Mapping the path of the nascent peptide chain through the 23S in the 50S ribosomal subunit

Abstract

Peptides of different lengths encoded by suitable mRNA fragments were biosynthesized in situ on Escherichia coli ribosomes. The peptides carried a diazirine derivative bound to their N-terminal methionine residue, which was photoactivated whilst the peptides were still attached to the ribosome. Subsequently, the sites of photo-cross-linking to 23S RNA were analyzed by our standard procedures. The N-termini of peptides of increasing length became progressively cross-linked to nucleotide 750 (peptides of 6, 9 or 13-15 amino acids), to nucleotide 1614 and concomitantly to a second site between nucleotides 1305 and 1350 (a peptide of 25-26 amino acids), and to nucleotide 91 (a peptide of 29-33 amino acids). Previously we had shown that peptides of 1 or 2 amino acids were cross-linked to nucleotides 2062, 2506 and 2585 within the peptidyl transferase ring, whereas tri-and tetrapeptides were additionally cross-linked to nucleotides 2609 and 1781. Taken together, the data demonstrate that the path of the nascent peptide chain moves from the peptidyl transferase ring in domain V of the 23S RNA to domain IV, then to domain II, then to domain III, and finally to domain I. These cross-linking results are correlated with other types of topographical data relating to the 50S subunit.

Symmetry at the active site of the ribosome: structural and functional implications

“…This apparent The PT center. Interacts with tip of A-and P-site tRNA 39-ends Yusupov et al, 2001;Bashan et al, 2003a) and the rotating CCA; suggested to anchor the rotating moiety (Agmon et al, 2003;Bashan et al, 2003a) A2602 H93 99 PT center; interacts with A-and P-site tRNA 39-ends Yusupov et al, 2001;Bashan et al, 2003a) and the rotating CCA, in the vicinity of the connection to the tRNA acceptor stems; suggested to anchor the rotating moiety, assisting passage of the tRNA 39-end from the A-to the P-site (Agmon et al, 2003;Bashan et al, 2003a) U2609 C loop, H73-93 96 The tunnel's upper part; cross-links to the C terminus of the elongating peptide (Stade et al, 1995) a Phylogenetic conservation across three phylogenetic domains and two organelles. Sample consisting of 930 species (Cannone et al, 2002).…”

Protein secondary structural types are differentially coded on messenger RNA

“…4 8, (calculated from a data set of soluble proteins), smaller folding units have sufficient space to adopt conformation in the solvent-filled ribosome channel. Protease protection studies (Blobel and Sebatini, 1970), antibody binding studies (Bernabeu and Lake, 1982), and crosslinking studies (Brimacombe, 1995;Stade et al, 1995) indicate that 30-40 residues of the nascent chain are protected inside the peptide channel. Given that the rise per helical residue is 1.5 8, and the maximal separation of successive main chain C, atoms in extended conformation is 3.3 8, (Robson and Gamier, 1986), residues in a 40-peptide nascent chain could adopt various helical, extended, and coil conformations in the 85 8, long ribosome channel.…”

The in situ architecture of Escherichia coli ribosomal RNA derived by electron spectroscopic imaging and three‐dimensional reconstruction

“…The red and yellow colours are intended only to assist visualization, and follow the scheme of Capel et al (1988), as does the numerical labelling of the proteins. In the first column, the intersubunit interface of the small subunit is facing the viewer, which interpretation is supported by the presence of the interface protein S20/L26 (the two designations represent the same protein (Stöffler-Meilicke et al, 1981;Stöffler-Meilicke & Stöffler, 1990;Stade et al, 1995)). The bottom of the small ribosomal subunit is protein-deficient (Capel et al, 1988;Stöffler-Meilicke & Stöffler, 1990) and here is seen to be phosphorus-rich (i.e.…”

“…The independent reconstructions seem to have a natural fit when portrayed in this way. The merger was guided using published data on neutron scattering and immunolocalization of proteins and rRNA sequence segments (Stöffler & Wittmann, 1971;Stöffler-Meilicke et al, 1981;Stöffler & Stöffler-Meilicke, 1983;Capel et al, 1988;Walleczek et al, 1988;Stöffler-Meilicke & Stöffler, 1990;Nierhaus, 1991;May et al, 1992;Stade et al, 1995). If we interpret our E. coli ribosome reconstruction as a biologically functional entity, then there is a large central cavity which provides sufficient room to contain two or three molecules of transfer RNA (tRNA) and the necessary ancillary protein factors.…”