Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the ®xation of the 3¢-ends of the tRNAs at the peptidyl-transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.
The arrival of high resolution crystal structures for the ribosomal subunits opens a new phase of molecular analysis and asks for corresponding analyses of ribosomal function. Here we apply the phosphorothioate technique to dissect tRNA interactions with the ribosome. We demonstrate that a tRNA bound to the P site of non-programmed 70 S ribosomes contacts predominantly the 50 S, as opposed to the 30 S subunit, indicating that codon-anticodon interaction at the P site is a prerequisite for 30 S binding. Protection patterns of tRNAs bound to isolated subunits and programmed 70 S ribosomes were compared. The results suggest the presence of a movable domain in the large ribosomal subunit that carries tRNA and reveal that only ϳ15% of a tRNA, namely residues 30 ؎ 1 to 43 ؎ 1, contact the 30 S subunit of programmed 70 S ribosomes, whereas the remaining 85% make contact with the 50 S subunit. Identical protection patterns of two distinct elongator tRNAs at the P site were identified as tRNA species-independent phosphate backbone contacts. The sites of protection correlate nicely with the predicted ribosomal-tRNA contacts deduced from a 5.5-Å crystal structure of a programmed 70 S ribosome, thus refining which ribosomal components are critical for tRNA fixation at the P site.Crystal structures for both 30 S (1, 2) and 50 S ribosomal subunits (3, 4) have been presented at molecular resolution, which has enabled certain ligand interactions with these subunits to be identified. On the 30 S subunit, interactions with initiation factors, numerous antibiotics (5-7), and tRNAs (8) have been determined, the latter of which has led to a detailed understanding of the mechanism of ribosome decoding at the A site. In addition to antibiotics, a transition state analogue for peptide bond formation, the "Yarus inhibitor," has been soaked into crystals of the large ribosomal subunit (9), the results of which have evoked intense discussion regarding the mechanism of peptide bond formation.The highest resolution structure for a complete 70 S ribosome is currently at 5.5 Å (10). At this resolution, molecular interactions with bound ligands cannot be directly visualized; instead, they are inferred by modeling based on the high resolution subunit structures. This has enabled the path of the mRNA through the ribosome, encompassing 31 nucleotides from positions Ϫ15 to ϩ16, to be determined (11), where the first nucleotide of the P site codon is defined as position ϩ1. Furthermore, the positions of A, P, and E site tRNAs were determined allowing contacts with the ribosomal components to be predicted (10), which were in good agreement with previous studies of tRNA-ribosome interactions.The general positions of tRNAs at the A, P, and E sites are well known from cryo-electron microscopy studies of functionally competent complexes (12), but at 11.5-Å resolution relatively little information pertaining to specific tRNA-ribosome interactions is available. Numerous chemical probing and cross-linking studies have been employed to map the contact sites...
The 23 S-type rRNA contains two phylogenetically conserved UGG sequences, which have the potential to bind the universal CCA-3-ends of tRNAs at the ribosomal peptidyltransferase center by base pairing. The first two positions, UG, of these sequences at the helix-loop 80 (U2249G2250) and helix-loop 90 (⌿2580G2581) and some related nucleotides were tested by site-directed mutagenesis for their involvement in ribosomal function, i.e. peptidyltransferase. The plasmid-derived mutated 23 S rRNA comprised about 50% of the total 23 S rRNA. None of the single mutations caused an assembly defect, and all 50 S subunits carrying an altered 23 S rRNA could freely exchange with the pools of 70S ribosomes and polysomes. The mutations at the helix-loop 80 region hardly affected bacterial growth. However, mutations at the helix 90 caused severe growth effects and severely impaired the in vitro protein synthesis, showing that this 23 S rRNA region is of high importance for ribosomal function.The central enzymatic activity of ribosomes is the formation of peptide bonds. The corresponding peptidyltransferase (PTF) 1 center is located on the large ribosomal subunit (1, 2). Reconstitution analyses have identified the ribosomal proteins L2, L3, and L4, and the 23 S rRNA as PTF candidates in Escherichia coli ribosomes (3-5). A complex derived from the large subunit of Thermus aquaticus ribosomes consisting of 23 S rRNA and only 3-8 proteins had significant PTF activity (6, 7). This observation underscores the possible involvement of 23 S rRNA in this activity.An impressive wealth of data points to a distinct feature of the secondary structure map of 23 S rRNA, the so-called "peptidyltransferase ring" of domain V (8, 9) as a component at or near the PTF center. The PTF ring comprises about 40 nucleotides and represents a cluster of universally conserved nucleotides (10, 11). It is satisfying that a highly divergent array of methods correspondingly identifies the same region of 23 S rRNA, methods such as cross-linking studies with substrates of the PTF center (12-15), mutations of the 23 S rRNA gene conferring resistance against inhibitors of the PTF activity (for review, see Refs. 16 and 17), and protection studies (18 -20).Some studies aimed to identify nucleotides at the PTF center. The nucleotides G2252 and G2253, which are at the helixloop 80, were protected against kethoxal by the 3Ј-terminal CCA of P-site bound tRNA (19). Mutation of G2252 generated a dominant lethal phenotype (21). A double mutant having both Gs altered was lethal and showed a reduced activity of peptide bond formation by approximately 50% (22). A recent analysis presented evidence that G2252 is involved in canonical base pairing with C74 at the acceptor end of tRNA, thus obviously playing a role in the binding of the donor substrate at the P site region of the PTF center (23).Another study applied a random mutagenesis of the "Southern half" of the PTF ring (residues 2493-2606). With an elegant screening procedure, 21 mutations of 18 positions were found. Mutatio...
We determined the positions and arrangements of RNA ligands within the ribosome with a new neutron-scattering technique, the proton-spin contrast-variation. Two tRNAs were bound to the ribosome in the pre-translocational and the post-translocational state. The mass centre of gravity of both tRNAs resides at the subunit interface of the body of the 30S subunit. Both tRNAs are separated by an angle of 50-55 degrees, and their mutual arrangement does not change during translocation. The mass centre of gravity moves by 13 +/- 3 A (1A = 0.1 nm) during translocation, corresponding well with the length of one codon. Using an RNase-digestion technique, the length of the mRNA sequence covered by the ribosome was determined to be 39 +/- 3 nucleotides before and after translocation. The ribosome moves like a rigid frame along the mRNA during translocation. In contrast, both tRNAs seem to be located on a movable ribosomal domain, which carries the tRNAs before, during, and after translocation, leaving the microtopography of the tRNAs with the ribosome unaltered. This conclusion was derived from an analysis of the contract patterns of thioated tRNAs on the ribosome. The results have led to a new model of the elongation cycle, which reinterprets the features of the previous "allosteric three-sites model" in a surprisingly simple fashion. Finally, a mutational analysis has identified a single nucleotide of the 23S rRNA essential for the peptidyltransferase activity.
The translocation reaction of two tRNAs on the ribosome during elongation of the nascent peptide chain is one of the most puzzling reactions of protein biosynthesis. We show here that the ribosomal contact patterns of the two tRNAs at A and P sites, although strikingly different from each other, hardly change during the translocation reaction to the P and E sites, respectively. The results imply that the ribosomal micro-environment of the tRNAs remains the same before and after translocation and thus suggest that a movable ribosomal domain exists that tightly binds two tRNAs and carries them together with the mRNA during the translocation reaction from the A-P region to the P-E region. These findings lead to a new explanation for the translocation reaction.Ribosomes contain three tRNA binding sites, the A, P, and E site, viz. the A site where the decoding takes place, the P site, where the peptidyl-tRNA is located before peptide bond formation, and the E site, which is specific for deacylated tRNA (1-6). During elongation of the nascent peptide chain, each tRNA passes through the ribosomal binding sites in the sequence A 3 P 3 E. To elongate the nascent peptide chain by one amino acid, the ribosome goes through a cycle of reactions, the socalled elongation cycle. The three basic reactions of an elongation cycle are 1) occupation of the A site by an aminoacyl-tRNA according to the corresponding codon at the A site, 2) peptidebond formation, which transfers the already synthesized peptidyl residue to the aminoacyl-tRNA so that the resulting peptidyl-tRNA, prolonged by an amino acid, now resides at the A site, and 3) the translocation reaction, which moves the peptidyl-tRNA to the P site and the deacylated tRNA to the E site.Cross-linking and footprinting studies have identified components of the ribosome that interact with the tRNAs in the specific sites (reviewed in Refs. 7 and 8). Recently, a technique developed by Eckstein and co-workers (9) has been used to investigate the interactions of tRNAs with ribosomal binding sites (10). This method exploits the fact that the addition of iodine (I 2 ) causes a breakage of the sugar-phosphate backbone of phosphorothioated RNA (here tRNA). The cleavage works equally well with phosphates in single or double strands. However, if tight contacts of a distinct phosphate group of a thioated tRNA with a synthetase or a ribosomal component prevents the access of iodine, the phosphate group under observation is protected, and a cleavage is not observed at this position. Highly differentiated and distinct cleavage patterns were found for A and P site-bound tRNAs in the pre-translocational (PRE) 1 state (10). Here we show that the protection patterns of both tRNAs at the A and P sites in the PRE state hardly change when translocated to P and E sites, respectively. Because probably most of the protection patterns are caused by interactions of the tRNA with the respective ribosomal binding sites, the data suggest the existence of a movable ribosomal domain that binds tightly the two ...
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