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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 subunitsCommon 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 (–CH3) 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 O’Farrell HC, Scarsdale JN, Rife JP. 2004.
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. 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%
40%
30S50S70S
Purification of Tagged 30S Mutants
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
After 30S mixture is purified:
MS2 is the connector between the column and the tagged 30S.
MS2 6xHis-tag
Ni-NTA Matrix
Tagged 30S
Image from Qiagen, 2003.
Scintillation Count Activity Assays
Combine 30S, KsgA, and 3H 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.
WT+
KsgA
A1518
C+KsgA
A1519
C+KsgA
cont
rol
0
2000
4000
6000
8000
10000
12000
14000
16000
Type of 30S and Condition
Counts
per
Min
ute
• 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. 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[5]: 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 dimethyl transferase 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 dimethyl transferase 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.