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To investigate the molecular chaperone function of trigger factor (TF) and its relationship with isomerase activity in vivo, the assisted folding of adenylate kinase (AK) by TF in Escherichia coli was examined by measuring the amounts of soluble AK produced during co-expression. When the mutant of chicken AK, P17G, is expressed in plasmid pBVAK, 95% of the protein is found in inclusion bodies. Co-expression of AK with TF was achieved using a plasmid pBVAT that allowed expression of TF and AK in the same plasmid under separate control. Co-expression with TF resulted in an increase in the amount of soluble AK, with a higher increase when TF was expressed at higher levels in the cell. Co-expression of AK with the two TF mutants, Y221G and F233Y, in which peptidyl-prolyl cis/trans isomerase activity was 1% of wild-type, gave the same results as wild-type TF. This provides in vivo evidence that the molecular chaperone activity of TF is distinct from its isomerase activity.

Covalent joining of the two half molecules of tRNA Ala by T 4 RNA ligase to form a reconstituted whole molecule was investigated. The two half molecules consisting, respectively, of residues 1–35 and 36–75 were prepared by partial degradation of tRNA Ala with RNAase T 1 . The 5′-half molecule was treated with alkaline phosphatase to remove the 3′-terminal phosphate group, and the 5′-OH group of the 3′-half molecule was phosphorylated with [γ- 32 P]ATP by polynucleotide kinase. The two terminal nucletides to be joined were identified as Guo and Cyd. Prior to the covalent joining reaction, the two modified half molecules in an equimolar mixture were annealed, and the rejoined half molecules, separated by gel electrophoresis, served as the substrate for T 4 RNA ligase. Optimum conditions for this ligation, such as RNA ligase concentration, pH, Mg 2+ concentration, reaction temperature and time of reaction, were investigated. Under the optimum conditions a yield of about 70% joining of the reconstituted whole molecule was obtained as shown by gel electrophoresis, resistance to hydrolysis by alkaline phosphatase, nearest neighbour analysis and alanine acceptor activity.