Presentation on theme: "BBSI 2010 Closing Symposium"— Presentation transcript:
1BBSI 2010 Closing Symposium Mentor: Jason RifeInvestigation into the mechanism and possible requirements for release of KsgA from rRNAMeganSilbaughBBSI 2010 Closing Symposium
2Ribosomes make proteins in the cell. 65% rRNA35% r-protein30S and 50S subunitsCommon antibiotic targetDifferent structures, parallel biogenesisrRNA formationr-proteinModificationsOne common modification factor50S30SE. coli ribosome; darker areas represent rRNA; lighter areas represent r-proteins.Image from Wikimedia Commons.
3KsgA is a conserved methyltransferase. Quick Facts:3-D representation (left) and ribbon model (right)Discovered from resistance to Kasugamycin4 methyl (–CH3) groups to 2 adenosinesS-adenosylmethionine (SAM)Minimal effect when not presentPerforms other rolesBiogenesis checkpointSuccessful complementation suggests high conservationNucleotide binding siteImages from O’Farrell HC, Scarsdale JN, Rife JP
4When can KsgA release from the rRNA? What is known:What can be learned:Dimethylation of A1518 and A1519Requires mostly formed 30SMust be catalytically inactiveBinds at helix 44, methylates on helix 45A1519 is preferredIs methylation processive or distributive?Which adenosine is first?Which dimethylation allows substrate release to occur?
5Experiment Produce 2 mutations in 30S of ΔKsgA E. coli cells A1518CA1519CPurify mutated 30S from wild typeHis-tagged proteinMonitor release of KsgA using fluorescent polarizationFluorescein tagged KsgA
6Making the Mutants Plasmid contains: Transform ΔKsgA cells.MS2 protein binds to the spur in the 16S.Plasmid contains:MS2 TagAdenosine point mutation (A1518C or A1519C)Cells will produce both wild type and mutant 30SInsertion 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
7Ribosome Purification Sucrose gradientPurify all 70S from gradientFraction using gradient machineLower concentration of Mg++Purify all 30S with new gradient10%40%30S50S70S
8Purification of Tagged 30S Mutants After 30S mixture is purified:MS2 is the connector between the column and the tagged 30S.Combine with MS2MS2 binds to 30S tagRun through Ni-NTA columnNi2+ binds to MS2Untagged 30S wash offElute tagged 30S from columnTagged 30SMS2 6xHis-tagNi-NTA MatrixImage from Qiagen, 2003.
9Scintillation Count Activity Assays Measure of radioactivityMutant activity was far below half of the wild type.Combine 30S, KsgA, and 3H SAMMethyl groups will be radioactiveCompare levels of radioactivity in wild type and mutantsExpect 2:1 ratioActivity of mutants was the same as controls.
10“Failure is only the opportunity to begin again more intelligently.” -Henry FordTo be continued:First:Resolve issues with purifying tagged 30SThen:More scintillation activity assaysControl experiments (PAGE gels, even more activity assays, etc.)Finally:Fluorescent polarizationLearn when KsgA releases from 16S rRNA
11Works CitedDNA 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:O’Farrell 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. 5th ed.