Genes VII

The basic message to remember about the ribosome is that it is a cooperative structure that depends on changes in the relationships among its active sites during protein synthesis. The active sites are not small, discrete regions like the active centers of enzymes. They are large regions whose construction and activities may depend just as much on the rRNA as on the ribosomal proteins. The crystal structures of the individual subunits of bacterial ribosomes, and (at lesser resolution) of the intact ribosome, give us a good impression of the overall organization and emphasize the role of the rRNA.

Figure 6.29 shows that the 30S subunit has an asymmetrical distribution of RNA and protein (931, 1184). The interface with the 50S subunit is composed almost entirely of RNA. Only two proteins (a small part of S7 and possibly part of S12) lie near the interface. This means that the association and dissociation of ribosomal subunits must depend on interactions with the 16S rRNA. Subunit association is affected by a mutation in a loop of 16S rRNA (at position 791) that is located at the subunit interface, and other nucleotides in 16S rRNA have been shown to be involved by modification/interference experiments. This behavior supports the idea that the evolutionary origin of the ribosome may have been in a particle consisting of RNA rather than protein.

The 50S subunit has a more even distribution of components than the 30S, with long rods of double-stranded RNA crisscrossing the structure (932, 1086). The RNA forms a mass of tightly packed helices. The exterior surface largely consists of protein, except for the peptidyl transferase center (see later). Almost all segments of the 23S rRNA interact with protein, but many of the protein are relatively unstructured

Figure 6.30 shows the 70S ribosome with the positions of tRNAs in the three binding sites (933). The tRNAs in the A and P sites are nearly parallel to one another. The aminoacyl end of the tRNA in the A site can reach the 3′ end of the tRNA in the P site, perhaps with a conformational change. The orientation of tRNA in the P site is determined largely by its anticodon stem, which is gripped tightly by the site in the 30S subunit. By contrast, the A site is much larger than the tRNA it holds. This fits with the roles of the two sites: by the time a tRNA arrives in the P site, it has been accepted for protein synthesis, and the main issue is to maintain integrity of the reading frame. But when a tRNA arrives in the A site, it is necessary to discriminate between appropriate (cognate) and inappropriate (noncognate) tRNAs, requiring some flexibility.

The tRNA in the P site contacts both rRNA and r-proteins, while the environment of the A site is largely made of rRNA (1185). Thus the process of decoding is largely RNA-mediated. By contrast, the E site, which is concerned just with expelling tRNA, has a protein environment.

The crystal structures show that both major rRNAs have considerable secondary structure. The most penetrating approach to analyzing secondary structure of such large RNAs is to compare the sequences of corresponding rRNAs in related organisms. Those regions that are important in the secondary structure retain the ability to interact by base pairing. So if a base pair is required, it can form at the same relative position in each rRNA. This approach has enabled detailed models to be constructed for both 16S and 23S rRNA.

16S rRNA forms four general domains, in which just under half of the sequence is base paired (see Figure 6.32). The individual double-helical regions tend to be short (<8 bp). Often the duplex regions are not perfect, but contain bulges of unpaired bases. Comparable models have been drawn for mitochondrial rRNAs (which are shorter and have fewer domains) and for eukaryotic cytosolic rRNAs (which are longer and have more domains). The increase in length in eukaryotic rRNAs is due largely to the acquisition of sequences representing additional domains (for review see 431). Each domain of 16S rRNA folds independently and has a discrete location in the 30S subunit.

Differences in the reactivity of 16S rRNA are found when 30S subunits are compared with 70S ribosomes; also there are differences between free ribosomes and those engaged in protein synthesis. Changes in the reactivity of the rRNA occur when mRNA is bound, when the subunits associate, or when tRNA is bound. Some changes reflect a direct interaction of the rRNA with mRNA or tRNA, while others are caused indirectly by other changes in ribosome structure. The main point is that ribosome conformation is flexible during protein synthesis.

A feature of the primary structure of rRNA is the presence of methylated residues. There are ~10 methyl groups in 16S rRNA (located mostly toward the 3′ end of the molecule) and ~20 in 23S rRNA. In mammalian cells, the 18S and 28S rRNAs carry 43 and 74 methyl groups, respectively, so ~2% of the nucleotides are methylated (about three times the proportion methylated in bacteria).

The large ribosomal subunit also contains a molecule of a 120 base 5S RNA (in all ribosomes except those of mitochondria). The sequence of 5S RNA is less well conserved than those of the major rRNAs. All 5S RNA molecules display a highly base-paired structure.

In eukaryotic cytosolic ribosomes, another small RNA is present in the large subunit. This is the 5.8S RNA. Its sequence corresponds to the 5′ end of the prokaryotic 23S rRNA.

Some ribosomal proteins bind strongly to isolated rRNA. Some do not bind to free rRNA, but can bind after other proteins have bound. This suggests that the conformation of the rRNA is important in determining whether binding sites exist for some proteins. As each protein binds, it induces conformational changes in the rRNA that make it possible for other proteins to bind. In E. coli, virtually all the 30S ribosomal proteins interact (albeit to varying degrees) with 16S rRNA. The binding sites on the proteins show a wide variety of structural features, suggesting that protein-RNA recognition mechanisms may be diverse.

Much of the structure of the ribosome is occupied by its active centers. The expanded view of the ribosomal sites drawn in Figure 6.31 shows they comprise about two thirds of the ribosomal structure. A tRNA enters the A site, is transferred by translocation into the P site, and then leaves the (bacterial) ribosome by the E site. The A and P sites must extend across both ribosome subunits, since tRNA is paired with mRNA in the 30S subunit, but peptide transfer takes place in the 50S subunit. The A and P sites are adjacent, enabling translocation to move the tRNA from one site into the other. The problem of how two bulky tRNAs fit into the ribosome is solved by a turn in the path for mRNA. The E site is located near the P site (representing a position en route to the surface of the 50S subunit). The peptidyl transferase center is located on the 50S subunit, close to the aminoacyl ends of the tRNAs in the A and P sites (see next section).

All of the G-proteins that function in protein synthesis (EF-Tu, EF-G, IF-2, RF1,2,3) bind to a factor-binding site (sometimes called the GTPase center), which probably triggers their hydrolysis of GTP. It is located at the base of the stalk of the large subunit, which consists of the proteins L7/L12. (L7 is a modification of L12, and has an acetyl group on the N-terminus.) In addition to this region, the complex of protein L11 with a 58 base stretch of 23S rRNA provides the binding site for some antibiotics that affect GTPase activity. Neither of these ribosomal structures actually possesses GTPase activity, but they are both necessary for it. The role of the ribosome is to trigger GTP hydrolysis by factors bound in the factor-binding site.

Initial binding of 30S subunits to mRNA requires protein S1, which has a strong affinity for single-stranded nucleic acid. It is responsible for maintaining the single-stranded state in mRNA that is bound to the 30S subunit. This action is necessary to prevent the mRNA from taking up a base-paired conformation that would be unsuitable for translation. S1 has an extremely elongated structure and associates with S18 and S21. The three proteins constitute a domain that is involved in the initial binding of mRNA and in binding initiator tRNA. This locates the mRNA-binding site in the vicinity of the cleft of the small subunit (see Figure 6.2). The 3′ end of rRNA, which pairs with the mRNA initiation site, is located in this region.

The initiation factors bind in the same region of the ribosome. IF-3 can be crosslinked to the 3′ end of the rRNA, as well as to several ribosomal proteins, including those probably involved in binding mRNA. The role of IF-3 could be to stabilize mRNA P30S subunit binding; then it would be displaced when the 50S subunit joins.

The incorporation of 5S RNA into 50S subunits that are assembled in vitro depends on the ability of three proteins, L5, L8, and L25, to form a stoichiometric complex with it. The complex can bind to 23S rRNA, although none of the isolated components can do so. It lies in the vicinity of the P and A sites.

The important functional sites of the ribosome consists of both RNA and protein. At both the A site and P site, the bound tRNA interacts with rRNA as well as with r-proteins. Similarly, a group of several proteins and the 23S rRNA are involved in creating the peptidyl transferase site. The catalytic activity of this site is exercised by the RNA. The site has been localized on the central protuberance by the binding of puromycin. We discuss the functions of RNA in these sites in the next section.

A nascent protein debouches through the ribosome, away from the active sites, into the region in which ribosomes may be attached to membranes (see 8 Protein localization). A polypeptide chain emerges from the ribosome through an exit channel, which leads from the peptidyl transferase site to the surface of the 50S subunit. It emerges ~15 Å away from the peptidyl transferase site. It probably extends through the ribosome as an unfolded polypeptide chain until it leaves the exit domain, when it is free to start folding.

This section updated 8-29-2000