Structure-function relationships for the small subunit (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase: Foreign S expression and characterization of engineered protein
AuthorGetzoff, Timothy Paul, 1964-
AdvisorBohnert, Hans J.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractThis dissertation addresses how small subunit (S) of higher plant Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC220.127.116.11) might influence Rubisco function. Toward this analysis a pea RbcS 3A cDNA expression cassette was introduced into Arabidopsis thaliana Landsberg by Agrobacterium tumefaciens mediated transformation. Analysis of RNA blots and 2-D gels indicate pea RbcS 3A is expressed and the S protein product is transported into Arabidopsis chloroplasts, processed and assembled into a stable chimeric holoenzyme. Incorporation of only one pea S per Arabidopsis Rubisco was sufficient to allow biochemical analyses. Biochemical analyses determined that chimeric enzymes displayed lower carboxylase activity (Vc) than WT Arabidopsis Rubisco coincident and consistent with the amount of pea S present in holoenzymes. Lower Vc is likely the result of reduced carbamylation following activation. Enhancement of Vc following temperature treatment at 42°C is kinetic evidence of increased activity of active sites which not due to differences in carbamylation. Unlike wild-type Rubisco, chimeric enzymes do not display the expected increase in carboxylase activity following activation at 42°C. This indicates S plays a role in allowing increased activity of neighboring L. Thus, both carbamylation and activity are disrupted by the interaction of pea S and Arabidopsis L. Kinetic data and formulae offered here support a structural model whereby S influences activity by allowing S-dependent interaction between L active sites. Also, high-temperature treated Rubisco shows a more pronounced fallover. This suggests that 42°C caused changes within Rubisco which either increase the synthesis of inhibitors or the response to inhibitors. To enhance abundance of pea S relative to Arabidopsis S pea S expressing plants were transformed with oligo-antisense cassettes targeting the 5'UTR and transit peptide of endogenous Arabidopsis RbcS transcripts. These doubly transformed plants were grown on media with 3% sucrose to cause metabolite repression which can further reduce endogenous Arabidopsis RbcS expression. However, both antisense and metabolite repression reduce the amount of pea S relative to Arabidopsis S protein. Genetic crosses between independently transformed plants expressing pea S suggest that expression of different amounts of pea S can be achieved.
Degree ProgramGraduate College
Molecular and Cellular Biology