The 3D arrangement of the 23 S and 5 S rRNA in the Escherichia coli 50 S ribosomal subunit based on a cryo-electron microscopic reconstruction at 7.5 A resolution.

The Escherichia coli 23 S and 5 S rRNA molecules have been fitted helix by helix to a cryo-electron microscopic (EM) reconstruction of the 50 S ribosomal subunit, using an unfiltered version of the recently published 50 S reconstruction at 7.5 A resolution. At this resolution, the EM density shows a well-defined network of fine structural elements, in which the major and minor grooves of the rRNA helices can be discerned at many locations. The 3D folding of the rRNA molecules within this EM density is constrained by their well-established secondary structures, and further constraints are provided by intra and inter-rRNA crosslinking data, as well as by tertiary interactions and pseudoknots. RNA-protein cross-link and foot-print sites on the 23 S and 5 S rRNA were used to position the rRNA elements concerned in relation to the known arrangement of the ribosomal proteins as determined by immuno-electron microscopy. The published X-ray or NMR structures of seven 50 S ribosomal proteins or RNA-protein complexes were incorporated into the EM density. The 3D locations of cross-link and foot-print sites to the 23 S rRNA from tRNA bound to the ribosomal A, P or E sites were correlated with the positions of the tRNA molecules directly observed in earlier reconstructions of the 70 S ribosome at 13 A or 20 A. Similarly, the positions of cross-link sites within the peptidyl transferase ring of the 23 S rRNA from the aminoacyl residue of tRNA were correlated with the locations of the CCA ends of the A and P site tRNA. Sites on the 23 S rRNA that are cross-linked to the N termini of peptides of different lengths were all found to lie within or close to the internal tunnel connecting the peptidyl transferase region with the presumed peptide exit site on the solvent side of the 50 S subunit. The post-transcriptionally modified bases in the 23 S rRNA form a cluster close to the peptidyl transferase area. The minimum conserved core elements of the secondary structure of the 23 S rRNA form a compact block within the 3D structure and, conversely, the points corresponding to the locations of expansion segments in 28 S rRNA all lie on the outside of the structure.

The NMR structure of the 5S rRNA E-domain-protein L25 complex shows preformed and induced recognition.

The structure of the complex between ribosomal protein L25 and a 37 nucleotide RNA molecule, which contains the E-loop and helix IV regions of the E-domain of Escherichia coli 5S rRNA, has been determined to an overall r.m.s. displacement of 1.08 A (backbone heavy atoms) by heteronuclear NMR spectroscopy (Protein Databank code 1d6k). The interacting molecular surfaces are bipartite for both the RNA and the protein. One side of the six-stranded beta-barrel of L25 recognizes the minor groove of the E-loop with very little change in the conformations of either the protein or the RNA and with the RNA-protein interactions occurring mainly along one strand of the E-loop duplex. This minor groove recognition module includes two parallel beta-strands of L25, a hitherto unknown RNA binding topology. Binding of the RNA also induces conversion of a flexible loop to an alpha-helix in L25, the N-terminal tip of which interacts with the widened major groove at the E-loop/helix IV junction of the RNA. The structure of the complex reveals that the E-domain RNA serves as a preformed docking partner, while the L25 protein has one preformed and one induced recognition module.

Direct identification of NH...N hydrogen bonds in non-canonical base pairs of RNA by NMR spectroscopy.

It is shown that the recently developed quantitative J(NN)HNN-COSY experiment can be used for the direct identification of hydrogen bonds in non-canonical base pairs in RNA. Scalar(2h)J(NN)couplings across NH.N hydrogen bonds are observed in imino hydrogen bonded GA base pairs of the hpGA RNA molecule, which contains a tandem GA mismatch, and in the reverse Hoogsteen AU base pairs of the E-loop of Escherichia coli 5S rRNA. These scalar couplings correlate the imino donor(15)N nucleus of guanine or uridine with the acceptor N1 or N7 nucleus of adenine. The values of the corresponding(2h)J(NN)coupling constants are similar in size to those observed in Watson-Crick base pairs. The reverse Hoogsteen base pairs could be directly detected for the E-loop of E.coli 5S rRNA both in the free form and in a complex with the ribosomal protein L25. This supports the notion that the E-loop is a pre-folded RNA recognition site that is not subject to significant induced conformational changes. Since Watson-Crick GC and AU base pairs are also readily detected the HNN-COSY experiment provides a useful and sensitive tool for the rapid identification of RNA secondary structure elements.

The NMR structure of Escherichia coli ribosomal protein L25 shows homology to general stress proteins and glutaminyl-tRNA synthetases.

The structure of the Escherichia coli ribosomal protein L25 has been determined to an r.m.s. displacement of backbone heavy atoms of 0.62 +/- 0.14 A by multi-dimensional heteronuclear NMR spectroscopy on protein samples uniformly labeled with 15N or 15N/13C. L25 shows a new topology for RNA-binding proteins consisting of a six-stranded beta-barrel and two alpha-helices. A putative RNA-binding surface for L25 has been obtained by comparison of backbone 15N chemical shifts for L25 with and without a bound cognate RNA containing the eubacterial E-loop that is the site for binding of L25 to 5S ribosomal RNA. Sequence comparisons with related proteins, including the general stress protein, CTC, show that the residues involved in RNA binding are highly conserved, thereby providing further confirmation of the binding surface. Tertiary structure comparisons indicate that the six-stranded beta-barrels of L25 and of the tRNA anticodon-binding domain of glutaminyl-tRNA synthetase are similar.

The mu- and delta-opioid pharmacophore conformations of cyclic beta-casomorphin analogues indicate docking of the Phe3 residue to different domains of the opioid receptors.

Cyclic beta-casomorphin analogues with a D-configured amino acid residue in position 2, such as Tyr-c[-Xaa-Phe-Pro-Gly-] and Tyr-c[-Xaa-Phe-D-Pro-Gly-] (Xaa = D-A2bu, D-Orn, D-Lys) were found to bind to the mu-opioid receptor as well as to the delta-opioid receptor, whereas the corresponding L-Xaa2 derivatives are nearly inactive at both. Low-energy conformers of both active and nearly inactive derivatives have been determined in a systematic conformational search or by molecular dynamics simulations using the TRIPOS force field. The obtained conformations were compared with regard to a model for mu-selective opiates developed by Brandt et al. [Drug Des. Discov., 10 (1993) 257]. Superpositions as well as electrostatic, lipophilic and hydrogen bounding similarities with the delta-opioid receptor pharmacophore conformation of t-Hpp-JOM-13 proposed by Mosberg et al. [J. Med. Chem., 37 (1994) 4371, 4384] were used to establish the probable delta-pharmacophoric cyclic beta-casomorphin conformations. These conformations were also compared with a delta-opioid agonist (SNC 80) and the highly potent antagonist naltrindole. These investigations led to a prediction of the mu- and delta-pharmacophore structures for the cyclic beta-casomorphins. Interestingly, for the inactive compounds such conformations could not be detected. The comparison between the mu- and delta-pharmacophore conformations of the cyclic beta-casomorphins demonstrates not only differences in spatial orientation of both aromatic groups, but also in the backbone conformations of the ring part. In particular, the differences on phi2 and psi2 (mu approximately 70 degrees, -80 degrees; delta approximately 165 degrees, 55 degrees) cause a completely different spatial arrangement of the cyclized peptide rings when all compounds are matched with regard to maximal spatial overlap of the tyrosine residue. Assuming that both the mu- and delta-pharmacophore conformations bind with the tyrosine residue in a similar orientation at the same transmembrane domain X of their receptors, the side chain of Phe3 as a second binding site has to dock with different domains.