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.