C for up to 15 min (data not shown). Identical reactions without GppNp were performed as a control for nonspecific binding of the labeled tRNA and subtracted from the respective assays as background. The amount of eIF-2 preparation in each reaction was adjusted to compensate for similar initial levels of ternary complex formation in the presence of GppNp. Total protein in each reaction was 1.25 μg for the wild-type eIF-2 and 5 μg for each of the mutant complexes. (C) The eIF-2γ^N135K complex (5 μg of total protein) was assayed for its ability to bind [³H]Met–tRNA_i^Met (60,000 cpm/pmole) in the presence of GppNp as compared with to the wild-type eIF-2 (5 μg of total protein). (Lanes 1–3) The amount of ternary complex formed by wild-type eIF-2 in the presence of GTP (25 μm), and 25 μm, and 1 mm GppNp, respectively, in a 5-min reaction. (Lanes 4–6) The amount of ternary complex formed by mutant eIF-2γ^N135K complex in the presence of GTP (25 μm); and 25 μm, and 1 mm GppNp, respectively, in a 10-min reaction. The 10-min time point used for the mutant complex analysis was to maximize the amount of initiator-tRNA binding. However, only a 10% increase in binding is observed by using a 10-min incubation vs. a 5-min incubation period. (D) Same as C except using purified mutant eIF-2β^S264Y complex (5 μg of total protein) compared with wild-type eIF-2 complex (2.5 μg of total protein). The different levels of total protein added to each reaction adjusts for the lower yields of eIF-2 in the former preparation as determined by Western blot analysis using antibodies directed against the α subunit of eIF-2. (Lanes 1,2) The amount of ternary complex formed by wild-type eIF-2 in the presence of GTP (25 μm); and 25 μm GppNp, respectively, in a 5-min reaction. (Lanes 3–5) The amount of ternary complex formed by mutant eIF-2β^S264Y complex in the presence of GTP (25 μm), and 25 μm, and 1 mm GppNp, respectively, in a 5-min reaction. (E) Same as C except using purified mutant eIF-2β^L254P complex (5 μg of total protein) compared with wild-type eIF-2 complex (0.17 μg of total protein). The different levels of total protein added to each reaction adjusts for the lower yields of eIF-2 in the former preparation as determined by Western blot analysis using antibodies directed against the α subunit of eIF-2. (Lanes 1,2) The amount of ternary complex formed by wild-type eIF-2 in the presence of GTP (25 μm), and 25 μm GppNp, respectively in a 5-min reaction. (Lanes 3,4) The amount of ternary complex formed by mutant eIF-2β^L254P complex in the presence of GTP (25 μm); and 25 μm GppNp, respectively, in a 5-min reaction. The data in panels B–E represent the average of two independent experiments with a standard deviation <15%.