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BBSI 2010 Closing Symposium

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1 BBSI 2010 Closing Symposium
Mentor: Jason Rife Investigation into the mechanism and possible requirements for release of KsgA from rRNA Megan Silbaugh BBSI 2010 Closing Symposium

2 Ribosomes make proteins in the cell.
65% rRNA 35% r-protein 30S and 50S subunits Common antibiotic target Different structures, parallel biogenesis rRNA formation r-protein Modifications One common modification factor 50S 30S E. coli ribosome; darker areas represent rRNA; lighter areas represent r-proteins. Image from Wikimedia Commons.

3 KsgA is a conserved methyltransferase.
Quick Facts: 3-D representation (left) and ribbon model (right) Discovered from resistance to Kasugamycin 4 methyl (–CH3) groups to 2 adenosines S-adenosylmethionine (SAM) Minimal effect when not present Performs other roles Biogenesis checkpoint Successful complementation suggests high conservation Nucleotide binding site Images from O’Farrell HC, Scarsdale JN, Rife JP

4 When can KsgA release from the rRNA?
What is known: What can be learned: 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?

5 Experiment 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

6 Making the Mutants Plasmid contains:
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

7 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

8 Purification of Tagged 30S Mutants
After 30S mixture is purified: MS2 is the connector between the column and the tagged 30S. Combine with MS2 MS2 binds to 30S tag Run through Ni-NTA column Ni2+ binds to MS2 Untagged 30S wash off Elute tagged 30S from column Tagged 30S MS2 6xHis-tag Ni-NTA Matrix Image from Qiagen, 2003.

9 Scintillation Count Activity Assays
Measure of radioactivity Mutant activity was far below half of the wild type. Combine 30S, KsgA, and 3H SAM Methyl groups will be radioactive Compare levels of radioactivity in wild type and mutants Expect 2:1 ratio Activity of mutants was the same as controls.

10 “Failure is only the opportunity to begin again more intelligently.”
-Henry Ford To be continued: 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

11 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[5]: 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.

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