Table 2.

Selection against modification of 23S rRNA bases in the large ribosomal subunits complexed with biotinylated tRNA substrates and captured on the avidin resin^*

Position	N-Ac-Tyr-tRNABiot†	N-Ac-Tyr-tRNABiot‡ (puromycin elution)
G2252	++	+
A2451	+++	+
U2506	++	+
U2585	+++	+
U2555	−	+

^*Band intensities were measured by scanning gels on phophorimager and, after subtraction of background, normalized to intensities of control bands. The Table summarizes the results of four independent experiments. +++, Strong selection (>75%) against base modification; ++, considerable selection (50–75%); +, moderate selection (25-50%); −, no selection. 

^†N-Ac-Tyr-tRNA_Biot^Tyr was bound to 50S ribosomal subunits in the fragment reaction conditions in the presence of 0.1 mM sparsomycin, and the complexes were captured on the avidin resin and eluted with SDS/EDTA-containing buffer. 23S rRNA was extracted and subjected to primer-extension analysis. 

^‡Same as †, but binding was performed in the absence of sparsomycin, and captured ribosomal subunits were eluted from the avidin complex by puromycin treatment.