Ribosomal crystallography: Peptide bond formation and its inhibition

Abstract

Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity. Crystallographic studies on complexes of the small and the large eubacterial ribosomal subunits with substrate analogs, antibiotics, and inhibitors confirmed that the ribosomal RNA governs most of its activities, and indicated that the main catalytic contribution of the ribosome is the precise positioning and alignment of its substrates, the tRNA molecules. A symmetry-related region of a significant size, containing about two hundred nucleotides, was revealed in all known structures of the large ribosomal subunit, despite the asymmetric nature of the ribosome. The symmetry rotation axis, identified in the middle of the peptide-bond formation site, coincides with the bond connecting the tRNA double-helical features with its single-stranded 3' end, which is the moiety carrying the amino acids. This thus implies sovereign movements of tRNA features and suggests that tRNA translocation involves a rotatory motion within the ribosomal active site. This motion is guided and anchored by ribosomal nucleotides belonging to the active site walls, and results in geometry suitable for peptide-bond formation with no significant rearrangements. The sole geometrical requirement for this proposed mechanism is that the initial P-site tRNA adopts the flipped orientation. The rotatory motion is the major component of unified machinery for peptide-bond formation, translocation, and nascent protein progression, since its spiral nature ensures the entrance of the nascent peptide into the ribosomal exit tunnel. This tunnel, assumed to be a passive path for the growing chains, was found to be involved dynamically in gating and discrimination.

Functional aspects of ribosomal architecture: symmetry, chirality and regulation

“…These are the multi-task inter-subunit bridge B2a [Plate 1(f)] that is likely to assist the A-to P-site tRNA acceptor-stem shift, 11,21 and the PTC nucleotide A2602 (Escherichia coli nomenclature is being used throughout) that seems to be involved in A-to P-translocation within the PTC. 8,9,11 The PTC itself possesses the dynamic properties required for facilitating the correctly bound A-site tRNA to the P-site and for assisting rearrangements of less well aligned substrates. 8,9,11,21 It is located close to the subunit interface, at the bottom of a cavity in the large ribosomal subunit.…”

“…8,9,11 The PTC itself possesses the dynamic properties required for facilitating the correctly bound A-site tRNA to the P-site and for assisting rearrangements of less well aligned substrates. 8,9,11,21 It is located close to the subunit interface, at the bottom of a cavity in the large ribosomal subunit. The peripheral upper rim of this cavity includes a ribosomal protein, L16, but the PTC itself is formed exclusively by ribosomal RNA [Plate (1b-d)].…”

“…Yet, a large symmetry-related region, containing about 180 nucleotides, was revealed in all known structures of the large ribosomal subunit [Plate 2(a)]. 8,9,11 This symmetry relates the backbone fold and nucleotides orientation [Plate 1(b)], rather than nucleotide type. The existence of a two-fold symmetry can be justified by the need to offer comparable supportive environments to two similar chemical moieties that have to face each other in order to allow participation of the A-site amine and the P-site carbonyl-carbon in peptide bond formation.…”

“…24 This rotatory motion creates favorable stereochemistry for peptide-bond formation [Plate 2(g)] and seems to be performed in conjunction with the chemical reaction. 8,9,11 Looking down the symmetry axis, the PTC appears as an arched void of a size sufficient to accommodate the tRNA-3 0 end. The shape of this void and its position seems to be designed as the path along which the rotatory motion of the A-site 3 0 end proceeds into the P-site [Plate 2(f)].…”

“…Crystallography of ribosomes, initiated over two decades ago 1 recently yielded several three-dimensional structures of free and complexes ribosomal particles [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] and enabled the assignments of the three tRNA binding sites on each of the ribosomal subunits to be made [plate 1(a) and (b)]. They also confirmed the notion that the main parts of the A-and P-site tRNA molecules are roughly parallel to each other, whereas the E-site tRNA has to rotate while exiting the ribosome.…”