Presentation on theme: "Investigation into the mechanism and possible requirements for release of KsgA from rRNA BBSI 2010 Closing Symposium Mentor: Jason Rife Megan Silbaugh."— Presentation transcript:
Investigation into the mechanism and possible requirements for release of KsgA from rRNA BBSI 2010 Closing Symposium Mentor: Jason Rife Megan Silbaugh
Ribosomes make proteins in the cell. 65% rRNA 35% r-protein 30S and 50S subunits Common antibiotic target Different structures, parallel biogenesis 1. rRNA formation 2. r-protein 3. Modifications One common modification factor E. coli ribosome; darker areas represent rRNA; lighter areas represent r-proteins. 30S 50S 70S Image from Wikimedia Commons.
KsgA is a conserved methyltransferase. Discovered from resistance to Kasugamycin 4 methyl (–CH 3 ) groups to 2 adenosines S-adenosylmethionine (SAM) Minimal effect when not present Performs other roles Biogenesis checkpoint Successful complementation suggests high conservation Quick Facts: 3-D representation (left) and ribbon model (right) Images from OFarrell HC, Scarsdale JN, Rife JP Nucleotide binding site
When can KsgA release from the rRNA? Dimethylation of A1518 and A1519 Requires mostly formed 30S Must be catalytically inactive Binds at helix 44, methylates on helix 45 A1519 is preferred Is methylation processive or distributive? Which adenosine is first? Which dimethylation allows substrate release to occur? What is known:What can be learned:
Produce 2 mutations in 30S of ΔKsgA E. coli cells A1518C A1519C Purify mutated 30S from wild type His-tagged protein Monitor release of KsgA using fluorescent polarization Fluorescein tagged KsgA Experiment
Making the Mutants Transform ΔKsgA cells. MS2 protein binds to the spur in the 16S. Plasmid contains: MS2 Tag Adenosine point mutation (A1518C or A1519C) Cells will produce both wild type and mutant 30S Insertion site for the MS2 tag in domain I of 16S rRNA: The green nucleotides were replaced by the blue nucleotides; the orange nucleotides denote the binding site of the MS2 protein. Image from Youngman EM, Green R
Ribosome Purification Sucrose gradient Purify all 70S from gradient Fraction using gradient machine Lower concentration of Mg ++ Purify all 30S with new gradient 10% 40% 30S 50S 70S
Purification of Tagged 30S Mutants Combine with MS2 MS2 binds to 30S tag Run through Ni-NTA column Ni 2+ binds to MS2 Untagged 30S wash off Elute tagged 30S from column After 30S mixture is purified: MS2 is the connector between the column and the tagged 30S. MS2 6xHis-tagNi-NTA Matrix Tagged 30S Image from Qiagen, 2003.
Scintillation Count Activity Assays Combine 30S, KsgA, and 3 H SAM Methyl groups will be radioactive Compare levels of radioactivity in wild type and mutants Expect 2:1 ratio Measure of radioactivity Mutant activity was far below half of the wild type. Activity of mutants was the same as controls.
First: Resolve issues with purifying tagged 30S Then: More scintillation activity assays Control experiments (PAGE gels, even more activity assays, etc.) Finally: Fluorescent polarization Learn when KsgA releases from 16S rRNA To be continued: Failure is only the opportunity to begin again more intelligently. -Henry Ford
Works Cited DNA and RNA Modification Enzymes: Structure, Mechanism, Function, and Evolution. Grosjean, H, ed. Chapter 35: Roles of the Ultra-Conserved Ribosomal RNA Methyltransferase KsgA in Ribosome Biogenesis. Rife, JP Molecular Biology Intelligence Unit. Landes Bioscience. Wikimedia Commons. Connolly K, Rife JP, Culver G Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA. Mol Microbiol. 70: Desai PM, Rife JP The adenosine dimethyltransferase KsgA recognizes a specific conformational state of the 30S ribosomal subunit. Biochem and Biophys. 449: Youngman EM, Green R Affinity purification of in vivo-assembled ribosomes for in vitro biochemical analysis. Methods. 36: Maki JA, Schnobrich DJ, Culver GM The DnaK chaperone system facilitates 30S ribosomal subunit assembly. Mol. Cell. 10: OFarrell HC, Scarsdale JN, Rife JP Crystal structure of KsgA, a universally conserved rRNA adenine dimethyl transferase in Escherichia coli. J. Mol. Biol. 339: Qiagen The QIAexpressionist: A handbook for high-level expression and purification of 6xHis- tagged proteins. 5 th ed.