Leishmania Ribosome Purification and Assembly

L. donovani (MHOM/ET/2009/GR356 clone IV) promastigote growth and 91S ribosome purification has been performed as previously described^{12 (link)}. Ribosome complexes with mRNA, three tRNA molecules and paromomycin were assembled by sequential addition of an mRNA fragment (CACCAUGUUCAAA, GE Dharmacon) containing the leishmanial Kozak sequence^{41 (link)} (highlighted in bold) a P-site start codon (AUG, underlined) and an A-site Phe codon (UUC), P-site tRNA_fmet (E.coli, Sigma), A-site tRNA_phe (E.coli, Sigma) and Paromomycin sulfate (Sigma) at 1:100:5:5:100 stoichiometric ratio. Complex assembly has been performed at 26 °C in ribosome conservation buffer (20 mM HEPES–KOH pH 7.6, 100 mM KOAc; 10 mM Mg(OAc)₂, 10 mM NH₄OAc, 2 mM β-mercaptoethanol and 1:40 dilution of RNAsin U (Promega)) with relaxation times of 30 min after the addition of each complex component. Ribosome final concentration was 125 nM.

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Shalev-Benami M., Zhang Y., Rozenberg H., Nobe Y., Taoka M., Matzov D., Zimmerman E., Bashan A., Isobe T., Jaffe C.L., Yonath A, & Skiniotis G. (2017). Atomic resolution snapshot of Leishmania ribosome inhibition by the aminoglycoside paromomycin. Nature Communications, 8, 1589.

Publication 2017

Paromomycin sulfate

2 mercaptoethanol Buffer Clone Codon Dilution E coli Hepes Mrna Paromomycin Paromomycin sulfate Promega Ribosome Start codon Trna Trnaphe

Corresponding Organization : Weizmann Institute of Science

Other organizations : Stanford University, Tokyo Metropolitan University, Hebrew University of Jerusalem

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Variable analysis

independent variables

Addition of mRNA fragment (CACCAUGTTCAAA)
Addition of P-site tRNA (E. coli tRNA_fmet)
Addition of A-site tRNA (E. coli tRNA_phe)
Addition of Paromomycin sulfate

dependent variables

Ribosome complex assembly

control variables

Temperature (26°C)
Ribosome conservation buffer (20 mM HEPES–KOH pH 7.6, 100 mM KOAc, 10 mM Mg(OAc)2, 10 mM NH4OAc, 2 mM β-mercaptoethanol, 1:40 dilution of RNAsin U (Promega))
Relaxation times (30 min after addition of each component)
Ribosome final concentration (125 nM)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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