Katrin Stade

Clustering of modified nucleotides at the functional center of bacterial ribosomal RNA.

An aryl trifluoromethyl diazirine photo‐reactive derivative was attached to the 2‐thiocytidine residue at position 32 of tRNAIArg and this derivatized tRNA was bound to Escherichia coli 70S ribosomes. After irradiation at 350 nm the site of cross‐linking to the 16S RNA was analyzed by our standard procedures and found to lie within the secondary structural element comprising bases 956‐983; this region contains two modified nucleotides at positions 966 and 967. Similarly, an aryl azido photoreactive derivative was attached to the phenylalanine residue of Phe‐tRNAPhe, and the derivatized aminoacyl tRNA was bound to the ribosome either at the A‐ or the P‐site. In both cases, after irradiation at 250 nm, the cross‐link site was localized to position 2439 of the 23S RNA; in the secondary structure of the latter the neighboring nucleotide 2442 is base‐paired to a modified nucleotide at position 2069. Taken together with other cross‐linking data, these results now directly implicate a total of 27 out of the 29 modified nucleotides in E. coli 16S and 23S RNA as lying within or close to the functional center of the ribosome.— Brimacombe, R., Mitchell, P., Osswald, M., Stade, K., Bochkariov, D. Clustering of modified nucleotides at the functional center of bacterial ribosomal RNA. FASEB J. 7: 161‐167; 1993.

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

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.

The three-dimensional structure and function of Escherichia coli ribosomal RNA, as studied by cross-linking techniques

A large number of intra-RNA and RNA-protein cross-link sites have been localized within the 23S RNA from E. coli 50 S ribosomal subunits. These sites, together with other data, are sufficient to constrain the secondary structure of the 23 S molecule into a compact three-dimensional shape. Some of the features of this structure are discussed, in particular, those relating to the orientation of tRNA on the 50 S subunit as studied by site-directed cross-linking techniques. A corresponding model for the 16S RNA within the 30 S subunit has already been described, and here a site-directed cross-linking approach is being used to determine the path followed through the subunit by messenger RNA.

Mapping the Functional Centre of the Escherichia Coli Ribosome

The principal functional components which are attached to the ribosome during the process of polypeptide chain elongation are the mRNA, two tRNA molecules (either at the A- and P-sites, or the P- and E-sites), and the nascent protein. When the two tRNAs are present at the A- and P-sites, they are tightly constrained by the concomitant requirements (i) that their respective CCA 3’-termini must be close together at the peptidyl transferase centre, in order to allow peptide bond formation to occur, and (ii) that their respective anti-codons must also be close, to enable base-pairing to take place with the appropriate adjacent codons on the mRNA. It is known from fluorescence measurements (Johnson et al, 1982; Paulsen et al, 1983) that in this situation the angle between the planes of the L-shaped tRNA molecules must be relatively small, and there are thus two basically different possible configurations for the two tRNAs; in one the angle between the tRNA planes is approximately 90° (the so-called ‘R’ configuration (Rich, 1974; Lim et al, 1992)) and in the other it is approximately 270° (the so-called ‘S’ configuration (Sundaralingam et al, 1975; Lim et al, 1992)). A tRNA molecule at the E-site is not subject to the same constraints, since - having lost its peptidyl residue - the CCA terminus of this tRNA need no longer be close to the peptidyl transferase centre. The anticodon loop of the E-site bound tRNA on the other hand either still undergoes codon-anticodon interaction (Rheinberger et al, 1986), or is at least still fairly close to its mRNA codon (Paulsen and Wintermeyer, 1986).