Reconstitution of 30S ribosomal subunits in vitro using ribosome biogenesis factors

Analysis of 30S particles reconstituted using conventional methods. (A) Reconstituted mixtures were analyzed by 10%–40% SDG analysis. Native 16S rRNA and native 30S subunits were analyzed as controls. The lines show the peaks of native 16S rRNA, native 30S subunits, and reconstituted 30S particles, using heat activation. (B) Purified reconstituted particles were analyzed by 15% SDS-PAGE and stained by SYPRO Orange (Invitrogen). Native 30S subunit proteins were loaded in the left lane, and 30S particles reconstituted via heat activation were loaded in the right lane. (C) Synthesized polyphenylalanine labeled with ¹⁴C was detected using a liquid scintillation counter. The values of ¹⁴C-phenylalanine incorporation were standardized (the value of ¹⁴C phenylalanine incorporation by native 30S and 50S subunits after 30 min was determined as 1). ¹⁴C-phenylalanine incorporation by various subunits is denoted as follows: native 70S, ○; native 30S and 50S subunits, ▴; reconstituted 30S subunits and native 50S subunits, Δ; native 50S subunits, ▪; and no ribosomes, ♦. The bar shows the standardized values. (D) Dihydrofolate reductase (DHFR) synthesis in the PURE system using 30S subunits reconstituted via heat activation.