A new model for the three-dimensional folding of Escherichia coli 16 s ribosomal RNA. III †. The topography of the functional centre 1 † Paper II in this series is an accompanying paper, Mueller & Brimacombe (1997b). 1 Edited by D. E. Draper (original) (raw)
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Journal of Molecular Biology, 1997
We describe the locations of sites within the 3D model for the 16 S rRNA (described in two accompanying papers) that are implicated in ribosomal function. The relevant experimental data originate from many laboratories and include sites of foot-printing, cross-linking or mutagenesis for various functional ligands. A number of the sites were themselves used as constraints in building the 16 S model. (1) The foot-print sites for A site tRNA are all clustered around the anticodon stem ± loop of the tRNA; there is no``allosteric'' site.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1990
A large number of intra-RNA and RNA-Iwotein cross-link sites have been localized within the 238 RNA from E. co//50 S ribosomal subunits. These sites, tngetlmr with other data, are sufficient to constrain the secondary ~ of the 23 S molecule into a compact three-dimensional shape. Some of the features of this s~ are discussed, in particular, those relating to the orientation of tRNA on the 50 S subunit as studied by site-directed cross-linking tedmklueS. A correspemling model for the 168 RNA within the 30 S subunit has already been described, and here a site-directed cross-linking altC¢oaeh is being used to determine the path followed through the sulmnit by messenger RNA. 0167-4781/90/$03.50
Structural organization of the 16S ribosomal RNA from E. coli. Topography and secondary structure
Nucleic Acids Research, 1981
Extensive studies in our laboratory using different ribonucleases ressulted in valuable data on the topography of the E.coli 16S ribosomal RNA within the native 30S subunit, within partially unfolded 30S subunits, in the free state, and in association with individual ribosomal proteins. Such studies gave precise details on the accessibility of certain residues and delineated highly accessible RNA regions. Furthermore, they provided evidence that the 16S rRNA is organized in its subunit into four distinct domains. A secondary structure model of the E.coZi 16S rRNA has been derived from these topographical data. Additional information from comparative sequence analyses of the small ribosomal subunit RNAs from other species sequenced so far has been used.
The modelling of the decoding site of the Escherichia coli ribosome
Nucleic Acids Research, 1984
Individual ribosomal proteins S4, S9 and S13 were tested for their ability to interact with tRNA and synthetic polynucleotides. All three proteins bind to immobilized to Sepharose poly(A) and poly(U), while S4 and S13 form stoichiometric (1:1) complexes with tRNA in solution. We show that only the polynucleotide S13 complexes are _ble to select their cognate tRNAs. In particular, the affinity of tRNAPne to the binary poly(U).S13 complex is about three orders of magnitude higher than that for poly(U) alone.
Nucleic Acids Research, 1990
Intramolecular RNA cross-links were induced within the large ribosomal subunit of E. coil by mild ultraviolet irradiation. Regions of the 23S RNA previously implicated in interactions with ribosomal-bound tRNA were then specifically excised by addressed cleavage using ribonuclease H, in conjunction with synthetic complementary decadeoxyribonucleotides. Individual cross-linked fragments within these regions released by such 'directed digests' were isolated by twodimensional gel electrophoresis and the sites involved in the cross-links determined using classical oligonucleotide analysis techniques. Using this approach, seven 'new' cross-links could be precisely localised, between positions in the 23S RNA sequence. These data, in conjunction with data from RNA-protein cross-linking studies carried out in our laboratory, were used to define a model for the tertiary organisation of the tRNA binding domain of 23S RNA 'in situ', in which the specific nucleotides associated with tRNA binding in the 'A' and 'P' sites are clustered at the base of the 'central protuberance' of the 50S subunit.
Topography of the E site on the Escherichia coli ribosome
The EMBO journal, 1993
Three photoreactive tRNA probes have been utilized in order to identify ribosomal components that are in contact with the aminoacyl acceptor end and the anticodon loop of tRNA bound to the E site of Escherichia coli ribosomes. Two of the probes were derivatives of E. coli tRNA(Phe) in which adenosines at positions 73 and 76 were replaced by 2-azidoadenosine. The third probe was derived from yeast tRNA(Phe) by substituting wyosine at position 37 with 2-azidoadenosine. Despite the modifications, all of the photoreactive tRNA species were able to bind to the E site of E. coli ribosomes programmed with poly(A) and, upon irradiation, formed covalent adducts with the ribosomal subunits. The tRNA(Phe) probes modified at or near the 3' terminus exclusively labeled protein L33 in the 50S subunit. The tRNA(Phe) derivative containing 2-azidoadenosine within the anticodon loop became cross-linked to protein S11 as well as to a segment of the 16S rRNA encompassing the 3'-terminal 30 nucl...
Role of conserved nucleotides in building the 16S rRNA binding site of E.coli ribosomal protein S8
Nucleic Acids Research, 1994
Ribosomal protein S8 specifically recognizes a helical and irregular region of 16S rRNA that is highly evolutionary constrained. Despite its restricted size, the precise conformation of this region remains a question of debate. Here, we used chemical probing to analyze the structural consequences of mutations in this RNA region. These data, combined with computer modelling and previously published data on protein binding were used to investigate the conformation of the RNA binding site. The experimental data confirm the model in which adenines A595, A640 and A642 bulge out in the deep groove. In addition to the already proposed non canonical U598 -U641 interaction, the structure is stabilized by stacking interactions (between A595 and A640) and an array of hydrogen bonds involving bases and the sugar phosphate backbone. Mutations that alter the ability to form these interdependent interactions result in a local destabilization or reorganization. The specificity of recognition by protein S8 is provided by the irregular and distorted backbone and the two bulged adenines 640 and 642 in the deep groove. The third adenine (A595) is not a direct recognition site but must adopt a bulged position. The U598 -U641 pair should not be directly in contact with the protein.
Proceedings of the National Academy of Sciences, 1991
Protein-nucleic acid interactions involved in the assembly process of the Escherichia coli 30S ribosomal subunit were quantitatively analyzed by high-resolution scanning transmission electron microscopy. The in vitro reconstituted ribonucleoprotein (core) particles were characterized by their morphology, mass, and radii of gyration. During the assembly of the 30S subunit, the 16S rRNA underwent signif-