The large ribosomal subunit catalyzes peptide bond formation and binds initiation, termination, and elongation factors. We have determined the crystal structure of the large ribosomal subunit from Haloarcula marismortui at 2.4 angstrom resolution, and it includes 2833 of the subunit's 3045 nucleotides and 27 of its 31 proteins. The domains of its RNAs all have irregular shapes and fit together in the ribosome like the pieces of a three-dimensional jigsaw puzzle to form a large, monolithic structure. Proteins are abundant everywhere on its surface except in the active site where peptide bond formation occurs and where it contacts the small subunit. Most of the proteins stabilize the structure by interacting with several RNA domains, often using idiosyncratically folded extensions that reach into the subunit's interior.

Using the atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with two substrate analogs, we establish that the ribosome is a ribozyme and address the catalytic properties of its all-RNA active site. Both substrate analogs are contacted exclusively by conserved ribosomal RNA (rRNA) residues from domain V of 23S rRNA; there are no protein side-chain atoms closer than about 18 angstroms to the peptide bond being synthesized. The mechanism of peptide bond synthesis appears to resemble the reverse of the acylation step in serine proteases, with the base of A2486 (A2451 in Escherichia coli) playing the same general base role as histidine-57 in chymotrypsin. The unusual pK(a) (where K(a) is the acid dissociation constant) required for A2486 to perform this function may derive in part from its hydrogen bonding to G2482 (G2447 in E. coli), which also interacts with a buried phosphate that could stabilize unusual tautomers of these two bases. The polypeptide exit tunnel is largely formed by RNA but has significant contributions from proteins L4, L22, and L39e, and its exit is encircled by proteins L19, L22, L23, L24, L29, and L31e.

Analysis of the 2.4-Å resolution crystal structure of the large ribosomal subunit from Haloarcula marismortui reveals the existence of an abundant and ubiquitous structural motif that stabilizes RNA tertiary and quaternary structures. This motif is termed the A-minor motif, because it involves the insertion of the smooth, minor groove edges of adenines into the minor groove of neighboring helices, preferentially at C-G base pairs, where they form hydrogen bonds with one or both of the 2 OHs of those pairs. A-minor motifs stabilize contacts between RNA helices, interactions between loops and helices, and the conformations of junctions and tight turns. The interactions between the 3 terminal adenine of tRNAs bound in either the A site or the P site with 23S rRNA are examples of functionally significant A-minor interactions. The A-minor motif is by far the most abundant tertiary structure interaction in the large ribosomal subunit; 186 adenines in 23S and 5S rRNA participate, 68 of which are conserved. It may prove to be the universally most important long-range interaction in large RNA structures. It is well known that single-stranded RNAs fold back on themselves to form short, double-stranded helices that are stabilized primarily by Watson-Crick and GU wobble base pairs. In recent years, as increasing numbers of RNA structures have been determined, additional, rarer elements of RNA secondary structure (1, 2) have been identified such as tetraloops (3, 4), bulged-G motifs (5-7), and cross-stand purine stacks (5,7,8).Less is known about the ways RNAs with complex secondary structures fold to form RNA tertiary structure because few of the RNA structures known previously were large enough to have sufficient tertiary structure to analyze that problem. In contrast, the recently determined structures of the large ribosomal subunit from Haloarcula marismortui (9, 10) and the small ribosomal subunit from Thermus thermophilus (11, 12) contain a large number of long-range interactions between regions of RNA that are distant in the secondary structure. The Ϸ3,000 nt of the two RNAs of the large ribosomal subunit form a compact structure stabilized by tertiary interactions between secondary structure elements that include about 100 double helical stems. The structure of this large polyanion is stabilized, in part, by interactions with metal ions and proteins, which will be discussed elsewhere. Here we address the interactions occurring between and among RNA helices and single strands that stabilize RNA tertiary and quaternary structure. MethodsFor our study, each adenosine residue in the structure of the H. marismortui 23S rRNA (Protein DataBank entry 1FFK) was assessed for occurrences of A-minor interactions by using the graphics program O (13). A-minor interactions were selected based on the following predetermined geometric criteria. The C2 atom of the adenosine had to be within 3.7 Å of one of its neighboring atoms. The interacting atom had to lie within 45°of the adenine plane. Finally, the C2 face of the adenosine had t...

Crystal structures of the Haloarcula marismortui large ribosomal subunit complexed with the 16-membered macrolide antibiotics carbomycin A, spiramycin, and tylosin and a 15-membered macrolide, azithromycin, show that they bind in the polypeptide exit tunnel adjacent to the peptidyl transferase center. Their location suggests that they inhibit protein synthesis by blocking the egress of nascent polypeptides. The saccharide branch attached to C5 of the lactone rings extends toward the peptidyl transferase center, and the isobutyrate extension of the carbomycin A disaccharide overlaps the A-site. Unexpectedly, a reversible covalent bond forms between the ethylaldehyde substituent at the C6 position of the 16-membered macrolides and the N6 of A2103 (A2062, E. coli). Mutations in 23S rRNA that result in clinical resistance render the binding site less complementary to macrolides.