- 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.
Helicase- models of DNA unwinding
- There are three different models of DNA unwinding:
- 1. The wedge model
- 2. The torsional model
- 3. The helix destabilizing model.
Wedge model of DNA unwinding
The hexamer at the unwiding junction interacts with one DNA strand through its central channel. The excluded strand and duplex DNA do not interact tightly. The force producing unidirectional translocation leads to separation of duplex base pairs .
Torsional model of DNA unwinding
The helicase interacts tightly with both the separated strands. The rotation of one strand with respect to the other results in unwinding of duplex DNA.
Helix destabilizing model of DNA unwinding
The hexamer interacs with one of the separated strands in the central channel, and also it interacts with the dsDNA on the outer parts of the hexamer. Helicases encounter a region of double helix they continue to move along their strand prying apart the double helix. The duplex region is melted by the helicase, and the helicase translocates unidirectionally along the DNA in the central channel.