Crystals ribosome

The only other E. coli ribosomal protein whose crystallization has so far been reported is L29 (Appelt et al., 1981). On the other hand, attempts to crystallize ribosomal proteins from the thermophilic Bacillus stearothermophilus have been more successful. Protein BL17, which according to its amino acid sequence (Kimura et al., 1980) corresponds to protein L9 from the E. coli ribosome (Kimura et al., 1982), was the first intact ribosomal protein to give crystals useful for X-ray structural analysis (Appelt et al., 1979). Several other B. stearothermophUus ribosomal proteins, namely BL6 and BL30 (Appelt eteU., 1981,1983) from the large and BS5 (Appelt et al., 1983) from the small subunit have been crystallized, and the determination of their three-dimensional structure at a resolution of better than 3 A is now in progress. Furthermore, crystals of aB. stearothermophilus ribosomal protein complex, which corresponds to the complex (L7/L12)4 LIO from E. coli ribosome, have been obtained (Liljas and Newcomer, 1981). 

Table 3 gives a summary of crystallized ribosomal proteins. 

The C-terminal fragment (CTF) of L7/L12 diffracts very well in contrast to all other crystallized ribosomal proteins. This can be explained by the almost globular shape of the stable domain while the flexible part (see below) is less than 10% of the structure. In addition the C-terminal region of L7/L12 is known to protrude from the 508 particle (Marquis et al., 1981) and can probably be regarded as a globular soluble protein. Fig. 5 shows two precission photographs of crystals of L7/L12 CTF. 

The atomic structure of this subunit and its complexes with substrate analogs revealed the enzymatic activity of the rRNA backbone. Thus, the ribosome is in fact a ribozyme P Nissen, J Hansen, N Ban, PB Moore, TA Steitz. Science 289 920-930, 2000. Atomic structure of the ribosome s small 30S subunit, resolved at 5 A WM Clemons Jr, JL May, BT Wimberly, JP McCutcheon, MS Capel, V Ramakrishnan. Nature 400 833-840, 1999. The 8-A crystal structure of the 70S ribosome reveals a double-helical RNA bridge between the 50S and the 30S subunit GM Culver, JH Cate, GZ Yusupova, MM Yusupov, HF Noller. Science 285 2133-2136, 1999. 

Khulbrandt W, Unwin PNT. Structural analysis of stained and unstained two-dimensional ribosome crystals, in Electron Microscopy at Molecular Dimensions, State of the Art and Strategies for the Future (Baumeister W, Vogell W, eds.), Springer-Verlag, Berlin, Germany, 1980, pp. 108-116. 

Since the X-ray structural analysis of crystallized proteins yields the most direct information on the tertiary structure, many attempts have been made in the last decade to crystallize individual ribosomal proteins. However, it was many years before any progress in this field was made. The N- and C-terminal fragments of the . coU protein L7/L12 have been crystallized, and the crystals diffract to 4 and 2.6 A, respectively (Liljas et ai, 1978). According to the X-ray analysis, the C-terminal fragment (positions 53-120) has a compact, plum-shaped tertiary structure with three a helices and three p sheets (Leijonmarck et ai, 1980). 

Three-dimensional crystals have been obtained with 50 S ribosomal subunits from B. stearothermophilus (Yonath et al, 1980, 1982a,b) and with 70 S ribosomes from E. coli (Wittmann et al, 1982) as shown in Fig. 7. 

Fig. 7. (a) Crystals of E. colt 70 S ribosomes, (b) and (c) Electron micrographs of sections through three-dimensional crystals shown in (a) in two orthogonal directions (Wittmann et al., 1982). (d) and (e) Crystals and computed filtered image of a section through a crystal of Bacillus stearothermophilus 50 S ribosomal subunits (Yonath et al., 1982a,b Leonard et al., 1983). (d) and (e) are related to two different crystal forms. Reproduced with permission from Wittmann (1983). 

Of great interest is the fact that ribosomal subunits and ribosomes themselves have now been crystallized, and low-resolution structural maps have already been obtained. However, to grow suitable crystals and to resolve the ribosomal structure at a sufficiently high resolution remains a great challenge and task to biochemists and crystallographers. 

The ribosome is a unique cellular machine in that its main functional component is RNA whereas proteins seem to play only a structural role. For a long time, it has been debated whether RNA or proteins contribute most to the ribosome s function. With the determination of high-resolution crystal structures, this question could finally be answered. Clearly, these structures have revolutionized the field of ribosome studies. Already in the 1980s, Yonath and coworkers had grown crystals of active ribosomes that diffracted to about 0.6 nm (6 A) (1 A = 0.1nm) resolution. However, owing to the large size of the ribosome of about 2 500 000 Da (lDa=lgmoP), the ribosome structure was not solved to atomic resolution until tbe year 2000. 

RF3)" " with the ribosome. However, the maximum resolution that can currently be obtained by cryo-EM is about 10 nm (8-12 A), far from the desired atomic resolution. Therefore, the crystal structures of the 30S subunit with initiation factors 1 and 3 (IFl IF3 ) and of the 70S subunit with release factors 1 and 2 (RFl/2 " ) as well as RRF have been important milestones toward understanding the interaction of the ribosome with protein factors. 

Peptide bond formation is the essential reaction catalyzed by the ribosome. Despite its importance, it was for a long time not the focus of ribosomal research, for several reasons. First, before the determination of the high-resolution ribosome crystal structures almost nothing was known about the active site. Second, under most experimental conditions accommodation of the incoming aminoacyl-tRNA is rate limiting for peptide bond... 

The new crystal structure of the ribosome—RFl complex sheds more light into the interactions between the GGQ motif and the peptidyltransferase center. This complex represents the product state of peptide release since a deacylated tRNA is bound to the P site. Importantly, the main chain amide of the conserved glutamine hydrogen bonds to the 3 OH of A76 in the P site, which is the leaving group of the hydrolysis... 

Figure 10 Alteration of the genetic code for incorporation of non-natural amino acids, (a) In nonsense suppression, the stop codon UAG is decoded by a non-natural tRNA with the anticodon CUA. In vivo decoding of the UAG codon by this tRNA is in competition with termination of protein synthesis by release factor 1 (RFl). Purified in vitro translation systems allow omission of RF1 from the reaction mixture, (b) A new codon-anticodon pair can be created using four-base codons such as GGGU. Crystal structures of these codon-anticodon complexes in the ribosomal decoding center revealed that the C in the third anticodon position interacts with both the third and fourth codon position (purple line) while the extra A in the anticodon loop does not contact the codon.(c) Non-natural base pairs also allow creation of new codon-anticodon pairs. Shown here is the interaction of the base Y with either base X or (hydrogen bonds are indicated by red dashes).

Vicens, Q. Westhof, E., Molecular recognition of aminoglycoside antibiotics by ribosomal RNA and resistance enzymes An analysis of x-ray crystal stmctures. 

E. E., Gittis, A. G. Crystal structure of a conserved ribosomal protein— RNA complex. Science 1999, 284, 1171-1174. 

---