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Interaction of BCAII and lysozyme with ribosome and its domain V RNA.

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posted on 07.05.2014, 03:39 by Bani Kumar Pathak, Surojit Mondal, Amar Nath Ghosh, Chandana Barat

Filter binding studies. Refolding of BCAII-m or reduced-denatured lysozyme was initiated in presence of radiolabeled various domain V RNA, was UV- crosslinked and filtered through nitrocellulose membrane (material and method). A) Time course of interactions of BCAII-m with radiolabeled bDV RNA (-○-), mDV RNA (-▴-), bDV RNA mutants U2585C (-▾-) and delG2252 (-▪-) are shown here. Experiments were repeated thrice and their average values were taken for final data plotting. B) Time course of interactions of reduced-denatured lysozyme and radiolabeled bDV RNA (-•-) and mDV RNA (-▴-) are shown. Size exclusion chromatography. Refolding of FITC labeled BCAII-m or reduced-denatured lysozyme was initiated in presence of 70S ribosome, was UV-crosslinked at 30 second of refolding, and the mix was loaded on Sephacryl S-300 column. The elution of the protein and the ribosome was monitored by fluorescence at 518 nm and absorbance at 260 nm. C) Detection of 70S- BCAII complex. The elution profiles of BCAII-m in presence of ribosome at 30 seconds of refolding (3), reduced denatured lysozyme in presence of ribosome at 30 seconds of refolding (5) are shown. The elution profiles of ribosome (1), native BCAII (2) and native lysozyme (4) are also shown for comparison.

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