Processing of transcribed precursor ribosomal RNA (pre-rRNA) to a mature state is a conserved aspect of ribosome biogenesis in vivo. We developed an affinity-purification system to isolate and analyze in vivo–formed pre-rRNA–containing ribonucleoprotein (RNP) particles (rRNPs) from wild-type E. coli. We observed that the first processing intermediate of pre–small subunit (pre-SSU) rRNA is a platform for biogenesis. These pre-SSU–containing RNPs have differing ribosomal-protein and auxiliary factor association and rRNA folding. Each RNP lacks the proper architecture in functional regions, thus suggesting that checkpoints preclude immature subunits from entering the translational cycle. This work offers in vivo snapshots of SSU biogenesis and reveals that multiple pathways exist for the entire SSU biogenesis process in wild-type E. coli. These findings have implications for understanding SSU biogenesis in vivo and offer a general strategy for analysis of RNP biogenesis.
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We thank D. Ermolenko and E. Phizicky for critical discussions during the preparation of the manuscript as well as the members of Culver laboratory for helpful discussions and technical advice. We thank F. Hagen and the University of Rochester Proteomics Facility for MS analysis. We thank G. Salahura for technical assistance. We thank R. Green (Johns Hopkins University) for 86M (Spur) and MS2 coat-protein overexpression constructs and M. O'Connor (University of Missouri–Kansas City) for the MC338 strain. This work was supported by US National Institutes of Health grant GM62432 to G.M.C.
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Integrated supplementary information
Supplementary Figure 1 Analysis of MS2-tag insertion on growth at low temperature and incorporation into 70S ribosomes.
(a) Schematics of 16S rRNA indicating the positions of different MS2 tags in leader, trailer and mature sequence of 16S rRNA. The tags are explained in Fig. 1b in main text, in addition, tag 86L is 86nts into the Leader and between the RNase III and RNase E cleavage sites. (b) Growth of E. coli harboring different tagged and non-tagged, wild-type (WT) plasmids at non-permissive temperature (25°C). (c) Sucrose gradient sedimentation profiles of ribosomes and ribosomal subunits from wild-type strain carrying non-tagged (WT) or 105L tagged rDNA plasmid. Allele specific primer extension analysis of rRNA (for details, see Supplementary Fig. 2) from 30S and 70S ribosomal fractions collected from sucrose gradients reveals the plasmid borne 16S rRNA in each fraction and is plotted as a histogram (n = 3, standard deviation is shown as error bars).
Supplementary Figure 2 Outline for the purification of tagged SSU assembly intermediates from wild-type E. coli.
MS2CP = MS2 coat protein fused with Maltose Binding Protein (MBP). Plasmid borne 16S rRNA carry the spectinomycin resistance mutation, C1192U, which is absent in genomic operons. Allele specific primer extension of 16S rRNA purified with tags at position 105L, 11L and 20T using Cy5 labeled primer starting reverse transcription at position 1193 in the presence of ddGTP and differential stops are detected on 20% polyacrylamide gel. 16S represents mature 16S rRNA from genomic operons. Quantification shows more than 95% rRNA is plasmid borne. 1192C is chromosomally encoded while 1192U is plasmid encoded 16S rRNA.
Supplementary Figure 3 Examination of nucleotides with altered reactivity in three SSU intermediates.
Nucleotides of mature 16S rRNA with altered reactivity in the three pre-SSU complexes purified with different tags are shown. The relative changes in the nucleotide reactivity intensity of the intermediates to mature subunits are plotted on a log2 scale (see Supplementary Data Set1). The dotted lines indicate cutoff values for relative change in nucleotide reactivity to be considered. The regions involved in pseudoknot formation, intersubunit bridges and tRNA interactions are marked.
(a, b) Interface side representation of SSU for Figs. 4a and 4b respectively in the main text. (c) The three groups emerged from the hierarchical clustering analysis are plotted on the in vitro assembly map. The dotted lines divide the three groups. The circles around the r-proteins indicate the free precursor pool of that protein in the wild-type cells as determined previously37.
Modified 3′5′ RACE products obtained during the processing of 17S rRNA associated with 11L and 20T assembly intermediates when incubated with different cell extract in vitro are separated on agarose gel. Different S100 extracts prepared from Δrng strain or a strain harboring a ts allele of rne13 are compared to wild-type S100 extract. Incubation time (in mins) is shown.
Supplementary Figures 1–5 (PDF 2881 kb)
Chemical modification of 17S rRNA–containing RNPs using kethoxal (XLSX 50 kb)
Relative levels of R proteins in SSU intermediates (XLSX 15 kb)
Uncropped gels and northern blots shown in the main figure (PDF 28616 kb)
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Gupta, N., Culver, G. Multiple in vivo pathways for Escherichia coli small ribosomal subunit assembly occur on one pre-rRNA. Nat Struct Mol Biol 21, 937–943 (2014). https://doi.org/10.1038/nsmb.2887
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