Ribosome exit site

In the 70 S initiation complex, formylme-thionine tRNA is initially located at a binding site known as the peptidyl site (P). A second binding site, the acceptor site (A), is not yet occupied during this phase of translation. Sometimes, a third tRNA binding site is defined as an exit site (E), from which uncharged tRNAs leave the ribosome again (see p. 252 not shown). 

Ban et al 7 Courtesy of T. A. Steitz. The peptidyltransferase center is marked by the green image of the transition state inhibitor shown in Fig. 29-13. (F) Model of three tRNAs bound to a ribosome from Thermus thermophilus in the A (a mi noacyl), P (pepti-dyl), and E (exit) sites. These are based on 0.75-nm X-ray data and a number of difference electron density maps. The 3-CCA end of the A-site tRNA is not modeled hut is... 

A careful stereochemical analysis has led to the conclusion that for all of the different aminoacyl groups to be able to react in the same way at the peptidyltransferase site and to all generate trans amide linkages, the torsion angles < ) and q/ of the resulting peptide must be approximately those of an a helix.388 Thus, the peptide emerging from the ribosome exit tunnel may be largely helical. 

Elongation factor EF-G and translocation. The third step in the elongation sequence on ribosomes (Fig. 29-12, step g) depends upon EF-G, a monomeric GTP-binding protein with a sequence homologous with that of other members of the G protein family. It apparently utilizes the Gibbs energy of hydrolysis of GTP to GDP to drive translocation of the peptidyl-tRNA from the A site to the P site (Fig. 29-12) and of the previously utilized (de-acylated) tRNA to the exit site. 

Fig. 2. Schematic of a prokaryotic 70S ribosome showing the peptidyl-tRNA site (P site), aminoacyl-tRNA site (A site) and exit site (E site).

Figure 29.22. Transfer RNA-Binding Sites. (A) Three tRNA-binding sites are present on the 70S ribosome. They are called the A (for aminoacyl), P (for peptidyl), and E (for exit) sites. Each tRNA molecule contacts both the SOS and the 50S subunit. (B) The tRNA molecules in sites A and P are base paired with mRNA.

The ribosome includes three sites for tRNA binding called the A (aminoacyl) site, the P (peptidyl) site, and the E (exit) site. With a tRNA attached to the growing peptide chain in the P site, an aminoacyl-tRNA binds to the A site. A peptide bond is formed when the amino group of the aminoacyl-tRNA nucleophically attacks the ester carbonyl group of the peptidyl-tRNA. On peptide-bond formation, the tRNAs and mRNA must be translocated for the next cycle to begin. The deacylated tRNA moves to the E site and then leaves the ribosome, and the peptidyl-tRNA moves from the A site into the P site. 

Translation of the genetic code from messenger RNA (mRNA) to proteins via appropriate selection of transfer RNAs (tRNA) is accomplished by the macro-molecular ribosome machine. The 70S ribosome is composed of two main subunits, the 50S and 30S, which are composed of ribosomal RNA (rRNA) and many individual ribosomal proteins (numerical values of the subunits relate to sedimentation/centrifugation rates). The main subunits of the 50S and 30S are the 23S and 16S, respectively. Spanning the contact surfaces of the 50S and 30S are the aminoacyl-, peptidyl-, and "exit"-sites, labeled the A-, P-, and E-sites... 

Since the distance between the nascent chain exit site from the ribosome and the peptidyl-transfer center is about 16 nm, it is possible that the SRP exerts its effect on protein translation by binding to both the signal sequence (near the exit site) and a component of the peptidyl-transfer site (Andrews et al., 1985). 

Ribosome function is complex numerous cofactors are required for initiation, elongation, and termination (for a detailed description please see ref 33). Independent functions can be ascribed to the two subunits. Peptide bond formation takes place on the 50S subunit within the peptidyl transferase center, whereas decoding of the mRNA takes place on the 308 subunit within the decoding A- and P-sites. tRNAs in the P-site and the A-site span both subunits and couple the two events. After each round of peptide bond formation, a translocation step takes place that involves the movement of the mRNA through the ribosome, transfer of the P-site tRNA to the E (or exit)-site, and transfer of the A-site tRNA to the P-site. Most antibiotics target one of the listed steps decoding at either the A-site or the P-site, peptide bond formation within the peptidyl transferase center, or translocation. 

Following peptide bond synthesis, the ribosome Is translocated along the mRNA a distance equal to one codon. This translocation step is promoted by hydrolysis of the GTP in eukaryotic EF2-GTP. As a result of translocation, tRNAj , now without its activated methionine, is moved to the E (exit) site on the ribosome concurrently, the second tRNA, now covalently bound to a dIpeptIde (a peptIdyl-tRNA), Is moved to the P site (Figure 4-26, step U). Translocation thus returns the ribosome conformation to a state in which the A site Is open and able to accept another amlnoacylated tRNA complexed with EFlct-GTP, beginning another cycle of chain elongation. 

Step 3 is the GTP-catalyzed translocation of the 70S ribosome one codon position in the 30 direction. This step requires the elongation factor EF-G and results in a vacancy in the A site, which is now ready to accept the next aminoacyl-tRNA. If the tRNA exit site (E site) were included in the figure, it would be... 

Three key steps in the elongation stage of protein synthesis are required for the addition of each amino acid. Because these steps are repeated for each peptide bond formed, this is sometimes called the elongation cycle. The central theme in elongation is that the fully assembled ribosomal complex functions as a ribonucleoprotein machine which rapidly moves 50 to 30 down the mRNA, much like a ratchet. At the center of this complex are two binding sites which line up over a pair of triplet codons, as shown in Figure 26.11. These two sites are called the P site, for peptidyl (or polypeptide), and the A site, for aminoacyl (or acceptor). A third site, called the E site for tRNA exit site, is also a functional component of the ribosome, but for reasons of clarity, it is not included in the figures. 

Deposition binding might cause a great conformational change in the overall three-dimensional structure essential for peptidyltransferase activity and the introduction of nascent peptide into the exit tunnel. Such modification also probably renders correct alignment of the peptidyl and aminoacyl substrates at the ribosome catalytic site, hindering peptide formation. In this connection, additional investigation is needed to obtain clear direct evidence. 

In chain elongation, the second amino acyl-tRNA binds to the A site. This amino acid s a-amino group performs a nucleophilic attack on the carbonyl group of the A-formylmethionine in the peptidyl transfer reaction. In a translocation step, the ribosome then moves one codon, leaving a dipeptidyl-tRNA in the A site and moving the unchained tRNA to the exit site. The process continues with a new aminoacyl-tRNA entering the P site. The uncharged tRNA is then ejected from the E site. 

The A site and the P site on the ribosome are both binding sites for charged tRNAs taking part in protein synthesis. The P (peptidyl) site binds a tRNA to which the growing polypeptide chain is bonded. The A (aminoacyl) site binds to an aminoacyl tRNA. The amino acid moiety is the next one added to the nascent protein. The E (exit) site binds the uncharged tRNA until it is released from the ribosome. 

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