Project description:In order to assess the prevalence of cotranslational assembly of protein complexes we performed RIp-chip experiments with many proteins that do not conatin RNA-binding motifs
Project description:We have used RIp-chip to identify mRNAs that coprecipitae with specific proteins, and later used the protein RNA interactions to predict protein-protein associations. To test whether some of the interactions we see require intact polysomes (that is, ribosomes translating mRNAs) we treated the extracts (and, in some cases, also the cells) with different compounds. In this case, we have a standard protocol, one in which the buffers contain EDTA, and one in which it includes puromycin.
Project description:Ribonucleoprotein immunoprecipitation microarray (RIp-chip) study using the RNA-binding protein Meu5 from the fission Schizosaccaromyces pombe
Project description:Ribonucleoprotein immunoprecipitation microarray (RIp-chip) study using the Rng3p myosin-specific chaperones from the fission Schizosaccaromyces pombe. Rng3p associates with RNAs encoding myosins. To investigate if the association of Rng3p with these RNAs occurs on ribosomes, Rng3-purified fractions were immunoprecipitated with antibodies against a ribosomal RNA or an unrelated antibody. The RNAs present in the second immunoprecipitate were analysed using DNA microarrays
Project description:BACKGROUND: Nitrogen is an essential element for bacterial growth and an important component of biological macromolecules. Consequently, responding to nitrogen limitation is critical for bacterial survival and involves the interplay of signalling pathways and transcriptional regulation of nitrogen assimilation and scavenging genes. In the soil dwelling saprophyte Mycobacterium smegmatis the OmpR-type response regulator GlnR is thought to mediate the transcriptomic response to nitrogen limitation. However, to date only ten genes have been shown to be in the GlnR regulon, a vastly reduced number compared to other organisms. RESULTS: We investigated the role of GlnR in the nitrogen limitation response and determined the entire GlnR regulon, by combining expression profiling of M. smegmatis wild type and glnR deletion mutant, with GlnR-specific chromatin immunoprecipitation and high throughput sequencing. We identify 53 GlnR binding sites during nitrogen limitation that control the expression of over 100 genes, demonstrating that GlnR is the regulator controlling the assimilation and utilisation of nitrogen. We also determine a consensus GlnR binding motif and identify key residues within the motif that are required for specific GlnR binding. CONCLUSIONS: We have demonstrated that GlnR is the global nitrogen response regulator in M. smegmatis, directly regulating the expression of more than 100 genes. GlnR controls key nitrogen stress survival processes including primary nitrogen metabolism pathways, the ability to utilise nitrate and urea as alternative nitrogen sources, and the potential to use cellular components to provide a source of ammonium. These studies further our understanding of how mycobacteria survive nutrient limiting conditions. [Data is also available from http://bugs.sgul.ac.uk/E-BUGS-143]
Project description:Ribonucleoprotein immunoprecipitation microarray (RIp-chip) study using various myosin proteins and myosin-specific chaperones from the fission Schizosaccaromyces pombe. Two strains were used. In rng3TAP the rng3 protein has been tagged with TAP to allow its detection and immunoprecipitation. The TAP strain expresses the TAP sequence alone (without being attached to an other protein)