A web services choreography scenario for interoperating bioinformatics applications

Background  

Very often genome-wide data analysis requires the interoperation of multiple databases and analytic tools. A large number of genome databases and bioinformatics applications are available through the web, but it is difficult to automate interoperation because: 1) the platforms on which the applications run are heterogeneous, 2) their web interface is not machine-friendly, 3) they use a non-standard format for data input and output, 4) they do not exploit standards to define application interface and message exchange, and 5) existing protocols for remote messaging are often not firewall-friendly. To overcome these issues, web services have emerged as a standard XML-based model for message exchange between heterogeneous applications. Web services engines have been developed to manage the configuration and execution of a web services workflow.     

SERF: in vitro election of random RNA fragments to identify protein binding sites within large RNAs

In vitro selection experiments have various goals depending on the composition of the initial pool and the selection method applied. We developed an in vitro selection variant (SERF, selection of random RNA fragments) that is useful for the identification of short RNA fragments originating from large RNAs that bind specifically to a protein. A pool of randomly fragmented RNA is constructed from a large RNA, which is the natural binding partner for a protein. Such a pool contains all the potential binding sites and is therefore used as starting material for affinity selection with the purified protein to find its natural target. Here we provide a detailed experimental protocol of the method. SERF has been developed for ribosomal systems and is a general approach providing a basis for functional and structural characterization of RNA-protein interactions in large ribonucleoprotein particles.     

Localization of the ribosomal protection protein Tet(O) on the ribosome and the mechanism of tetracycline resistance

Tet(O) belongs to a class of ribosomal protection proteins that mediate tetracycline resistance. It is a G protein that shows significant sequence similarity to elongation factor EF-G. Here we present a cryo-electron microscopic reconstruction, at 16 A resolution, of its complex with the E. coli 70S ribosome. Tet(O) was bound in the presence of a noncleavable GTP analog to programmed ribosomal complexes carrying fMet-tRNA in the P site. Tet(O) is directly visible as a mass close to the A-site region, similar in shape and binding position to EF-G. However, there are important differences. One of them is the different location of the tip of domain IV, which in the Tet(O) case, does not overlap with the ribosomal A site but is directly adjacent to the primary tetracycline binding site. Our findings give insights into the mechanism of tetracycline resistance.     

Dissection of the mechanism for the stringent factor RelA

During conditions of nutrient deprivation, ribosomes are blocked by uncharged tRNA at the A site. The stringent factor RelA binds to blocked ribosomes and catalyzes synthesis of (p)ppGpp, a secondary messenger that induces the stringent response. We demonstrate that binding of RelA and (p)ppGpp synthesis are inversely coupled, i.e., (p)ppGpp synthesis decreases the affinity of RelA for the ribosome. RelA binding to ribosomes is governed primarily by mRNA, but independently of ribosomal protein L11, while (p)ppGpp synthesis strictly requires uncharged tRNA at the A site and the presence of L11. A model is proposed whereby RelA hops between blocked ribosomes, providing an explanation for how low intracellular concentrations of RelA (1/200 ribosomes) can synthesize (p)ppGpp at levels that accurately reflect the starved ribosome population.     

DnaK-facilitated ribosome assembly in Escherichia coli revisited

Assembly helpers exist for the formation of ribosomal subunits. Such a function has been suggested for the DnaK system of chaperones (DnaK, DnaJ, GrpE). Here we show that 50S and 30S ribosomal subunits from an Escherichia coli dnaK-null mutant (containing a disrupted dnaK gene) grown at 30 degrees C are physically and functionally identical to wild-type ribosomes. Furthermore, ribosomal components derived from mutant 30S and 50S subunits are fully competent for in vitro reconstitution of active ribosomal subunits. On the other hand, the DnaK chaperone system cannot circumvent the necessary heat-dependent activation step for the in vitro reconstitution of fully active 30S ribosomal subunits. It is therefore questionable whether the requirement for DnaK observed during in vivo ribosome assembly above 37 degrees C implicates a direct or indirect role for DnaK in this process.     

Structure of 5S rRNA within the Escherichia coli ribosome: iodine-induced cleavage patterns of phosphorothioate derivatives.

The protection patterns of 5S rRNA in solution, within the ribosomal 50S subunit, 70S ribosomes, and functional complexes, were assessed with the phosphorothioate method. About 20% of the analyzed positions (G9-G107) showed strong assembly defects: A phosphorothioate at one of these positions significantly impaired the incorporation of 5S rRNA into 50S particles. The reverse has also been observed: A phosphorothioate is preferred over a phosphate residue in the assembly process at a few positions. The results further demonstrate that 5S rRNA undergoes conformational changes during the assembly in the central protuberance of the 50S subunit and upon association with the small ribosomal subunit forming a 70S ribosome. In striking contrast, when the 70S ribosomes are once formed, the contact pattern of the 5S rRNA is the same in various functional states such as initiation-like complexes and pre- and posttranslocational states.     

Ribosome reactivation by replacement of damaged proteins

Ribosomal functions are vital for all organisms. Bacterial ribosomes are stable 2.4 MDa particles composed of three RNAs and over 50 different proteins. Accumulating damage to ribosomal RNA or proteins can disturb ribosome functioning. Organisms could benefit from degrading or possibly repairing inactive or partially active ribosomes. Reactivation of chemically damaged ribosomes by a process of protein replacement was studied in vitro. Ribosomes were inactivated by chemical modification of Cys residues. Incubation of modified ribosomes with total ribosomal proteins led to reactivation of translational activity. Intriguingly, ribosomal proteins extracted by LiCl are equally active in the restoration of ribosome function. Incubation of 70S ribosomes with isotopically labelled r‐proteins followed by separation of ribosomes was used to identify exchangeable proteins. A similar set of proteins was found to be exchanged in vivo under stress conditions in the stationary phase. We propose that repair of damaged ribosomes might be an important mechanism for maintaining protein synthesis activity following chemical damage.     

Immobilized pH gradient-driven paper-based IEF: a new method for fractionating complex peptide mixtures before MS analysis

Introduction  

The vast difference in the abundance of different proteins in biological samples limits the determination of the complete proteome of a cell type, requiring fractionation of proteins and peptides before MS analysis.     

Deacylated tRNA is released from the E site upon A site occupation but before GTP is hydrolyzed by EF-Tu

The presence or absence of deacylated tRNA at the E site sharply influences the activation energy required for binding of a ternary complex to the ribosomal A site indicating the different conformations that the E-tRNA imparts on the ribosome. Here we address two questions: (i) whether or not peptidyltransferase—the essential catalytic activity of the large ribosomal subunit—also depends on the occupancy state of the E site and (ii) at what stage the E-tRNA is released during an elongation cycle. Kinetics of the puromycin reaction on various functional states of the ribosome indicate that the A-site substrate of the peptidyltransferase center, puromycin, requires the same activation energy for peptide-bond formation under all conditions tested. We further demonstrate that deacylated tRNA is released from the E site by binding a ternary complex aminoacyl-tRNA•EF-Tu•GDPNP to the A site. This observation indicates that the E-tRNA is released after the decoding step but before both GTP hydrolysis by EF-Tu and accommodation of the A-tRNA. Collectively these results reveal that the reciprocal linkage between the E and A sites affects the decoding center on the 30S subunit, but does not influence the rate of peptide-bond formation at the active center of the 50S subunit.