In vitro protein folding by E. coli ribosome: Unfolded protein splitting 70S to interact with 50S subunit Arunima Basu, Dibyendu Samanta, Debasis Das, Saheli Chowdhury, Arpita Bhattacharya, Jaydip Ghosh, Anindita Das, Chanchal DasGupta * Department of Biophysics, Molecular Biology and Genetics, University College of Science, 92 A.P.C. Road, Kolkata 700009, India Received 21 November 2007 Abstract Folding of unfolded protein on Escherichia coli 70S ribosome is accompanied by rapid dissociation of the ribosome into 50S and 30S subunits. The dissociation rate of 70S ribosome with unfolded protein is much faster than that caused by combined effect of translation and polypeptide release factors known to be involved in the dissociation of ribosome into subunits. The protein then reaches a ‘‘folding competent’’ state on 50S and is released to take up native conformation by itself. Release before attaining the folding competent state or prevention of release by cross-linking it with ribosome, would not allow the protein to get back to its native conformation. Ó 2007 Elsevier Inc. All rights reserved. Keywords: 50S subunit; Ribosomal subunit dissociation; Protein folding Ribosomes from eubacteria, archaea, eukaryotes and mitochondria have been shown to fold proteins in vitro and in vivo [1–12]. The experiments were done on a large number of proteins including the chaperone DnaK [13]. While searching for the active site of protein folding on ribosome, we found that it was in the large subunit. Fur- ther investigation revealed that the activity lies on the pep- tidyl transferase centre (PTC) of large ribosomal RNA present at the large subunit [8–12]. For example, in eubac- teria, the folding centre is in the domain V of 23S rRNA from the 50S subunit. In Escherichia coli, the domain V of 23S rRNA is in the interface of 50S and 30S ribosomal subunits [14] and could be easier to access in the free 50S rather than in the 70S ribosome. As a matter of fact, it turned out that while folding, the unfolded protein bovine carbonic anhydrase (BCA) quickly dissociated 70S ribo- some to associate itself with the 50S subunit [15]. The dis- sociation of 70S by unfolded proteins turned out to be much faster than GTP dependent subunits dissociation by elongation factor EFG and release factor RRF. It also turned out that before getting back the activity which was lost due to unfolding, the protein dissociated from the 50S subunit in a state that could attain the active form by a slow rate limiting step by itself [12]. Detailed studies on these steps of folding are reported here. Materials and methods Preparation of ribosome and proteins. 70S ribosome was prepared as described earlier [1–3]. Translation factors EFG, RRF, IF3 etc were purified from E. coli BL21 (DE3) transformed with plasmid containing their genes (under T7 promoter). The proteins were over-expressed in presence of IPTG (Sigma). All proteins were purified to single band for 5 lg protein in SDS–PAGE stained with silver. Bovine carbonic anhydrase (BCA) was purchased from Sigma. The homogeneity of the protein was confirmed by a single band for 10 lg of it in SDS–PAGE following silver staining. Synthesis of domainV of E. coli 23S rRNA. Domain V of 23S rDNA was cloned under T7 promoter in plasmid pTZ57R/T (Fermentas). The domain V RNA was synthesized by run off transcription from the line- arized plasmid using T7 RNA polymerase (Bangalore Genei, India). It was labelled with a trace of a- 32 P UTP for gel detection and estimation. Unfolding and refolding of BCA. BCA (30 KDa) was denatured with 6 M guanidine hydrochloride (Sigma) for 3 h at 25 °C. Loss of secondary 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.11.143 * Corresponding author. E-mail address: chanchaldg2000@yahoo.com (C. DasGupta). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 366 (2008) 598–603