Creation and analysis of chimeras of spinach and tobacco Rubisco activase

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco: EC 4.1.1.39) catalyzes the initial step in photosynthesis, by combining CO$\sb2$ with ribulose bisphosphate to form two molecules of PGA. Rubisco activase is a protein which regulates the activity of Rubisco in vivo and hydrolyzes ATP. Regulation is achieved by promoting carbamylation of Rubisco (required for catalytic activation) as well as freeing Rubisco of various other sugar phosphates which either interfere with carbamylation or diminish the activity of carbamylated enzyme. Previously spinach Rubisco activase had been cloned into the pPlex expression vector and the resulting polypeptide purified and characterized. I modified this clone to obtain protein truncated at the N and C terminus. Truncation at the N terminus had no effect until a conserved tryptophan was removed. Deletion of this residue reduced Rubisco activation with no effect on ATP hydrolysis. Truncation of the C terminus, on the other hand, had no effect on ATP hydrolysis but rather increased Rubisco activation. It was concluded that the ratio of Rubisco activation to ATP hydrolysis, the efficiency of activation, could be altered by modification of the Rubisco activase polypeptide.Previously it was found that Rubisco activase isolated from species in the Solanaceae family activated Rubisco isolated from non-Solanaceae species poorly and vice versa. A comparison of the sequences of Rubisco activase isolated from spinach and tobacco reveals many amino acid differences. Two partial clones of tobacco Rubisco activase were isolated which were truncated at either end. Using a combination of five unique restriction sites and cloning, constructs were created in which sequences of tobacco were exchanged with the corresponding sequence from spinach. Chimeric polypetides were obtained in which the N terminal, C terminal and middle portions from spinach were introduced into tobacco and vice versa. ATP hydrolysis and activation of both spinach and tobacco Rubisco were determined for each. From these results the preference for spinach and tobacco Rubisco and both the specificity of activation (spinach or tobacco) and the efficiency of activation (ratio of Rubisco activation to ATP hydrolysis) were calculated. Depending on their composition these polypeptides displayed a range of Rubisco activation and ATP hydrolysis rates as well as varying Rubisco preference and efficiency of activation. Comparative analysis of the results with the chimera indicate that the C terminus is the most important part of the protein in determining Rubisco preference. However, the importance of interactions among regions of the polypeptide distant in the primary structure in determining Rubisco preference could not be ruled out.U of I OnlyETDs are only available to UIUC Users without author permissio