The publication of the crystal structures of the ribosome offers an opportunity to retrospectively evaluate the information content of hundreds of qualitative biochemical and biophysical studies of these structures. We assessed the correspondence between more than 2,500 experimental proximity measurements and the distances observed in the ribosomal crystals. Although detailed experimental procedures and protocols are unique in almost each analyzed paper, the data can be grouped into subsets with similar patterns and analyzed in an integrative fashion. We found that, for crosslinking, footprinting, and cleavage data, the corresponding distances observed in crystal structures generally did not exceed the maximum values expected (from the estimated length of the agent and maximal anticipated deviations from the conformations found in crystals). However, the distribution of distances had heavier tails than those typically assumed when building three-dimensional models, and the fraction of incompatible distances was greater than expected. Some of these incompatibilities can be attributed to the experimental methods used. In addition, the accuracy of these procedures appears to be sensitive to the different reactivities, flexibilities, and interactions among the components. These findings demonstrate the necessity of a very careful analysis of data used for structural modeling and consideration of all possible parameters that could potentially influence the quality of measurements. We conclude that experimental proximity measurements can provide useful distance information for structural modeling, but with a broad distribution of inferred distance ranges. We also conclude that development of automated modeling approaches would benefit from better annotations of experimental data for detection and interpretation of their significance.

The wild-type transcript of Escherichia coli tRNASec, characterized by a peculiar core architecture and a large variable region, was shown to be aspartylatable by yeast AspRS. Similar activities were found for tRNASec mutants with methionine, leucine, and tryptophan anticodons. The charging efficiency of these molecules was found comparable to that of a minihelix derived from tRNAAsp and is accounted for by the presence of the discriminator residue G73, which is a major aspartate identity determinant. Introducing the aspartate identity elements from the anticodon loop (G34, U35, C36, C38) into tRNASec transforms this molecule into an aspartate acceptor with kinetic properties identical to tRNAAsp. Expression of the aspartate identity set in tRNASec is independent of the size of its variable region. The functional study was completed by footprinting experiments with four different nucleases as structural probes. Protection patterns by AspRS of transplanted tRNASec and tRNAAsp were found similar. They are modified, particularly in the anticodon loop, upon changing the aspartate anticodon into that of methionine. Altogether, it appears that recognition of a tRNA by AspRS is more governed by the presence of the aspartate identity set than by the structural framework that carries this set.

This is the first study in which the complex of a monoclonal autoantibody fragment and its target, stem loop II of U1 snRNA, was investigated with enzymatic and chemical probing. A phage display antibody library derived from bone marrow cells of an SLE patient was used for selection of scFvs specific for stem loop II. The scFv specificity was tested by RNA immunoprecipitation and nitrocellulose filter binding competition experiments. Immunofluorescence data and immunoprecipitation of U1 snRNPs containing U1A protein, pointed to an scFv binding site different from the U1A binding site. The scFv binding site on stem loop II was determined by footprinting experiments using RNase A, RNase V1, and hydroxyl radicals. The results show that the binding site covers three sequence elements on the RNA, one on the 5′ strand of the stem and two on the 3′ strand. Hypersensitivity of three loop nucleotides suggests a conformational change of the RNA upon antibody binding. A three-dimensional representation of stem loop II reveals a juxtapositioning of the three protected regions on one side of the helix, spanning approximately one helical turn. The location of the scFv binding site on stem loop II is in full agreement with the finding that both the U1A protein and the scFv are able to bind stem loop II simultaneously. As a consequence, this recombinant monoclonal anti-U1 snRNA scFv might be very useful in studies on U1 snRNPs and its involvement in cellular processes like splicing.