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- Translocation of the acceptor arm of t-RNA on the large subunit.
- Translocation of the m-RNA on the small subunit.
- 1. The first step is that EF-G, in the GTP bound conformation, enters the ribosome. It binds and catalyses translocation, after that GTP hydrolysis is triggered on the EF-G.
- 2. After the conformational change of Ef-G’s, domain IV of Ef-G separates the m-RNA anticodon helix from the decoding center. t-RNA and m-RNA are translocated by one codon on the ribosome. The next codon will move into the A site of the ribosome being recognized by amynoacyl-t-RNA.
Ef-G vs Ef-Tu binding to the ribosome
Both EF-Tu and EF-G bind near the L7/L12 stalk on the large ribosomal subunit.
The elongation binding sites on the large ribosomal subunit are composed of
- 1. SRL (sarcin-ricin loop) a fixed structure on the ribosome. The GTP biding region of both factors contacts SRL which is less flexible.
- 2. GAC (GTP-ase associated center) a mobile structure on the ribosome. GAC is found in either opened or closed conformation.
- The orientation of the GAC relative to the SRL determines whether EF-G or EF-Tu will bind to the ribosome. The smaller the distance between GAC relative to SRL the higher the chance of binding to the ribosome.
- EF-Tu and EF-G binding sites on the ribosome is more shaped specific than electrostatic.
GTP hydrolysis by EF
- GTP hydrolysis occurs by translation of guanosine triphosphatases (trGTPases). It provides the energy necessary for binding of t-RNA to EF-Tu and then the bind of the complex to the A site. GTP hydrolysis is necessary for the fast release of the EF from the ribosome.
- GTP acts in a similar manner with initiation factors and release factors.
Mechanism by which GTP is activated by the ribosome in eukaryotes and in prokaryotes.
- Highly conserved His84 acts as a general base to activate the catalytic water molecule, which is positioned by interactions with Thr61, Gly83, and His84.
- A 2662 is part of the SRL (sarcin ricin loop), a binding site on the ribosome. A2662 interacts with His84 and it will move it into position to place the water molecule in the vicinity of the gamma-phosphate of the GTP molecule.
- His activates the water molecule attacking the gamma-phosphate and hydrolyze GTP to GDP.
- Glutamine in a specific loop of switch II is essential as it places a water molecule at gamma-phosphate of the GTP molecule.
- Gamma-phopshate of GTP molecule removes a proton of the water molecule hydrolyzing the phosphate ester bond. The gamma and beta phophatases interact with a Mg2+ ion and a lysine from Thr 61 from the swich 1 loop.
- The water molecule donates two H+ bonds: one to the carboxyl oxygen of Thr 61 and one to the O2 of the gamma phosphate.