A deterministic model for non-monotone relationship between translation of upstream and downstream open reading frames.

Totally asymmetric simple exclusion process (TASEP) modelling was shown to offer a parsimonious explanation for the experimentally confirmed ability of a single upstream open reading frames (uORFs) to upregulate downstream translation during the integrated stress response. As revealed by numerical simulations, the model predicts that reducing the density of scanning ribosomes upstream of certain uORFs increases the flow of ribosomes downstream. To gain a better insight into the mechanism which ensures the non-monotone relation between the upstream and downstream flows, in this work, we propose a phenomenological deterministic model approximating the TASEP model of the translation process. We establish the existence of a stationary solution featuring the decreasing density along the uORF for the deterministic model. Further, we find an explicit non-monotone relation between the upstream ribosome density and the downstream flow for the stationary solution in the limit of increasing uORF length and increasingly leaky initiation. The stationary distribution of the TASEP model, the stationary solution of the deterministic model and the explicit limit are compared numerically.

Identification of A-to-I RNA editing: dotting the i's in the human transcriptome.

The phenomenon of adenosine-to-inosine (A-to-I) RNA editing has attracted considerable attention from the scientific community due to its potential relationship to the evolution of cognition in animals. While A-to-I editing exists in all organisms with neurons, including those with primitive neuronal systems (hydra and nematodes), it is particularly frequent in organisms with a highly developed central nervous system (primates, especially humans). Diversification of RNA transcript sequences via A-to-I editing serves a number of different functional roles, such as altering the genome-templated identity of particular amino acids in proteins or altering splice site junctions and modulating regulation of alternatively spliced mRNA variants. Here we provide an overview of current computational and experimental methods for the high-throughput discovery of edited RNA nucleotides in the human transcriptome, as well as a survey of the existing RNA editing bioinformatics resources and an outlook of future perspectives.

RECODE: a database of frameshifting, bypassing and codon redefinition utilized for gene expression.

The RECODE database is a compilation of 'programmed' translational recoding events taken from the scientific literature and personal communications. The database deals with programmed ribosomal frameshifting, codon redefinition and translational bypass occurring in a variety of organisms. The entries for each event include the sequences of the corresponding genes, their encoded proteins for both the normal and alternate decoding, the types of the recoding events involved, trans-factors and cis-elements that influence recoding. The database is freely available at http://recode.genetics. utah.edu/.

The Database of Ribosomal Cross-links: an update.

The Database of Ribosomal Cross-links (DRC) was created in 1997. Here we describe new data incorporated into this database and several new features of the DRC. The DRC is freely available via World Wide Web at http://visitweb.com/database/ or http://www. mpimg-berlin-dahlem.mpg.de/ approximately ag_ribo/ag_brimacombe/drc/

New features of 23S ribosomal RNA folding: the long helix 41-42 makes a "U-turn" inside the ribosome.

23S rRNA from Escherichia coli was cleaved at single internucleotide bonds using ribonuclease H in the presence of appropriate chimeric oligonucleotides; the individual cleavage sites were between residues 384 and 385, 867 and 868, 1045 and 1046, and 2510 and 2511, with an additional fortuitous cleavage at positions 1117 and 1118. In each case, the 3' terminus of the 5' fragment was ligated to radioactively labeled 4-thiouridine 5'-,3'-biphosphate ("psUp"), and the cleaved 23S rRNA carrying this label was reconstituted into 50S subunits. The 50S subunits were able to associate normally with 30S subunits to form 70S ribosomes. Intra-RNA crosslinks from the 4-thiouridine residues were induced by irradiation at 350 nm, and the crosslink sites within the 23S rRNA were analyzed. The rRNA molecules carrying psUp at positions 867 and 1117 showed crosslinks to nearby positions on the opposite strand of the same double helix where the cleavage was located, and no crosslinking was detected from position 2510. In contrast, the rRNA carrying psUp at position 384 showed crosslinking to nt 420 (and sometimes also to 416 and 425) in the neighboring helix in 23S rRNA, and the rRNA with psUp at position 1045 gave a crosslink to residue 993. The latter crosslink demonstrates that the long helix 41-42 of the 23S rRNA (which carries the region associated with GTPase activity) must double back on itself, forming a "U-turn" in the ribosome. This result is discussed in terms of the topography of the GTPase region in the 50S subunit, and its relation to the locations of the 5S rRNA and the peptidyl transferase center.

The Database of Ribosomal Cross links (DRC).

The Database of Ribosomal Cross-links (DRC) provides a complete collection of all the published data produced by cross-linking studies on the Escherichia coli ribosome, as well as on its components and functional ligands. The DRC currently includes data on 986 cross-links from >100 research papers, yielded by >40 different reagents. For each cross-link, information is given concerning its location in the ribosome, the chemical or photochemical reagent applied, a brief description of the method(s) used to locate the cross-link, and the literature reference. The DRC is freely available via the World Wide Web at: http://Ribosome.Genebee.MSU.SU/DRC/ or at http://WWW:MPIMG-Berlin-Dahlem.MPG.DE/[symbol: see text]baranov/DRC/

A new technique for the characterization of long-range tertiary contacts in large RNA molecules: insertion of a photolabel at a selected position in 16S rRNA within the Escherichia coli ribosome.

A new approach for inserting a photo-label at a selected position within the long ribosomal RNA molecules has been developed. The Escherichia coli 16S rRNA was cleaved at a single internucleotide bond, 1141-1142, with RNase H in the presence of a complementary chimeric oligonucleotide. 4-Thiouridine 5', 3'-diphosphate was ligated to the 3'-end of the 5'fragment at the cleavage site with T4 RNA ligase. The 16S rRNA fragments containing this added photo-reactive nucleotide were assembled together with total 30S ribosomal proteins into small ribosomal subunits. The ability of such 30S particles containing fragmented rRNA to form 70S ribosomes has been demonstrated previously. Crosslinks were induced within the 30S subunits by mild UV irradiation. The sites of crosslinking within the 16S rRNA were then analyzed using RNase H digestion and reverse transcription. Two crosslinks from the thio-nucleotide attached to nt C1141 of 16S rRNA were observed, namely to nt U1295 and G1272. These results are in agreement with the established proximity of helix 39 and 41 in the 3D structure of the 30S ribosomal subunit, as shown by other intra RNA crosslinking data. These data furthermore allow us to refine the structural arrangement of helices 41 and 39 relative to one another.

[Directed cleavage of the 16S rRNA molecule at a single internucleotide bond]. / Napravlennoe razrezanie molekul 16S rRNK po edinichnoi mezhnukleotidnoi sviazi.

Cleavage of 16S ribosomal RNA (rRNA) from E. coli "hammerhead" type ribozymes as well as by RNAase iI in the presence of "hymeric" (2'-deoxy-F-thymidine containing) oligonucleotides has been studied. The conditions for the cleavage of a desired single internucleotide bond have been found for a large molecule with a very complicated secondary and three-dimensional structure.