# Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock
## 2 Results
### Isolation and Affinity Purification of Tagged 21S Intermediates  
We submitted the E6006 strain to a heat shock of 45 \({}^{\circ}\)C for 60 min. We then isolated the ribosomes by ultracentrifugation on a 10-40% sucrose gradient. To pinpoint the location of 21S ribosomal particles, we performed hybridization of the appropriate biotinylated oligonucleotides as well as RNA gel electrophoresis (Figure 2). The oligonucleotides targeting the non-mature rRNAs (21S-115 and 21S-33) hybridize to the RNAs from fractions 10 to 19 (Figure 2C), and a supplementary band above the 16S rRNA is recovered in fractions 10 to 16 (Figure 2D). This reveals the presence of 17S rRNA, a pre-maturation form of 16S rRNA [22], in the first peak (fractions 10 to 22, Figure 2A) of the ribosome profile. Figure 2: Identification of ribosomal precursors in sucrose-gradient fractions. (**A**) Sucrose density gradient profiles of purified ribosomal particles from the Escherichia coli E6006 strain before (top) and after (bottom) heat shock. The fractions 7 to 25 in 10–40% sucrose gradients (framed box) were used for purifying 21S ribosomal particles. The fractions with blue circles are those analyzed by slot blot hybridization (**B**,**C**) or RNA gel electrophoresis (panel D). (**B**) Schematic based on Rene and Alix [22] showing non-mature 16S rRNAs after cleavage by RNAse III. The cleavage generates the 115 nt and 33 nt extremities (ends). The ribonuclease cleavage sites are indicated: III for RNAse III; G for RNAse G; E for RNAse E; and Y for YbeY [27]. The biotinylated oligonucleotides used for hybridization against the dot blots (see panel C) are marked with arrows. (**C**) Hybridizations with the biotinylated oligonucleotides 21S-115, 21S-33, and 16S-F27C, target the non-mature SSU rRNA’s 115 nt and 33 nt ends as well as mature 16S rRNAs, respectively. The numbers on the top indicate the fractions shown after 2 ug of RNAs were applied on Amersham Hybond-N + nylon membranes. (**D**) Electrophoresis of RNA extracted from the fractions as numbered on top, with RNA sizing produced using Ambion Millenium RNA markers. The 17S precursor rRNA suggests that 21S early ribosomal particles must be present just before the 30S peak. We therefore collected these fractions containing 21S and discarded the others. We eliminated the remaining 30S mature particles by three successive nickel affinity chromatography passes. The resulting fractions contained the expected ribosomal proteins as well as various contaminating proteins, but these were easily eliminated by purifying the 21S fractions on stret  
Figure 3: Purification of 21S particles. (**A**) _Escherichia coli_ E6006 cells were grown in M63 medium and subjected to heat shock for 1 h at 45 \({}^{\circ}\)C. The clarified supernatants were loaded onto a 10–40% (\(w/v\)) linear sucrose gradient, and the sucrose density gradient profile is shown here. The fractions corresponding to 21S are outlined in red. (**B**) These fractions (“a”) were pooled, while 30S subunits were retained on NiTa resin and eluted with imidazole (“b”). The flow-through was applied to streptactin resin and eluted with desthiobittion (21S), with the non-retained fraction (“c”) and the molecular weight (MW) markers shown. The bands corresponding to the ribosomal proteins bS1, uS2, uS3, uS4, uS5, and uS7 are also indicated. the 'atypical' ribosomal protein bS1, weakly associated with the 30S subunit [28], and the levels of uS3, uS5, and uS7 were reduced. In order to compare 21S protein composition with that of 30S mature subunits, we then used relative quantification using mass spectrometry-based proteomics analysis (Supplementary Figure S2). By comparing the protein ratios (21S/30S), we were able to define three groups based on their occupancies [29], (Figure 4A). The first set of proteins contains bS20 (which was an expected result, since it was used to fish out the particles), bS6, uS8, uS12, and uS15, with all of these found at just about equal stoichiometry as that of mature 30S.