A secondary structure model for 23S ribosomal RNA has been constructed on the basis of comparative sequence data, including the complete sequences from E. coli. Bacillus stearothermophilis, human and mouse mitochondria and several partial sequences. The model has been tested extensively with single strand-specific chemical and enzymatic probes. Long range base-paired interactions organize the molecule into six major structural domains containing over 100 individual helices in all. Regions containing the sites of interaction with several ribosomal proteins and 5S RNA have been located. Segments of the 23S RNA structure corresponding to eucaryotic 5.8S and 25 RNA have been identified, and base paired interactions in the model suggest how they are attached to 28S RNA. Functionally important regions, including possible sites of contact with 30S ribosomal subunits, the peptidyl transferase center and locations of intervening sequences in various organisms are discussed. Models for molecular 'switching' of RNA molecules based on coaxial stacking of helices are presented, including a scheme for tRNA-23S RNA interaction.

Synthetic mRNA analogues were prepared by T7 transcription, each containing several thio‐uridine residues at selected positions. After binding to the ribosome in the presence of cognate tRNA, the thio‐U residues were activated by UV irradiation and the resulting sites of cross‐linking to 16S RNA analysed. Three distinct cross‐links were consistently observed: (i) from position ‘+6’ of the mRNA (the 3′‐base of the A‐site codon) to base 1052 of 16S RNA; (ii) from position ‘+7’ of the mRNA to base 1395; and (iii) from ‘+11’ to base 532. Individual yields of the cross‐links were strongly dependent on the particular mRNA sequence in each case. The ‘+11/532’ and ‘+6/1052’ cross‐links were always entirely tRNA‐dependent, whereas the ‘+7/1395’ cross‐link was observed at lower intensity in the absence of tRNA. In the presence of a second (A‐site bound) tRNA the +6/1052 cross‐link was markedly reduced. A cross‐link to the 1050 region was again observed when a message carrying a thio‐U at position ‘+9’ was translocated on the ribosome so as to bring the thio‐U to position +6. Taken together, the data are incompatible with some current models both for the three‐dimensional arrangement of 16S RNA and for the orientation of the tRNA‐mRNA complex in the ribosome.

To cite this article: Protopopova AD, Barinov NA, Zavyalova EG, Kopylov AM, Sergienko VI, Klinov DV. Visualization of fibrinogen aC regions and their arrangement during fibrin network formation by high-resolution AFM. J Thromb Haemost 2015; 13: 570-9.See also Rocco M, Weisel JW. Exposed: the elusive aC regions in fibrinogen, fibrin protofibrils and fibers. This issue, pp 567-9.Summary. Background: Fibrinogen has been intensively studied with transmission electron microscopy and x-ray diffraction. But until now, a complete 3D structure of the molecule has not yet been available because the two highly flexible aC regions could not be resolved in fibrinogen crystals. This study was aimed at determining whether the aC regions can be visualized by high-resolution atomic force microscopy. Methods: Atomic force microscopy with super high resolution was used to image single molecules of fibrinogen and fibrin associates. The key approach was to use a graphite surface modified with the monolayer of amphiphilic carbohydrate-glycine molecules and unique supersharp cantilevers with 1 nm tip diameter. Results: Fibrinogen aC regions were visualized along with the complete domain structure of the protein.In almost all molecules at pH 7.4 the D domain regions had one or two protrusions of average height 0.4 AE 0.1 nm and length 21 AE 6 nm. The complex, formed between thrombin and fibrinogen, was also visualized. Images of growing fibrin fibers with clearly visible aC regions have been obtained. Conclusions: Fibrin aC regions were visible in protofibrils and large fibers; aC regions intertwined near a branchpoint and looked like a zipper. These results support the idea that aC regions are involved in the thickening of fibrin fibers. In addition, new details were revealed about the behavior of individual fibrin molecules during formation of the fibrin network. Under the diluted condition, the positioning of the aC regions could suggest their involvement in long-range interactions between fibrin but not fibrinogen molecules.

A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 µs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange.

By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brainspecific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 59 domain of BC1 RNA, derived from tRNA Ala , does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNA Ala domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 39 end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA.