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Translation Factors (translation + factor)
Selected AbstractsExpression, purification, and analysis of unknown translation factors from Escherichia coli: A synthesis approachBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2010Justin D. Walter Abstract New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity of their unknown (from a list of potential candidates). Subsequent to the identification of their unknown translation factor, a series of protein unfolding exercises are performed employing circular dichroism and fluorescence spectroscopies, allowing students to directly calculate thermodynamic parameters centered around determining the equilibrium constant for unfolding as a function of denaturant (temperature or chemical). The conclusion of this multi-part laboratory exercise consists of both oral and written presentations, emphasizing synthesis of the roles of each translation factor during the stepwise process of translation. [source] Interaction of ribosome recycling factor and elongation factor EF-G with E. coli ribosomes studied by the surface plasmon resonance techniqueGENES TO CELLS, Issue 12 2000Tetsuya Ishino Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the post-termination complex of a ribosome after the release of polypeptides. How RRF dissociates the complex has long been puzzling. Crystal structures of RRF molecules have been solved recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does. The formation of ribosome complexes on the surface-coupled RRF and elongation factor EF-G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique. RRF interacted with 70S ribosomes as well as 50S and 30S subunits, although it interacted preferentially with 50S subunits, which was clearly seen under high but physiological ionic conditions. This 50S interaction was diminished by a single amino acid substitutions for Arg132 of RRF, which did not appreciably affect the protein folding but nullified the activity in vivo and in vitro. Moreover, a set of antibiotics that inhibited the RRF,50S interaction were also inhibitory to the polysome breakdown activity of RRF in vitro. The BIACORE technique also worked very well in demonstrating the action of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre- and post-translocation intermediates catalysed by EF-G. These results suggest that the preferential interplay of RRF with the 50S subunit may be of biological significance, probably reflecting the mode of RRF action. The BIACORE technique proved useful for real-time monitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling factor. [source] Technical issues affecting the implementation of US Environmental Protection Agency's proposed fish tissue-based aquatic criterion for selenium,INTEGRATED ENVIRONMENTAL ASSESSMENT AND MANAGEMENT, Issue 4 2007A Dennis Lemly Abstract The US Environmental Protection Agency is developing a national water quality criterion for selenium that is based on concentrations of the element in fish tissue. Although this approach offers advantages over the current water-based regulations, it also presents new challenges with respect to implementation. A comprehensive protocol that answers the "what, where, and when" is essential with the new tissue-based approach in order to ensure proper acquisition of data that apply to the criterion. Dischargers will need to understand selenium transport, cycling, and bioaccumulation in order to effectively monitor for the criterion and, if necessary, develop site-specific standards. This paper discusses 11 key issues that affect the implementation of a tissue-based criterion, ranging from the selection of fish species to the importance of hydrological units in the sampling design. It also outlines a strategy that incorporates both water column and tissue-based approaches. A national generic safety-net water criterion could be combined with a fish tissue,based criterion for site-specific implementation. For the majority of waters nationwide, National Pollution Discharge Elimination System permitting and other activities associated with the Clean Water Act could continue without the increased expense of sampling and interpreting biological materials. Dischargers would do biotic sampling intermittently (not a routine monitoring burden) on fish tissue relative to the fish tissue criterion. Only when the fish tissue criterion is exceeded would a full site-specific analysis including development of intermedia translation factors be necessary. [source] ,4 phosphoprotein interacts with EDD E3 ubiquitin ligase and poly(A)-binding proteinJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2010William J. McDonald Abstract Mammalian ,4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target-of-rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell-cycle progression. ,4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using ,4 as bait in yeast two-hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of ,4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)-binding protein (PABP), suggesting that PABP may also interact with ,4. PABP recruits translation factors to the poly(A)-tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between ,4 and EDD in rat Nb2 T-lymphoma and human MCF-7 breast cancer cell lines. ,4 also interacted with PABP in Nb2, MCF-7 and the human Jurkat T-leukemic and K562 myeloma cell lines. COS-1 cells, transfected with Flag-tagged-pSG5-EDD, gave a (Flag)-EDD,,4 immunocomplex. Furthermore, deletion mutants of ,4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C-terminal region of ,4, independent of the ,4-PP2Ac binding site. Therefore, in addition to PP2Ac, ,4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell-cycle progression. J. Cell. Biochem. 110: 1123,1129, 2010. Published 2010 Wiley-Liss, Inc. [source] Ribosome rescue: tmRNA tagging activity and capacity in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 2 2005Sean D. Moore Summary When protein synthesis stalls in bacteria, tmRNA acts first as a surrogate tRNA and then as an mRNA in a series of reactions that append a peptide tag to the nascent polypeptide and ,rescue' the ribosome. The peptide tag encoded by wild-type tmRNA promotes rapid degradation of rescued proteins. Using a mutant tmRNA that encodes a tag that does not lead to degradation, we demonstrate that the synthesis of approximately 0.4% of all proteins terminates with tagging and ribosome rescue during normal exponential growth of Escherichia coli. The frequency of tagging was not significantly increased in cells expressing very high levels of tmRNA and its binding protein SmpB, suggesting that recognition of ,stalled' ribosomes does not involve competition between tmRNA and other translation factors for A-sites that are unoccupied transiently during protein synthesis. When the demand for ribosome rescue was increased artificially by overproduction of a non-stop mRNA, tmRNA levels did not increase but tmRNA-mediated tagging increased substantially. Thus, the ribosome-rescue system usually operates well below capacity. [source] Expression, purification, and analysis of unknown translation factors from Escherichia coli: A synthesis approachBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2010Justin D. Walter Abstract New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity of their unknown (from a list of potential candidates). Subsequent to the identification of their unknown translation factor, a series of protein unfolding exercises are performed employing circular dichroism and fluorescence spectroscopies, allowing students to directly calculate thermodynamic parameters centered around determining the equilibrium constant for unfolding as a function of denaturant (temperature or chemical). The conclusion of this multi-part laboratory exercise consists of both oral and written presentations, emphasizing synthesis of the roles of each translation factor during the stepwise process of translation. [source] | ||||||||||