Mrna 5 aaauuu3

We purified and crystallized 30S ribosomal subunits from T. thermophilus HB8 strain essentially as described^{29 (link),30 (link)}. The 2′-O-methylated mRNA fragments (with codon sequences underlined), 5′AmAAUUU3′, 5′AAmAUUU3′, 5′AAAmUUU3′ and native mRNA 5′AAAUUU3′ were purchased from Dharmacon. The presence of 2′-O-methylation was confirmed by mass spectrometry. The
${\text{ASL}}_{\mathit{UUU}}^{\mathit{Lys}3}$ , (with anticodon sequence underlined GCAGACU(_mcm5s2U)UU(_ms2t6A)AΨCUGC ), is a generous gift of Paul Agris (University of Albany). 30S crystals were sequentially transferred to the final buffer with 26% v/v 2-methyl-2,4-pentanediol (MPD) for cryoprotection and soaks were performed in the final buffer supplemented with 200 μM of each mRNA oligos,
${\text{ASL}}_{\mathit{UUU}}^{\mathit{Lys}3}$ and in the presence or absence of 80 μM paromomycin for 48 hours, as indicated. Crystals were flash-frozen for data collection by plunging them directly into liquid nitrogen.