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Update (January 2, 2004). This project has never been populated. Interested students are encouraged to enquire with any of the investigators who may still have an interest in this or related research. Carboxylation of Biotin by Catalytic RNA This project will: focus on the synthesis, in vitro selection and characterization of a catalytic RNA macromolecule capable of carboxylating the vitamin biotin. Primary Faculty Advisors:
Prospectus: The oceans and atmosphere contain 5.2 x 1012 kilograms of CO2. The CO2 in the geosphere is exchanged with living organisms via biological carboxylation reactions. The enzyme ribulose-1,5-bisphosphate carboxylase catalyzes the key reaction in photosynthesis and as such is the most abundant protein on the planet. Carboxylation reactions are also essential in mammals; these reactions differ in that the mammalian carboxylases require the vitamin biotin which serves as the carrier of activated CO2. Biotin-dependent carboxylases are involved in some of the most important metabolic processes in mammalian physiology, such as fatty acid synthesis and gluconeogenesis. Biotin-dependent carboxylation reactions: The reactions catalyzed by this family of enzymes follow the same two-step pathway shown below:
The first partial reaction involves the carboxylation of biotin at the N1' position. This is accomplished by the ATP-dependent phosphorylation of bicarbonate, the source of CO2, forming a reactive carboxyphosphate intermediate. The carboxyl group is then transferred to biotin, which is covalently linked to the enzyme through an amide linkage to the side chain of a specific lysine residue. In the second half of the reaction, the carboxylate group is transferred from carboxybiotin to an acceptor molecule. The acceptor will vary depending on the enzyme; for example, pyruvate carboxylase utilizes pyruvate as an acceptor while acetyl CoA carboxylase utilizes acetyl CoA as its substrate. Waldrop's laboratory is currently studying the catalytic mechanism of biotin carboxylase, which is one component of acetyl CoA carboxylase which, in turn, catalyzes the regulated step in long-chain fatty acid synthesis. Biotin carboxylase catalyzes the first half-reaction in Scheme 1.
Recently, the Waldrop and Strongin laboratories synthesized a reaction intermediate analog of biotin carboxylase that incorporated the carboxyphosphate intermediate and biotin. This analog can be used to select for RNA molecules capable of carboxylating biotin. This is the first step in making "designer" catalysts for the important caboxylation reactions. In vitro selection of catalytic RNA: RNA molecules that bind a variety of small molecules have been isolated from large pools of random sequences, including RNA molecules that bind tightly to transition state analogs of certain chemical reactions (Morris et al., 1994). This reiterative selection and amplification process (SELEX: Systematic Evolution of Ligands by EXponential enrichment; Tuerk & Gold, 1990) will be used to develop RNA molecules that are capable of specific carboxylation of biotin. A single-stranded DNA template for in vitro transcription will be created: the DNA molecules will contain known sequences at either end to serve as priming sites for reverse transcription, for subsequent amplification by PCR and for sequencing, as well as to initiate in vitro transcription by T7 RNA polymerase. The intervening DNA sequence will contain randomized sequence. The analog BP1 will be used initially to select for catalytic RNA molecules. The reaction intermediate analog will be coupled to activated agarose beads via its carboxyl group; the presence of a hydrophilic spacer arm is designed to render the attached molecule accessible to binding of RNA molecules. Successive rounds of selection on the reaction intermediate analog will be carried out to identify RNA molecules with high affinity for the reaction intermediate analog. The identified molecules will be amplified, sequenced, and assayed for catalytic activity. Once a catalytic RNA is selected it will be characterized using steady-state kinetic methodology. It is conceivable that a catalytic RNA molecule may not be identified by selection on the existent reaction intermediate analog. The entire ATP substrate may be required to select for an RNA molecule capable of catalyzing a biotin-dependent carboxylation reaction. We therefore propose to synthesize a multisubstrate analog of the biotin carboxylation reaction. The configuration of the multisubstrate analog is shown at right.
This approach has been very successful in the generation of both catalytic antibodies (Schultz & Lerner, 1993) and catalytic RNA molecules (Morris et al., 1994), and is expected to yield RNA molecules with pre-determined catalytic activity. References:
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