Ribosome exit tunnel

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. 

Macrolides are a group of antibiotics, produced in nature by many actinomycetes strains, that are composed of a 12- to 16-membered lactone ring, to which one or more sugar substituents is attached. They target the peptidyl transferase center on the 50S ribosomal subunit and function primarily by interfering with movement of the nascent peptide away from the active site and into the exit tunnel. 

This exit tunnel through the 50S subunit was first revealed by 3-D image reconstruction 20 years ago (Yonath et al., 1987) by two giants of ribosome research, Ada Yonath and the late Heinz-Giinther Wittmann (in whose laboratory in Berlin I spent a very fruitful stay 1970-1973). 

The peptidyl transferase centre of the ribosome is located in the 50S subunit, in a protein-free environment (there is no protein within 15 A of the active site), supporting biochemical evidence that the ribosomal RNA, rather than the ribosomal proteins, plays a key role in the catalysis of peptide bond formation. This confirms that the ribosome is the largest known RNA catalyst (ribozyme) and, to date, the only one with synthetic activity. Adjacent to the peptidyl transferase centre is the entrance to the protein exit tunnel, through which the growing polypeptide chain moves out of the ribosome. 

Most of the chemical activity of ribosomes occurs in the interface between the 30S and 50S subunits. Entrance and exit tunnels for both mRNA and the amino-acylated tRNAs are formed between these subunits. The mRNA apparently moves across the platform as the tRNAs move from A to P to E sites experiencing codon selection (decoding) and peptidyltransferase activity. Many loop ends from 16S RNA interact with those of 23S RNA.41 88... 

Most antibiotics that inhibit the function of the SOS subunit bind near its peptidyl transferase center (Fig. 4.4) and block peptide bond formation. Crystal structures are available for several such antibiotics bound to the ribosome (Fig. 4.5). They appear to inhibit the peptidyl transferase reaction either by competing directly with its substrates for binding, or indirectly by blocking the exit tunnel. 

Fig. 4.5 Overview of antibiotics bound at the peptidyl transferase center. A surface representation of the large subunit of H. marismortui includes the P-site, A-site and entrance to the peptide exit tunnel. Most of these antibiotics contact either the active site hydro-phobic crevice (green surface, upper center) or the hydrophobic crevice at the entrance to the exit tunnel (green surface, lower right). In addition, many of these antibiotics occupy an elongated pocket (dark surface, center) in the wall of the exit tunnel between these two crevices. The antibiotics shown are all from complexes with H. marismortui ribosomes and overlap the binding site of A-site substrates (red sticks) or of a P-site substrates (orange sticks). Fig. modified from...

In H. marismortui, the 15-membered and 16-membered macrolides studied so far bind to the ribosome almost identically. When rRNA portions of these structures are superimposed, the lactone rings of the macrolides become superimposed on an almost atom by atom basis (Fig. 4.7). At the center of the macrolide binding site is the hydrophobic crevice at the entrance to the peptide exit tunnel between Hm G2099 and A2100 Ec A2058 and A2059) (Fig. 4.8). 

The bound macrolides almost completely occlude the peptide exit tunnel, which explains the two distinctive characteristics of macrolide inhibition. Firstly, the reason that macrolides do not inhibit ribosomes that are already actively making protein is that the nascent peptide in the exit tunnel blocks access to the macrolide binding site. Secondly, the reason macrolides do not directly inhibit the peptidyl transferase reaction is that they bind near to, but not directly at, the active site. Only after a few peptide bonds are formed will the peptide contact the bound macrolide and be blocked from further elongation. As a result, short di-, tri- and tetra-peptides will accumulate. 

When tylosin binds to the ribosome, the mycinose extension from C14 of the lactone ring extends down the exit tunnel and interacts with domain II of rRNA (Fig. 4.8 B). Nucleotide modification by N1 methylation of Ec G748 (Hm A841) in... 

Two crystal structures of chloramphenicol bound to the ribosome are available. In one structure, chloramphenicol is observed to bind only at the active site hydro-phobic crevice of the bacterial (D. radiodurans) ribosome [4]. In the other structure chloramphenicol binds only at the hydrophobic crevice at the entrance to the exit tunnel of an archaeal (H. marismortui) ribosome [7]. Both of these sites are surrounded by nucleotides implicated in chloramphenicol binding either by nucleotide protection studies or by mutational studies (Fig. 4.12). They probably correspond to the two sites inferred from biochemical experiments. 

Currently, it seems likely that the high affinity binding site of chloramphenicol in bacterial ribosomes is the active site hydrophobic crevice and that the low affinity site corresponds to the crevice at the entrance to the peptide exit tunnel. In addition, it seems reasonable that binding of chloramphenicol at the active site crevice might have a greater inhibitory effect on protein translation than binding at the more distant site. 

Fig. 4. 12 Chloramphenicol. A cutaway view of a space filled representation of the H. marismortui ribosome chloramphenicol bound at two sites, the active site hydrophobic crevice [4] and at the hydrophobic crevice at the entrance to the exit tunnel [7]. Both of these binding sites are surrounded by nucleotides that upon mutation confer resistance to chloramphenicol (orange spheres) or that are protected by chloramphenicol from chemical modification (green spheres). Fig. from [7].

According to Nissen et al. [27], proteins L4, L22, and L39e (the letter e represents a protein in a bacterial SOS ribosomal subunit, a protein that belongs to one of the homologs in eukaryotic 60S ribosomal subunit) have been shown to be present in the polypeptide exit tunnel [average diameter, about IS A length of the tunnel, 100 A (Fig. 3B)] present in the SOS ribosomal subunit from H. maris-mortui. Six other proteins (L19, L22, L23, L24, L29, and L31e) are known to be located in the exit area of the polypeptide tunnel [26, 27]. If these proteins are involved in the inhibitory actions of macrolide antibiotics, mutant bacteria resistant to macrolides will develop in the future. 

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. 

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