N

Figure 11.4. Alanylprolyladenosine residue as a donor substrate in the peptidyl transferase center of the ribosome: ball-and-stick skeletal model (without hydrogens). Atom designations are the same as in Fig. 11.11. Atoms of the C-terminal prolyl residue are marked by the index i, those of the preceding alanyl residue by the index i-1.

apexes of a tetrahedron, while the orbital of the unshared pair of electrons is directed toward the fourth apex. It follows from stereochemical analysis that, during the nucleophilic attack, the free valency of the attacking nitrogen atom should have a strictly defined direction: the plane formed by this direction and the Ni+1-Ca bond should be at an angle of about 120* to the plane

(Ni+1-Cai+1-C'i+1) of the acceptor aminoacyl residue (Fig. 11.5 A); with any other orientations there can be no nucleophilic attack on the carbonyl group because of steric hindrances. After the peptide bond is formed, an angle j of about -60* is set in the newly added aminoacyl residue of the product (Fig. 11.5 C).

Taking all of the above into consideration, stereochemical analysis demonstrates that an effective nucleophilic attack in the ribosomal peptidyl transferase center can take place only if the angle between the plane of the ester group COO and the plane (C'i-Cai-Ni) defined by the rotation around the Cai-C'i-bond in the attacked aminoacyl residue of the donor substrate (peptidyl-tRNA) is about 60* (Fig. 11.5 A). After transpeptidation, it becomes an angle y (with an approximate value of -60* (60* counterclockwise rotation around the Ca-C'-bond when viewed from the Ca -atom) (Fig. 11.5 C).

Thus, the conformation of the donor aminoacyl residue in the ribosomal peptidyl transferase center should be similar to that of the aminoacyl residue in the a-helix (j = -50*, y = -60*). Moreover, the acceptor aminoacyl residue attacks the donor residue in such an orientation that the transpeptidation reaction yields an elongated backbone Ni-Cai-C'i-Ni+1-Cai+1 with parameters of the a-helical conformation.

Independent of this result of stereochemical analysis, the rule of the equivalent (universal) positioning of any aminoacyl residue in the ribosomal peptidyl tranferase center leads to the conclusion that the residue newly incorporated into the polypeptide chain always acquires a standard conformation of its backbone, i.e. standard values of torsion angles Ca-C' and Ca-N. This implies that the peptidyl transferase center will generate a helical conformation of the synthesized peptide. It would not come as a surprise, therefore, if the peptide is built in a sterically and energetically favorable helical conformation, e.g. the a-helix. This initial conformation later rearranges into a unique three-dimensional structure. That the polypeptide chain folding in vivo does not, perhaps, begin from a random or extended state but rather is the result of a rearrangement of the helical structure may provide unique opportunities for a quick and accurate search for the final conformations of the protein molecule.

Figure 11.5. Stereochemistry of the transpeptidation reaction: ball-and-stick drawing (without hydrogens). Atom designatians are the same as in Fig. 11.12. Atoms of the acceptor aminoacyl residue are marked by the index i+1. A is the adenine residue in both the donor and acceptor substrate.

A: Mutual positions of the donor (lower) and acceptor (upper) substrates in the peptidyl transferase center; the acceptor substrate nitrogen (Ni+1) attacks the donor carbonyl carbon (C'i). B: Tetrahedral intermediate resulting from the attack.

C: Products of the reaction: the elongated peptidyl adenosine residue (left) and the deacylated adenosine residue (right).

(V.I. Lim & A.S. Spirin, J. Mol. Biol. 188, 565-574, 1986).

Figure 11.5. Stereochemistry of the transpeptidation reaction: ball-and-stick drawing (without hydrogens). Atom designatians are the same as in Fig. 11.12. Atoms of the acceptor aminoacyl residue are marked by the index i+1. A is the adenine residue in both the donor and acceptor substrate.

A: Mutual positions of the donor (lower) and acceptor (upper) substrates in the peptidyl transferase center; the acceptor substrate nitrogen (Ni+1) attacks the donor carbonyl carbon (C'i). B: Tetrahedral intermediate resulting from the attack.

C: Products of the reaction: the elongated peptidyl adenosine residue (left) and the deacylated adenosine residue (right).

(V.I. Lim & A.S. Spirin, J. Mol. Biol. 188, 565-574, 1986).

11.4. Movement of Transpeptidation Products

The consideration of the stereochemistry of transpeptidation reaction (Fig. 11.5) implies that the decay of the tetrahedral untermediate should be accompanied by moving the deacylated ribose group aside. Moreover, in order to prevent reversibility of the decay, and so reversibility of transpeptidation on the ribosome, the deacylated group should be immediately removed from the reaction site. This is probably the function of the e-site that fixes the terminus of the deacylated tRNA outside the peptidyl transferase center.

At the same time the ester group with the amino acid residue which had been bound at the d-site before transpeptidation becomes destroyed as a result of the tetrahedral intermediate decay, and thus this residue loses its affinity to the J-site. Instead, the amino acid residue which occupied the a-site before transpeptidation acquires now the feature (amide group) that provides for its affinity to the d-site. As a consequence, the newly added amino acid residue with its ester group and amide (peptide) group should displace the previous amino acid residue, i.e. move from the a-site to the J-site of the peptidyl transferase center.

Indeed, the foot-printing analysis by Noller et al. (1990) fully confirms these expectations. It was shown that peptidyl-tRNA bound in the P site of the ribosome (pre-transpeptidation P/J state) protects G2252-G2253, U2506 and U2584-U2585 by its CCA terminus on the 50S subunit; thus these positions can be attributed to the J-site. Aminoacyl-tRNA bound in the A site (pre-transpeptidation A/a state) specifically shields G2553, U2555, A2602 and U2609 on the 50S subunit, so that these residues seem to belong to the a-site. After transpeptidation, prior to translocation, the deacylated tRNA still occupying the P site on the 30S subunit does not protect the J-site residues anymore and switches its CCA terminus to the residues characteristic of the e-site: G2112, G2116, A2169 and C2394; this is the pre-translocation P/e state of the deacylated tRNA. The newly formed peptidyl-tRNA continues to occupy the A site with its tRNA moiety on the 30S subunit, but now it protects the J-site residues on the 50S subunit, with the exception of A2602 that is still protected as before transpeptidation; this is called the pre-translocation A/J state of the peptidyl-tRNA. Hence, despite the fixation of the two tRNA moieties at the P and A sites where they have been bound prior to transpeptidation, the CCA termini of the tRNA move on the 50S subunit during transpeptidation: the deacylated A76 of the P-site-bound tRNA is displaced out of the peptidyl transferse center and caught by the e-site, while the esterified A76 with C-terminal amino acid of the A-site-bound tRNA shifts within the peptidyl transferase center from its a-site to the J-site (Fig. 11.6).

The potential flexibility of the CCA terminus of the tRNA molecule, and especially the mobility of the 3'-terminal adenosine relative to the rest of the tRNA body, should play the decisive role in the above-mentioned movements of the tRNA end on the large ribosomal subunit. At the same time, conformational changes in the region of the peptidyl transferase center in response to the formation and breakdown of contacts with ligand groups during transpeptidation are very plausible. Also large-block movements, such

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