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.