Drummond:Paromomycin

Introduction

Error spectrum

• binds to A-site of ribosome
• in yeast, paromomycin acts by blocking aminoacyl-tRNA binding to A-site of ribosome after initiation
• interrupts with elongation step of translation [1]

Dose response

Stock solution

• Paromomycin is water soluble (50 mg/mL) (Sigma Aldrich)
• in the lab, a 100 mg/mL stock solution of paromomycin was synthesized (some vortexing was necessary)

Paromomycin dilution series

• 100X paromomycin stock solution (for 140 μM)

1. 140 μM paromomycin for 50% cell growth inhibition [1]
2. (1.4*10^-4 mol paromomycin/L solution)(713.71 g/mol)(1000 mg/g)(1 L/1000 mL) = 0.0991 mg/mL ≈ 0.1 mg/mL
3. (0.1 mg paromomycin/mL sln.)(50 cultures)(2 mL sln./culture) = 10 mg paromomycin (for 100 mL solution [2 mL*50])
4. 100 mL/100 = 1 mL
5. 10 mg paromomycin/1 mL nanopure water for 100X stock solution

• 2-fold dilution series (1:1 to 1:64)

1. 500 μL 100X paromomycin stock solution into tube 1 (1:1 dilution)
2. 250 μL nanopure water into 6 tubes (1:2 to 1:64)
3. Pipet 250 μL from 1:1 dilution into 1:2 dilution
4. Pipet 250 μL from 1:2 dilution into 1:4 dilution
5. " 1:4 to 1:16, 1:16 to 1:32, 1:32 to 1:64

For the second paromomycin trial, we adjusted the range of concentrations of paromomycin in which the yeast cells were grown. Instead of a 100X stock solution of paromomycin, a 1000X stock was used.

• 1000X paromomycin stock solution (for 140 μM)

1. 62.0 mg paromomycin/620 μL H₂O
2. vortex solution to dissolve paromomycin
3. 1:1 to 1:2048 serial dilutions

• 100X Yeast preculture stock solution

1. Maximum OD after 21 hours (14 doublings) = 10
2. 10/2¹⁴ = 6.10*10^-4
3. 6.10*10^-4/Initial preculture OD = X mL preculture (in 1 mL YPD solution)

• Yeast growth with paromomycin

1. 1.8 mL YPD
2. 200 μL paromomycin dilution (1:1 to 1:2048) or H₂O for controls
3. 20 μL 100X yeast culture
4. incubate overnight at 30°C
5. Record OD as a percentage of the control (0 μM paromomycin)for each dilution

cooperativity: 1.329
half max: 305.968
(using R and hill equation)

References

1. Tselika S, Konstantinidis TC, and Synetos D. Two nucleotide substitutions in the A-site of yeast 18S rRNA affect translation and differentiate the interaction of ribosomes with aminoglycoside antibiotics. Biochimie. 2008 Jun;90(6):908-17. DOI:10.1016/j.biochi.2008.02.010 |

2. http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/P5057