Large-Scale Motions within Ribosomal 50S Subunits as Demonstrated Using Photolabile Oligonucleotides

Photolabile anticodon stem–loop analogs of tRNAPhe as probes of ribosomal structure and structural fluctuation at the decoding center

“…6B). An earlier photocrosslinking study of ours (Seo and Cooperman 2002) identified specific regions of the 50S subunit, in particular those involved with the GTPase activation center and with tRNA binding, that form "incompatible" crosslinks that are also in accord with the conclusions of Gabashvili et al (2003). The crosslinks identified in these two studies may result from movements that exaggerate those that are normally accessible to ribosomes in solution, because of the driving force provided by covalent bond formation.…”

“…RNA extraction from 70S ribosomes, and PAGE analysis of RNase H fragments were carried out essentially as described (Cooperman et al 2000;Seo and Cooperman 2002). Interestingly, the time required to photolyze the s 4 U of Ph-ASL 33 (t 1/2 ∼3-4 min), as determined by the decrease in A 330 , was significantly longer than to photolyze the s 4 U of Ph-ASL 37 (t 1/2 < 1 min).…”

“…Conversion of these results into photoincorporation yields required determination of the yield of fragment formation (i.e., cleavage efficiency) in the presence of either one or two added complementary oligonucleotides. Yields of the larger fragments (>22 nt) were determined essentially as described (Seo and Cooperman 2002), using staining intensity (methylene blue or silver) relative to a tRNA Phe standard. Estimates of the yields of small labeled RNA fragments within the 3Ј-terminal region of 16S rRNA were made by comparing the radioactivity in the 1495-1542 fragment on RNase H digestion in the presence of chimera 1494 alone (the cutting efficiency of oligonucleotide chimera 1494, which generates the 1495-1542 fragment, is ∼100%) with that remaining in the presence of an added second oligonucleotide.…”

“…Kethoxal modification on ribosomal complexes containing P-site bound tRNA f Met or ASL was performed as described (Moazed and Noller 1986) with slight modifications (Bukhtiyarov et al 1999;Seo and Cooperman 2002). The sites of 16S rRNA modifications were analyzed by primer extension assay (Cooperman et al 2000) using the oligodeoxynucleotide complementary to 16S nucleotides 984-969 as the primer.…”

Thiostrepton inhibition of tRNA delivery to the ribosome

“…1; Stark et al 1997;Valle et al 2003). While these data provide an excellent biochemical and static structural representation of the GTPase center of the ribosome, dynamic information linking the known structural elements with the biochemical functions of the dynamic GTPase center (Bushuev et al 1989;Porse et al 1998;Ban et al 2000;Seo and Cooperman 2002) is not available.…”