Abstract
Bovine mitochondrial elongation factor Ts (EF-Tsmt) stimulates the activity of Escherichia coli elongation factor Tu (EF-Tu). In contrast, E. coli EF-Ts is unable to stimulate mitochondrial EF-Tu. EF-Tsmt forms a tight complex with E. coli EF-Tu governed by an association constant of 8.6 x 10(10). This value is 100-fold stronger than the binding constant for the formation of the E. coli EF-Tu.Ts complex. To test which domain of EF-Tsmt is important for its strong binding with EF-Tu, chimeras were made between E. coli EF-Ts and EF-Tsmt. Replacing the N-terminal domain of E. coli EF-Ts with that of EF-Tsmt increases its binding to E. coli EF-Tu 2-3-fold. Replacing the N-terminal domain of EF-Tsmt with the corresponding region of E. coli EF-Ts decreases its binding to E. coli EF-Tu approximately 4-5-fold. A chimera consisting of the C-terminal half of E. coli EF-Ts and the N-terminal half of EF-Tsmt binds to E. coli EF-Tu as strongly as EF-Tsmt. A chimera in which Subdomain N of the core of EF-Ts is replaced by the corresponding region of EF-Tsmt binds E. coli EF-Tu approximately 25-fold more tightly than E. coli EF-Ts. Thus, the higher strength of the interaction between EF-Tsmt and EF-Tu can be localized primarily to a single subdomain.
Highlights
The classical model for the elongation cycle of protein biosynthesis was developed on observations made with Escherichia coli elongation factor Tu (EF-Tu).1 EF-Tu forms a ternary complex with aminoacyl-tRNA and GTP that promotes the binding of the aminoacyl-tRNA with the A-site of the ribosome
The activity of Chimera V is slightly higher than that of EF-Tsmt but significantly lower than E. coli EF-Ts. These results indicate that EF-Tsmt and the chimeras that bind to EF-Tu more tightly have lower activities in promoting guanine nucleotide exchange
The N-terminal Domain and Subdomain N Determine the Tight Binding of EF-Tsmt to E. coli EF-Tu—The data presented here show that EF-Tsmt binds to E. coli EF-Tu ϳ100-fold more tightly than E. coli EF-Ts
Summary
The classical model for the elongation cycle of protein biosynthesis was developed on observations made with Escherichia coli EF-Tu. EF-Tu forms a ternary complex with aminoacyl-tRNA and GTP that promotes the binding of the aminoacyl-tRNA with the A-site of the ribosome. When EF-Tsmt is expressed and purified from E. coli, it forms a 1:1 complex with E. coli EF-Tu (EFTuEco1⁄7Tsmt) (9) This heterologous complex is very resistant to dissociation by guanine nucleotides even at high concentrations of GDP or GTP (10). This feature of the heterologous complex is quite reminiscent of the native EF-Tu1⁄7Tsmt complex It is apparently the nature of EF-Ts that determines the strength of its interaction with EF-Tu. it is apparently the nature of EF-Ts that determines the strength of its interaction with EF-Tu It is not clear what features of EF-Tsmt modulate its tight interaction with EF-Tu. The crystal structures of trypsin-modified E. coli EF-Tu1⁄7GDP and of Thermus aquaticus EF-Tu complexed with a nonhydrolyzable GTP analogue have been determined (11, 12). This structure indicates that EF-Ts consists of 4 structural modules: the N-terminal domain (residues 1–54); the core domain (residues 55–179 and 229 –263); the dimerization domain
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