Mentor: Jason Rife Investigation into the mechanism and possible requirements for release of KsgA from rRNA Megan Silbaugh BBSI 2010 Closing Symposium
Ribosomes make proteins in the cell. 70S 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.
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. 2004.
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?
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
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. 2005.
Ribosome Purification • Sucrose gradient • Purify all 70S from gradient • Fraction using gradient machine • Lower concentration of Mg++ • Purify all 30S with new gradient 10% 30S 50S 70S 40%
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.
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.
“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
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. 2009. Molecular Biology Intelligence Unit. Landes Bioscience. • Wikimedia Commons. http://en.wikipedia.org/wiki/File:Ribosome_shape.png • Connolly K, Rife JP, Culver G. 2008. Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA. Mol Microbiol. 70: 1062-1075 • Desai PM, Rife JP. 2006. The adenosine dimethyltransferase KsgA recognizes a specific conformational state of the 30S ribosomal subunit. Biochem and Biophys. 449: 57-63. • Youngman EM, Green R. 2005. Affinity purification of in vivo-assembled ribosomes for in vitro biochemical analysis. Methods. 36: 305-312. • Maki JA, Schnobrich DJ, Culver GM. 2002. The DnaK chaperone system facilitates 30S ribosomal subunit assembly. Mol. Cell. 10:129-138. • O’Farrell HC, Scarsdale JN, Rife JP. 2004. Crystal structure of KsgA, a universally conserved rRNA adenine dimethyltransferase in Escherichia coli. J. Mol. Biol. 339: 337-353. • O’Farrell HC, Scarsdale JN, Rife JP. 2004. Crystal structure of KsgA, a universally conserved rRNA adenine dimethyltransferase in Escherichia coli. J. Mol. Biol. 339: 337-353. • Qiagen. 2003. The QIAexpressionist: A handbook for high-level expression and purification of 6xHis-tagged proteins. 5th ed.