Ribosome crystallized proteins

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). 

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. 

On the technical side, synchrotron X-ray radiation is necessary for most protein complexes as they have large unit cells and weak diffraction patterns. It may be necessary to protect the crystds from the beam by freezing them this was particularly valuable for obtaining data from ribosome crystals. ... 

Another important point is radiation damage. Thermal neutrons appear to destroy protein crystal structures much less than X-rays do. This is so at room temperature. Recently, it has been found that cooling of ribosome crystals to liquid nitrogen temperature makes the crystal structure extremely resistant to the intense synchrotron... 

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). 

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. 

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).

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

Naturally, the question of whether or not a 27-nucleotide fragment could faithfully mimic the complete 30S subunit had to be answered. Are there nuances to this short stem of RNA binding with paromomycin that do not exist when bound to the intact subunit What role, if any, do neighboring RNA and protein moieties of the ribosome play in binding when the 30S subunit is whole Answers to these questions have become much clearer recently with the ouqtouring of X-ray crystal structures containing aminoglycosides bound to the complete 30S ribosomal subunit. ... 

During the previous few years, the number of RNA crystal structures has increased in an exponential manner. This is mainly due to the fact that RNA is increasingly viewed as a predominant part of biological processes such as translation, ribozyme catalysis, and gene regulation (RNAi (Kamath et ah, 2003), riboswitches (Barrick et ah, 2004), and mRNA-protein interactions (Lescure et ah, 2002)), for which the gap in structural knowledge is still deep despite the determination of the crystal structure of the ribosome (Clemons et ah, 2001 Ban et ah, 2000 Yusupov et ah, 2001). 

Radioactive lateling of this cluster and neutron activation analysis of the g)ld enabled us to determine the extent of Nnding of the cluster to the particles. The results of both analytical methods show that a spacer of minimum length of about 10 A between the -SH group of a ribosomal protein and the N-atom on the cluster is n ed for significant binding. Preliminary experiments indicate that the producte of the derivatization reaction with SOS particles can be crystallized. 

Most ribosomal proteins are rich in lysine and arginine and, therefore, carry a substantial net positive charge. Proteins S20, L7/12, and L10 have over 20% alanine, while L29 is almost as rich in leucine. Proteins S10, S13, L7/L12, L27, L29, and L30 are surprisingly low (<2 mol %) in aromatic amino acids. Proteins S5, S18, and L7 have acetylated N termini while Lll, L3, L7/12, Lll, L16, and L33 contain methylated amino acids. Lll contains nine methyl groups.22 Protein S6 is the major phosphoprotein of eukaryotic ribosomes.103104 Most ribosomal proteins have no known enzymatic activity. Although often difficult to crystallize, high-resolution three-dimensional structures are known for many free ribosomal proteins.24 Most of them have shapes resembling those previously found... 

Many specific parts of ribosomal RNA molecules and specific proteins within the intact ribosome were located prior to the determination of high resolution crystal structures. One major approach was the use of immunoelectron microscopy. Antibodies to specific ribosomal proteins or to special sites in the RNA were prepared, and electron microscopy was used to map the binding sites of the antibodies on the ribosomal... 

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