The recent impressive progress in ribosomal crystal- lography has yielded insights into the mechanism of protein biosynthesis. Analysis of the high-resolution structures (Ban et al. 2000; Schluenzen et al. 2000; Wimberly et al. 2000) has led to the identification of dynamic aspects of this process and highlighted strategies adopted by the ribosomes for maintaining their structural integrity and for their survival under extreme conditions (Gluehmann et al. 2001; Harms et al. 2001). Naturally, the crystallographic studies have expanded far beyond the presentation of still pictures and are rapidly progress- ing toward the elucidation of snapshots describing spe- cific functional stages during the biosynthetic process. Structures of complexes with analogs of transfer RNA and messenger RNA (Weinstein et al. 1999; Auerbach et al. 2000; Brodersen et al. 2000; Yusupov et al. 2001), compounds believed to be substrate analogs (Nissen et al. 2000), translation initiation factors (Carter et al. 2001; Pi- oletti et al. 2001), and antibiotics (Brodersen et al. 2000; Carter et al. 2000; Pioletti et al. 2001; Schlunzen et al. 2001) are rapidly emerging. The ribosome is a precisely engineered molecular ma- chine performing an intricate multistep process that re- quires smooth and rapid switches between different con- formations. As such, it contains structural elements that allow global motions together with local rearrangements that create a defined sequence of events at the functional centers. Large-scale movements were detected by cryo- electron microscopy (Frank et al. 1995; Stark et al. 1995; Gabashvili et al. 1999), by surface RNA probing (Alexander et al. 1994), by monitoring the ribosomal ac- tivity, by numerous attempts at crystallization (see, e.g., Berkovitch-Yellin et al. 1992), and by the analysis of the high-resolution structures (Schluenzen et al. 2000; Harms et al. 2001; Ogle et al. 2001; Pioletti et al. 2001). The small ribosomal subunit (30S in prokaryotes) is heavily involved in decoding and translocation—the dy- namic aspects of protein biosynthesis—and its significant conformational variability has been correlated with its function. Analysis of the crystal structures of this subunit indicated its mobile structural elements (Gluehmann et al. 2001). Consequently, special efforts were made to iden- tify (Wimberly et al. 2000) or to promote (Tocilj et al. 1999; Carter et al. 2000, 2001; Schluenzen et al. 2000; Pi- oletti et al. 2001) selected conformations within its crys- tals. The structures of complexes of this subunit with ini- tiation factors, antibiotics, and mRNA or tRNA analogs showed that the decoding process is accomplished mainly by the 16S ribosomal RNA, and that both the proteins and the RNA features involved in the dynamic functions can assume various conformations. The large subunit (50S in prokaryotes) is responsible for peptide-bond formation. It is known to show less con- formational variability than that found for the small one, but significant mobility can be assigned to some of its fea- tures, especially those directly involved in its functions. Both subunits may undergo reversible alterations be- tween active and inactive conformations that may be in- duced by the environmental conditions. We have previ- ously shown that only functionally active ribosomal particles yield crystals and that the dissolved crystallized material is usually highly active when tested under near- physiological conditions (Berkovitch-Yellin et al. 1992). Nevertheless, within the crystals, the ribosomes may as- sume a non-active conformation, if maintained under far- from-physiological conditions. Thus, there are reasons to believe that the 2.4 Å structure of the large ribosomal sub- unit from Haloarcula marismortui (H50S), which was determined under far-from-physiological conditions (Ban et al. 2000), reflects less active conformations. In this paper, we describe our analyses on the structures of the two ribosomal subunits in several conformational states. These studies indicate the strategies that the ribo- some adopts for enhancing and directing the binding of factors and substrates. They may also show how the ribo- some takes advantage of the built-in flexibility of its com- ponents for preventing nonproductive interactions. THE SMALL RIBOSOMAL SUBUNIT The small ribosomal subunit (30S) is responsible for the decoding of the genetic information and plays a key role in the initiation phase of protein synthesis. The re- fined 3.2 Å structure of the functionally activated form of this subunit from Thermus thermophilus contains >99% of its 16S RNA chain and most of the amino acids of the subunit’s 20 proteins (Schluenzen et al. 2000; Pioletti et al. 2001). The overall fold of the RNA chain, as traced in High-resolution Structures of Ribosomal Subunits: Initiation, Inhibition, and Conformational Variability A. BASHAN,* I. AGMON,* R. ZARIVACH,* F. SCHLUENZEN,† J. HARMS,† M. PIOLETTI,†¶ H. BARTELS,† M. GLUEHMANN,† H. HANSEN,† T. AUERBACH,†¶ F. FRANCESCHI,‡ AND A. YONATH*† *Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel; † Max-Planck-Research Unit for Ribosomal Structure, Hamburg, Germany; ‡ Max-Planck-Institute for Molecular Genetics, Berlin, Germany; ¶ Department of Biology, Chemistry, Pharmacology, Free University of Berlin, Germany Cold Spring Harbor Symposia on Quantitative Biology, Volume LXVI. © 2001 Cold Spring Harbor Laboratory Press 0-87969-619-2/01. 43