Point of
VIEW
Deepening Ribosomal Insights Anders Liljas*
Department of Molecular Biophysics, Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-221 00, Lund, Sweden
T
ranslation of messenger RNA (mRNA) to protein occurs on the ribosome. The mRNA binds around the neck of the small (30S) subunit, between the head and the body, whereas the peptidyl transfer occurs at the peptidyl transfer center (PTC) on the large (50S) subunit (Figure 1). The ribosome has three major sites for transfer RNA (tRNA) molecules, the A-, P-, and E-sites. The A-site is where the codon–anticodon interactions are scrutinized to ensure that the correct amino acid will be incorporated. The acceptor end of the tRNA is composed of three conserved single-stranded nucleotides, C74, C75, and A76. The aminoacyl moiety attached to A76 of the tRNA in the A-site accepts the growing polypeptide from the tRNA in the P-site during peptidyl transfer. The new peptidyl-tRNA is subsequently translocated from the A-site to the P-site. The deacylated tRNA is translocated from the P-site to the exit (E)-site, where it finally dissociates. Translation is catalyzed by several GTPases, primarily elongation factors Tu (EF-Tu) and G (EF-G). Whereas EF-Tu with GTP delivers the charged tRNA to the ribosome and dissociates after GTP hydrolysis, EF-G with GTP catalyzes the translocation of mRNA and tRNAs (see below). Two new structures of 70S ribosomes (1, 2) add significantly to the information gained by the high-resolution structures of separate 30S subunits from Thermus thermophilus and 50S subunits (3, 4) from Haloarcula marismortui (5) and Deinococcus radiodurans (6), the 5.5-Å structure of 70S T. thermophilus ribosomes (7), and the two www.acschemicalbiology.org
complete Escherichia coli ribosomes analyzed at 3.5-Å resolution (8). For a full understanding of ribosomal function, we need to study complete ribosomes and as many of their complexes as possible at high resolution. Even though the ribosomes in the most recent studies come from the same species, the varying crystallization conditions have led to different crystal packing and diffraction power (1, 2). With improved resolution, new details can be identified and old observations can be confirmed or contradicted and are of great general interest. With regard to the general structure, certain features remain too flexible to be seen. In both structures, proteins L10, L7/L12, and L11 at the GTPase center are not seen, and a few additional poorly visible proteins differ between the two structures. In the new 2.8-Å resolution structure from Selmer et al. (1), the authors fitted protein L28 into the density that previously had been interpreted as L31. This gave a better agreement with both the electron density and biochemical observations. Protein L31 could instead be placed in a density close to protein L5 in the 50S subunit. Furthermore, they could see no density for protein L36 where it had been placed previously in Deinococcus (1), whereas Korostelev et al. (2) seem to have seen it. L36 has been found to organize a conserved region of the 50S subunit (9). These observations do not lead to any alteration of the functional mechanism. The mRNA and the Sites for tRNAs. The two structures provide snapshots of the ribosome bound to different components. In
A B S T R A C T The remarkable progress of cryoelectron microscopy and crystallography in elucidating ribosomal structure and function continues. Most recently, two papers about complete 70S ribosomes from Thermus thermophilus at 2.8and 3.7-Å resolution give us more details about the conformations of bound transfer RNA (tRNA) molecules; the bridges between subunits; the locations and roles of proteins, magnesium ions, and water molecules; and the dynamics of ribosomes. Very significant new insights have been gained, particularly for the tRNAs, which can only be studied in their entirety in full ribosomes.
*Corresponding author,
[email protected].
Published online October 20, 2006 10.1021/cb600407u CCC: $33.50 © 2006 by American Chemical Society
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This magnesium ion is also held in place by the 30S subunit. In the 2.8-Å structure, only the anticodon stem and loop (ASL) of the A-site tRNA was visible. Its conformation would allow the acceptor arm to be located in the PTC (1). The details of the interaction support the mechanism for decoding established earlier from studies of 30S subunits alone (10). In this mechanism, the aminoacyl-tRNA is severely bent between the ASL and the D-stem during the initial binding of aminoacyl-tRNA in complex with EF-Tu (11, 12). The fact that only the ASL is Figure 1. a) A schematic illustration of the organization of seen suggests that the tRNA the ribosome. The large subunit (50S) is seen behind, and retains its flexibility at this the small subunit (30S) is in the foreground. The ribosomal region. functional sites are between the ribosomal subunits. The mRNA is bound around the neck of the small subunit Compared with the tRNA in between the head and the body. Three sites for tRNA are the A-site, the P-site tRNA is shown, the A-, P-, and E-sites. The binding sites for the bound firmly and surrounded catalyzing translational factors, the GTPases, are also by ribosomal RNA (rRNA) and shown. Several bridges (B1–B7) between the subunits are ribosomal proteins from both also illustrated. Two RNA helices are of special interest, h44 (30S) and H69 (50S). They make a functionally important subunits (1, 2). This prevents interaction at bridge B2a, which is at the decoding site (Aloss of the peptidyl-tRNA and site) for tRNA. b) This top view of the ribosome shows the also helps to maintain the mRNA, the sites for tRNA, and the PTC. reading frame. The P-site tRNA the 3.7-Å ribosomal structure from Korosis distorted in both new structures comtelev et al. (2), a 10-nucleotide mRNA and pared with isolated tRNA. This seems to be 2 tRNA molecules bound: a tRNAPhe in the due to opposing interactions by the head of P-site and a mixture of deacylated tRNAs in the small subunit and helix H69 of the large the E-site. The 2.8-Å structure contains an subunit on the ASL. An interesting observamRNA, an aminoacyl-tRNAPhe in the A-site, tion is the fact that releasing the distortion a deacylated initiator tRNAfMet in the P-site, drives the tRNA toward the E-site (1). and a noncognate tRNA in the E-site (1). The The question of whether the E-site tRNA antibiotic paromomycin was bound to stabi- contacts its codon on the 30S subunit is lize the interactions at the A-site and to close to an answer. In the 2.8-Å structure, inhibit translocation. this anticodon is closer to the rRNA than to As seen before, the mRNA makes a sharp the mRNA (1). Even though the codon–antikink between the codons in the A- and codon pair is noncognate, it would not seem P-sites (1, 2). This bend is now seen to be possible to form a cognate codon–antistabilized by a Mg2⫹ ion between the codon interaction without significant movements (Figure 1, panel b). The opposite, or closest phosphates of the two codons (1). 568
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acceptor, end of the tRNA is firmly held against the large subunit. The terminal A76 is intercalated between G2421 and A2422, and the 3=-OH is surrounded by elements of the 50S subunit. It is obvious from the narrow space that only a deacylated tRNA can be bound at this site. Also, the E-site tRNA is distorted, but less so than what is seen for the A- and P-sites. The strain placed on the tRNA from the initial binding to the ribosome in complex with EF-Tu and GTP could thus be gradually decreased in the subsequent sites. The PTC. Peptidyl transfer occurs on the 50S subunit (Figure 1, panel b). The acceptor ends of the two tRNAs in the A- and P-sites with their aminoacyl and peptidyl moieties are closely bound to promote the transfer of the peptide on the tRNA in the P-site to the aminoacyl residue of the tRNA in the A-site. Unlike other polymerases, RNA forms vital parts of the functional sites (2). However, the claim that the ribosome is a ribozyme is now less certain for several reasons. First, no group from the rRNA seems to be directly involved with the central activity of the ribosome, peptidyl transfer (13, 14). Second, Maguire et al. (15) found that the three N-terminal residues of L27 were important for full peptidyl transferase activity. In the 3.7-Å structure, the nine N-terminal residues of L27 could not be seen (2), but in the 2.8-Å structure, the L27 N-terminus was close enough to interact with A76 of the P-site tRNA (1). Archaea are different from bacteria in that they do not have protein L27 (5, 6). The rRNA performs no major catalytic role, but with the aid of the ribosomal proteins, it provides selective binding sites for mRNA, tRNAs, and protein factors (13, 14). The Bridges between the Subunits. A primary interaction between the subunits is the three tRNAs, in particular the P-site tRNA. In addition, 12 subunit bridges exist between different ribosomal components (7). These bridges, initially identified from cryo-electron microscopy studies (16), are now seen with improved clarity. One additional bridge is seen in the 2.8-Å structure www.acschemicalbiology.org
Point of
VIEW toward the bottom of helix h44 (1). Helix h44 (30S) is part of the decoding site, and H69 (50S) is part of the peptidyl transfer site. They are also part of the ribosomal interface and the areas where the changes of subunit orientation take place during the translocation of mRNA and tRNAs. One important path for communicating correct codon–anticodon interactions in the decoding site to the GTPase binding site on the 50S subunit has been identified. In bridge B2a (Figure 1, panel a), A1913 of H69 is inserted into a pocket between h44 and the A-site tRNA and makes a hydrogen bond to the 2=OH of nucleotide 37 next to the two ribosomal bases A1492 and A1493, which participate in discriminating noncognate from cognate tRNAs. Two magnesium ions further stabilize the contacts between H69, h44, and A-site tRNA in this bridge. This interaction is not present in the empty E. coli ribosomes and requires a significant conformational change. Three other bridges (B5, B6, and B8) are also stabilized by magnesium ions. Bridge B2c is purely magnesiummediated. Bridge B6 is mediated by a single solvent molecule. Ribosomal Dynamics. Scientists have long known that translation is a dynamic process. The codons of the mRNA and the tRNA molecules are translocated successively from the A- to the P- to the E-sites. In the process, they change conformation. The neck of the small subunit allows movements of the head in different states of the functional cycle. Individual ribosomal components also undergo dynamic structural changes that can be inferred from the lack of density for several components. It is now clear that nucleotides A1339 and G1338 in the head of the small subunit, as well as nucleotide 790 on the platform of the small subunit, interact with the ASL of the P-site tRNA and prevent it from moving into the E-site (1, 2, 8). Obviously, this gate needs to open during translocation. Protein L1 interacts with the E-site tRNA (1, 2, 5–7). To release this deacylated tRNA, L1 undergoes www.acschemicalbiology.org
a large conformational change to the one observed in empty ribosomes (1, 2, 8). This is seen in greater detail in the new structures. Moving forward. Researchers in the field of ribosomes have reached yet another goal: to see, at high resolution, the complete ribosome with bound mRNA and tRNAs. Many of the previous findings are now more firmly established. The improved resolution also leads to greater detail, corrections of details, and identification of distorted or strained structures. Thus, the tRNAs do not conform to the structure seen in isolation but deviate to a decreasing extent from the A-site to the E-site. The E-site tRNA does not seem to interact with its codon. Crystallographic data shows that in the PTC, a protein, L27, interacts with A76 of the P-site tRNA. The improved detail of the 70S structures will provide a richer source for precise biochemical experimentation. Much is known, but much remains to be explored by crystallography. Several translation factors have been studied on the ribosome, but key factors remain to be analyzed, primarily the GTPases. So far, no useful crystals with any of the translational GTPases have been reported. Their binding site seems to be an important site for crystal contacts with 70S ribosomes. A crystal structure with one of these GTPases would most likely also elucidate some of the now invisible proteins at the L12 stalk in functional interactions. REFERENCES 1. Selmer, M., Dunham, C. M., Murphy, F. V., IV, Weixlbaumer, A., Petry, S., Kelley, A. C., Weir, J. R., and Ramakrishnan, V. (2006) Structure of the 70S ribosome complexed with mRNA and tRNA, Science 313, 1935–1942. 2. Korostelev, A., Trakhanov, S., Laurberg, M., and Noller, H. (2006) Crystal structure of a 70S ribosometRNA complex reveals functional interactions and rearrangements, Cell 126, 1065–1077. 3. Wimberly, B. T., Brodersen, D. F., Clemons, W. M., Jr., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Structure of the 30S ribosomal subunit, Nature 407, 327–339.
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