Ribosomal Mechanics, Antibiotics, and GTP Hydrolysis

Institute of Molecular Biology proteins dimerize through their N-terminal domains, proCopenhagen University ducing a “stalk” structure on the 50S subunit with a Sølvgade 83 flexible hinge region that probably allows the C-terminal DK1307-Copenhagen K domains of the dimers to move relative to one another Denmark and to the ribosomal surface (Traut et al., 1995). The rRNA region is also involved, together with L11, in the binding of two thiopeptide antibiotics, thiostrepton and micrococcin, which can impede ribosomal factor–depenThe ribosome is an extremely ancient, highly complex, dent processes. The second component is the ribotoxin molecular machine in which rRNAs and ribosomal prostem-loop (nucleotides 2645–2675 of E. coli 23S rRNA), teins have coevolved over some 3 billion years in order where cytotoxic proteins, including a-sarcin and ricin, to produce proteins efficiently and accurately. After four modify the rRNA loop and block ribosomal factor– decades of dedicated, frequently demoralizing, but ofdependent processes, at least for elongation factors ten very creative experimental work, the ribosome may that produce overlapping chemical footprints in this finally be starting to reveal the secrets of its molecular rRNA region (Munishkin and Wool, 1997; Wilson and mechanics. For those who have persevered in this enNoller, 1998). This highly conserved site composed of deavour, this year signals the beginning of an exciting two rRNA regions and their associated proteins reguperiod of enlightenment. Evidence for this is presented lates binding and GTPase activity of the ribosomal facin the current issue of Cell, where Wimberly et al. (1999) tors (Figure 1). have determined the crystal structure of a complex of The Structure of the L11–rRNA Complex ribosomal protein L11 and an rRNA fragment from the The crystal structure of the L11–rRNA complex is a landhyperthermophilic bacterium Thermotoga maritima, which mark costructure of a ribosomal protein and an rRNA is an essential part of an important functional center on fragment. Moreover, as an integral part of an important the 50S subunit. It will be followed, shortly, by highfunctional site on the 50S subunit, its structural and resolution structures of the ribosomal subunits. functional properties have been investigated in detail. It has long been known for bacterial ribosomes that Gratifyingly, the crystal structure reinforces much of the the L11–rRNA complex is involved, in some way, in earlier binding and footprinting data and correlates regulating several ribosomal factor–dependent proclosely with the genetic results. However, what was not cesses, including (1) elongation factor Tu (EF-Tu)– predictable from the earlier studies is the large number dependent aminoacyl-tRNA binding; (2) elongation facof tertiary interactions present in the rRNA component tor G (EF-G)–mediated translocation; (3) initiation factor and the high degree of complexity of the interactions 2–dependent fMet-tRNA binding; and (4) release factor– between the C-terminal domain of L11 and the rRNA. The secondary structure of the rRNA was derived eardependent termination, as well as (5) stringent factorlier from compensatory base change analyses, and it dependent synthesis of the “magic spots” ppGpp and contains four double helical elements located around a ppGppp (Gale et al., 1981). Most of these factors are four-way junction. In the crystal structure, the coaxial known to be G proteins, and their intrinsic GTPase activistacking of the terminal stem with the A1095 stem-loop, ties are stimulated by a ribosomal site that includes the and of the U1082 hairpin with the A1067 stem-loop, L11-rRNA. It is widely termed the “GTPase-associated produces a two-domain rRNA structure with very tight site” and is assumed to undergo conformational changes interdomain packing (Figure 2A). This packing is mainthat regulate ribosomal factor binding and GTP hydrolytained first by a minor groove “ribose zipper,” involving sis (Cundliffe, 1986). Although much progress has been multiple hydrogen bond interactions between 29-hydroxyl made recently in characterizing functional conformagroups of riboses and bases that link the terminal stem tional transitions that occur within the elongation factors and the U1082 hairpin and, second, by tertiary interacEF-Tu and EF-G on the ribosome, little is known about tions between the major grooves of the A1067 and A1095 those that occur in the ribosome (Nyborg and Liljas, stem-loops (Figure 2A). Several divalent metal ions are 1998). observed in the crystal structure, and, in particular, a The “GTPase-associated site” has been characterized cadmium ion (which may be replaced by another divaextensively by a variety of genetic, cross-linking, and lent ion in vivo) is located at the four-way junction and chemical footprinting approaches, and it consists of two may correspond to one of two divalent cations that were main ribosomal components. The first is a protein–rRNA previously shown to stabilize the tertiary structure of cluster containing protein L11, an rRNA region extending the rRNA (Bukhman and Draper, 1997). from nucleotides 1030–1125 of Escherichia coli 23S Protein L11 exhibits two globular domains connected by a linker sequence. The structure of the C-terminal domain, which was solved earlier by NMR (Xing et al., 1997), makes extensive main-chain contacts with the * To whom correspondence should be addressed (e-mail: garrett@