Multiple in vivo pathways for E. coli small ribosomal subunit assembly occur on one pre-rRNA

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 particles (rRNPs) from wild-type E. coli. We observed that the first processing intermediate of pre-SSU rRNA is a platform for biogenesis. These pre-SSU containing RNPs have differing ribosomal protein and auxiliary factors association and rRNA folding. Each RNP lacks the proper architecture in functional regions 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 important implications in understanding SSU biogenesis in vivo and offer a general strategy for analysis of RNP biogenesis.


Introduction
Ribosome biogenesis, production of mature ribosomal subunits, is indispensable and essential in all living organisms. Ribosome biogenesis involves proper coordination of transcription, processing, modification and structural rearrangement of ribosomal RNA (rRNA), ribosomal protein (r-protein) synthesis, modification and association with rRNA, to form functional ribonucleoprotein complexes (RNPs) 1 . The process of forming mature rRNA by the action of several nucleases on precursor rRNA (pre-rRNA) is conserved in all domains of life; while these events in eukaryotes have been studied in detail 2 , such studies in prokaryotes have been lacking. This lack of knowledge hampers not only understanding of an essential cellular process but also, as changes in bacterial ribosome biogenesis have recently been linked to altered virulence and drug resistance in pathogens [3][4][5][6] , understanding this process is of particular importance in the discovery of new antimicrobials 7 .
Like most cellular RNAs, rRNAs are transcribed as a primary transcript, which undergoes maturation to a functional state 8 . In all domains of life, a primary rRNA transcript is processed to release fragments containing the individual rRNA species. Early pre-rRNA species contain mature rRNA sequences and additional spacer sequences at both ends (5′ and 3′) 9 . In E. coli, seven rDNA operons encode the three rRNAs [16S (SSU), 23S (Large subunit or LSU) and 5S (LSU) rRNAs]. Ribonuclease (RNase) III (a double stranded endonuclease) cleavage is believed to be the first step in the release of pre-rRNAs 10,11 . RNase III action on pre-16S rRNA resolves a pre-rRNA product,17S rRNA, with 115 nucleotides upstream of the mature 5′ end (Leader) and 33 nucleotides downstream of the mature 3′ end (Trailer) (see Fig. 1a) 12 . 5′ end maturation involves two cleavage events catalyzed by RNases E and G 13,14 . Maturation of the 3′ end of 16S rRNA is less well understood as several distinct RNases have been linked to this process 15,16 . Thus, while 16S rRNA maturation enzymes and the length of pre-rRNA processing intermediates have been identified, substrates, timing of enzyme action and coupling of 16S rRNA maturation to other SSU biogenesis events are not well understood.
An integrated understanding of critical in vivo bacterial ribosome biogenesis events is still lacking after nearly five decades of study. Mutant strains and chemical perturbations have yielded some information on the SSU biogenesis pathway and intermediates in E. coli [17][18][19] . Small populations of ribosomes undergoing biogenesis (2-5%), rapid kinetics and asynchronous transcription from rDNA loci greatly complicates identification and characterization of bacterial SSU biogenesis intermediates 20 . To overcome these deficiencies, we developed an RNA tagging and affinity purification system to isolate SSU assembly intermediates from the wild-type cells. This system allowed purification of SSU intermediates, which were essentially free of contaminating mature SSUs, thus allowing detailed characterization of the pre-SSUs. One pre-SSU rRNA (17S rRNA) was the predominant species associated with all purified assembly intermediates. Yet, these 17S rRNA containing RNPs had distinct rRNA architectures and protein composition. These findings suggest that distinct RNPs form on a 17S rRNA platform during SSU biogenesis in vivo and that different SSU biogenesis snapshots can be characterized using this strategy. While each pre-SSU RNP was distinct, they lacked mature architecture at several functional sites, thus indicating preclusion of pre-rRNPs from involvement in translation. Several independent pathways for maturation of 17S rRNA in these RNPs were observed suggesting that flux through SSU biogenesis involves multiple pathways. Additionally, these data suggest that similar analysis will be productive with most dynamic cellular RNPs. 105 nucleotides into the Leader sequence (Fig. 1b). As a control and comparison, a tag at position 86 in mature 16S rRNA (86M) was used to isolate mature SSUs 22 .
As disruption of ribosome biogenesis or rRNA maturation can result in slow growth and cold sensitivity 18,23 , we determined if the MS2 tag insertions effected growth and therefore by extension SSU biogenesis. The three MS2 rDNA tagged plasmids (105L, 11L and 20T), as the sole copy of rDNA in an E. coli strain lacking all chromosomal rDNA copies 24 , were able to complement growth at levels indistinguishable from wild-type, non-tagged constructs. These results indicate that insertion of these tags does not affect ribosome biogenesis (Fig. 1c). A second independent in vivo assay demonstrated that expression of 105L, 11L and 20T tagged rDNA plasmids did not affect growth at non-permissive temperatures; thus, these MS2 tagged rRNAs do not result in cold-sensitivity, a hallmark of ribosome biogenesis defects ( Supplementary Fig. 1b). In contrast, an rDNA plasmid with an MS2 tag at position 86L exhibited cold-sensitivity; this result indicates that defects associated with MS2 tagging of pre-16S rRNA can be observed in our assay but given this defect, further work with this construct was not undertaken. Moreover, 16S rRNA derived from plasmids expressing untagged pre-16S rRNA or the pre-rRNA tagged at positions 105L, 11L and 20T was detected in 70S ribosomes at similar levels (Supplementary Fig. 1c and data not shown). These experiments also revealed no discernible change in ribosomal subunit distribution when pre-16S rRNA contains the MS2 tag or mature 16S rRNA was plasmid derived. The complementation analysis, lack of cold sensitivity and incorporation of tagged rRNA in mature ribosomes suggest that insertion of the tags at positions 105L, 11L and 20T does not affect ribosome biogenesis. These tagged pre-16S rRNA transcripts allowed affinity purification of pre-SSU RNPs from wild-type E. coli with less than 5% contamination with untagged rRNA (Supplementary Fig. 2). Thus, this system offers a robust and largely unperturbed means of isolating pre-SSU particles from exponentially growing E. coli.

17S rRNA is a predominant pre-16S rRNA species in vivo
Examination of pre-rRNA from the purified RNPs by denaturing gel electrophoresis (Fig.  2a), Northern blotting ( Fig. 2b), modified 3′5′ RACE (Rapid amplification of cDNA ends) (Fig. 2c) and primer extension (data not shown) revealed that the predominant purified rRNA was 17S rRNA, the pre-SSU species liberated from the primary rRNA transcript by RNase III cleavage. Purification of assembly intermediates tagged at position 11L from an E. coli strain lacking RNase G, a 16S rRNA maturation enzyme, resulted in isolation of significant amounts of 16.3S rRNA (compared to the 17S rRNA) (Fig. 2d), consistent with previous results on pre-rRNA processing in this strain 12,22 . These data indicate that affinity purification can yield many pre-16S rRNA containing rRNPs and thus suggests that isolation of 17S rRNA from several tagged positions in wild-type E. coli is not due to other species being refractory to purification and suggest that 17S rRNA is a platform for assembly of pre-SSUs in the wild-type E. coli.
conformations, likely revealing different biogenesis snapshots occurring on the same 1690 nucleotide rRNA scaffold. We observed reactivity differences in both mature and leader regions of 17S rRNA (Fig. 3). Thus purification of architecturally diverse intermediates with the same length pre-rRNA could be due to availability of MS2 tags for MS2 coat protein binding as a result of distinct RNP conformations during biogenesis, i.e. the tags could be differentially available during pre-rRNA transcription, folding, processing and incremental protein binding. Notably, leader nucleotides exhibited different reactivity in the three intermediates (Fig. 3a); hence during biogenesis different conformations exist in intermediates. The reactivity of the MS2 tag nucleotides indicated that stem loop structure was properly formed, suggesting no significant interactions with the pre-rRNA molecule. Moreover, some of these changes are in agreement with prior work where the leader (from untagged 17S rRNA) interacts with mature 16S rRNA sequence 26,27 suggesting that tagged 17S rRNA can form these conformations and that tagging does not globally perturb rRNA conformation.
Additionally, many other conserved, functional nucleotides of mature 16S rRNA showed altered reactivity in the intermediates compared to mature SSUs, indicating that these 17S rRNA containing intermediates should be unable to enter the translational cycle ( Fig. 3b, Supplementary Fig. 3). Some of the observed changes occur at nucleotides involved in pseudoknots 28 . These results suggest that there are some common folding pathways and that certain regions achieve "final" conformational states early in biogenesis ( Supplementary Fig. 3). The probing data suggest that 20T tagged rRNPs most closely resembled mature SSUs while 105L tagged rRNPs were the most distinct from mature SSUs. Additionally, it was clear that the body of the pre-SSUs was more structurally similar to mature SSUs than the other domains in all three intermediates. These data reveal that the 17S rRNA containing RNPs are structurally distinct but all lack the formation of functional sites.

R-proteins are differentially associated with the pre-SSUs
To further investigate differences between the 17S rRNA containing RNPs, we examined rprotein association by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and spectral counting (Fig. 4, Supplementary Fig. 4). We used mature ribosomes, purified with an MS2 tag in the mature 16S rRNA (86M), as a normalization standard for rprotein association (Supplementary Data Set 2), allowing direct comparison of r-protein levels in intermediates to mature SSUs. No r-protein was found in any of the three 17S rRNA containing RNPs, at levels observed in mature SSUs (Fig. 4a, Supplementary Fig.  4a). R-protein, S4, which binds to the 5′ domain of 16S rRNA, corresponding to the SSU body, had approximately the same occupancy in all three intermediates. Three r-proteins, S2, S3 and S21 were highly underrepresented in all intermediates as compared to mature SSUs (Fig. 4a, ≤38%). 105L tagged rRNPs had the lowest total r-protein association compared to the other two pre-SSUs and mature SSUs. 11L and 20T tagged rRNP had distinct r-protein association. Thus, distinct r-protein composition corroborates the different conformations observed in probing experiments and further supports that each affinity purified intermediate is distinct.
To further analyze the differential association of r-proteins to purified intermediates and examine whether r-proteins associate in groups to pre-SSUs, we calculated relative protein abundance (RPA, see Methods) and performed hierarchical clustering analysis (Fig. 4b, c, Supplementary Fig. 4b). This allowed calculation of relative protein association between intermediates (not relative to mature SSUs) in an unbiased, comprehensive manner, allowing r-protein binding to 17S rRNA to be studied in detail. We observed multiple pathways for rprotein association to the body and platform of 11L and 20T tagged rRNPs. R-proteins associating with the platform, S8 and S18 were relatively more abundant in 11L tagged RNPs as compared to 20T tagged rRNPs while S15 and S11 levels were elevated in 20T tagged RNPs when compared to 11L tagged pre-SSUs (Fig. 4b, c). In addition, this analysis revealed three r-protein groups (Fig. 4b, Supplementary Fig. 4c). Group 1 largely included rproteins (r-protein S4 is an exception) enriched in 20T tagged pre-SSUs (including rproteins S15 and S11). Group II represented the r-proteins that were more abundant in 11L tagged rRNPs (includes r-proteins, S8 and S18) as compared to the other two intermediates. Interestingly, 11L tagged pre-SSUs had increased r-protein S7 association (Group II) and several other r-proteins (Group III) that bind to the SSU head. This analysis reveals multiple pathways of r-protein association and domain assembly on 17S rRNA in wild-type E. coli. Although each intermediate had distinct r-protein association, the clustering analysis indicated that overall r-protein addition is in agreement with previous in vitro and in vivo work [36][37][38] (Supplementary Fig. 4c). Interestingly, late binding r-proteins S5 (Group III) and S12 (Group II), which play an important role in translational fidelity 39 and ribosome assembly 40,41 , fall in two separate groups and were previously shown to assemble at similar rates 38,42 . However, S5 aids in the formation of the head region and was grouped with most r-proteins that bind the head (Fig. 4b) while S12 influences SSU body formation. The predominance of 17S rRNA, distinct architecture and r-protein complements in the in vivo formed intermediates suggest that 17S rRNA can be a major platform for SSU assembly in wild-type E. coli.

Independent 5′ and 3′ end maturation of 16S rRNA in vivo
Our results, and data from other groups, suggest multiple pathways for r-protein association and rRNA folding during SSU assembly in vivo 43,44 . However, whether further maturation of 17S rRNA to 16S rRNA also follows multiple or an obligatory pathway was not known. Modified 3′5′ RACE revealed a predominant 17S rRNA species purified using all three tags; however, minor, but detectable products of lengths between 17S rRNA and 16S rRNA were also observed (Fig. 2c). These minor products represented additional pre-16S rRNA maturation products (Fig. 5a, b), with either a mature 3′ end (in 105L and 11L tagged intermediates) or a mature 5′ end (in tag 20T intermediates). These data suggest that further maturation of 17S rRNA can be initiated at either the 5′ or 3′ end of 17S rRNA in wild-type E. coli. These results show that post-RNase III cleavage, 17S rRNA processing in vivo can initiate at either end and like r-protein addition and rRNA folding, rRNA maturation can follow multiple pathways.

In vitro rRNA maturation of 17S rRNA containing RNPs
The authenticity of these 17S rRNA containing intermediates was further evaluated by incubation with wild-type extracts and examination of resulting rRNA species by modified 3′5′RACE (Fig. 5c), denaturing agarose gel and Northern analysis (data not shown). The 17S rRNA associated with the three different intermediates was processed into several shorter 16S rRNAs with maturation occurring at either or both the 5′ or 3′ end. The conversion of three different purified 17S rRNA complexes into primarily 16S rRNA suggests that these RNPs are capable of further maturation. The shorter pre-16S rRNA products observed during 17S rRNA maturation to 16S rRNA in vitro were consistent with those observed in purified intermediates in vivo (compare Fig. 5c with 5a, as depicted in Fig.  5b) suggesting 17S rRNA can undergo multiple maturation pathways in vitro and the purified rRNPs are most likely on-path SSU intermediates. Delayed rRNA processing, with accumulation of pre-16S rRNA, upon incubation of these intermediates with cell extracts lacking pre-16S rRNA processing enzymes, RNase G or RNase E 13 , indicates that the processing of these purified 17S rRNA containing RNPs is defined ( Supplementary Fig. 5). These data provide evidence that these purified RNPs are bona fide assembly intermediates in wild-type E. coli.

Discussion
An RNP affinity purification strategy was used to isolate E. coli SSU assembly intermediates formed in a wild-type strain. Our data revealed that 17S pre-rRNA acts as a major scaffold for SSU biogenesis in wild-type E. coli. 17S rRNA containing RNPs formed in vivo have distinct rRNA folding and r-protein association; these findings are consistent with the multiple SSU biogenesis pathways in vivo. Moreover, we observed additional rRNA maturation events at the 3′ or 5′ ends of 17S rRNA, both in vivo and in vitro, indicating that final end maturation can follow multiple pathways. Our results suggest that there are multiple pathways for SSU formation in vivo in wild-type E. coli for all aspects of biogenesis. We have established a system that allows examination of snapshots of RNP formation in vivo and characterization of various biogenesis processes.
A requirement for processing and maturation of cellular RNAs is common throughout all kingdoms of life. Initial processing of double stranded precursor RNAs is conserved for many cellular RNAs including microRNAs and rRNAs 8,12,45 . For bacterial rRNAs, RNase III, a double stranded RNA endonuclease, likely acts early in biogenesis once transcription of pre-16S rRNA is completed and RNase III cleavage sites become available (see Fig. 1a); longer pre-rRNA transcripts, uncut by RNase III, are rarely detected in vivo 10,11 . Our data, suggesting that this RNase III cleavage product, 17S rRNA, is a platform for biogenesis and these data are consistent with results from mutant bacterial strains where 17S rRNA accumulation is observed upon disruption of most genes associated with SSU biogenesis 17,18 . Our data in wild-type E. coli along with previous data indicates that 17S rRNA is the major platform for SSU biogenesis.
Maturation of 17S rRNA and stable association of a full complement of r-proteins are likely rate limiting during SSU biogenesis. In a primary transcript, a double stranded helix formed by the leader and trailer is required for RNase III cleavage (17S rRNA production). Helical elements might persist in 17S rRNA and could be inhibitory to further 17S rRNA maturation 12 . Our data suggest that the leader can adopt different conformations at various stages of biogenesis (Fig 3a). Mutations or deletions in the leader affect SSU formation 27,46,47 and the leader can interact with mature 16S rRNA sequences at different stages of biogenesis 48 . Moreover, cryo-electron microscopy studies have failed to detect densities for unprocessed sequences [49][50][51] further suggesting that it might be a flexible and multi-structured during biogenesis and explains why there is no defined structure for pre-rRNA elements unlike mature 16S rRNA. The different conformations of pre-rRNA could influence the solvent accessibility of MS2 tag present in this region, thus allowing us to purify distinct intermediates with differently positioned tags.
Several lines of evidence suggest that the affinity purified pre-rRNPs are on-path. First, insertion of tags in precursor regions of 16S rRNA do not perturb ribosome biogenesis as these rDNA constructs were able to complement growth at wild-type untagged levels as a sole source of rDNA 24 and did not exhibit cold-sensitivity. Additionally, rRNA from rDNA plasmids containing tags in precursor regions were incorporated into 70S ribosomes at similar levels as untagged plasmid-derived rRNA (Fig. 1c, Supplementary Fig. 1). Moreover, these 17S rRNAs can be converted into 16S rRNA in vitro (Fig. 5). This maturation follows multiple pathways and is dependent on pre-16S rRNA processing enzymes ( Supplementary Fig. 5). 17S rRNA containing pre-SSU intermediates in single deletion strains ΔrsgA 49 , ΔrimM 50-52 and ΔrbfA 52 and double deletion strain ΔrbfAΔrsgA 51 showed several snapshots of SSU assembly with differential levels of r-proteins association and distinct architectures suggesting multiple pathways of SSU formation. Our chemical probing and r-protein addition data are consistent with the changes observed in intermediates accumulated in these deletion strains. However, our work offers the first analysis in wild-type E. coli growing at 37°C affording a detailed dissection of SSU biogenesis in vivo.
Chemical probing with kethoxal showed varying degrees of susceptibility of nucleotides to modification (Fig. 3, Supplementary Fig. 3). One possibility is that each intermediate is composed of a consortium of heterogeneous but related intermediates that yield differential levels of reactivity. Another possibility is that conformational changes occur at different stages, yet ultimately achieve a functional structure. This later possibility is supported by differential r-protein association to 17S rRNA in vivo (Fig. 4, Supplementary Fig. 4). In addition, strong thermodynamic dependencies observed in vitro and illustrated in the SSU assembly map do not always hold true in vivo (see Supplementary Fig. 4c). Several rproteins (S6, S9, S13, S15, S17 and S20) are non-essential in vivo 53 whereas they are indispensable for in vitro reconstitution 54,55 . R-protein, S15 is essential for assembly of the platform in vitro but the gene encoding S15 (rpsO) can be deleted in vivo and SSUs can still appropriately assemble the platform 56 . Additionally, contrary to predictions from in vitro data, the 5′ domain of 16S rRNA can fold correctly in the absence of r-proteins S17 and S20 (ref. 57). The kinetic analysis of r-protein addition in vitro as well as in vivo 38,43 and concurrent binding of r-proteins to several regions of 16S rRNA during in vitro assembly further suggest the existence of multiple assembly pathways 44 . We cannot rule out the possibility that some r-proteins are weakly bound in the intermediates and may dissociate during purification. This would suggest that these r-proteins exhibit stability differences among intermediates, which are consistent with induced-fit and multi-phasic SSU assembly 44 . While it has been shown that r-proteins have complex kinetics and that parallel pathways exist, it was unclear if these parallel pathways were dependent on different pre-rRNA scaffolds or if these pathways were obligate depending on the pre-rRNA to which they bind during the cascade. Our analysis suggests that 17S rRNA is a major pre-16S rRNA species for these multiple pathways.
We have established that intermediates have distinct conformations and r-protein association but the general functional regions in the intermediates are not in mature conformations. Sites involved in the process of translation showed altered conformation in isolated intermediates suggesting that 17S rRNA containing rRNPs are not fully functional (Fig. 3b). The nonfunctional state of pre-rRNPs is supported by r-protein data: two translation-important rproteins (S5 and S12) are differentially associated with the distinct pre-rRNPs, suggesting that while distinct pathways are followed there are similarities in the overall capacity of SSU intermediates. As bacteria lacks nuclear compartmentalization, the retention of the spacer sequences may aid in sequestration of immature SSUs from the translational cycle and act in SSU quality control, which occurs in the nucleolus in eukaryotes. A U3 boxA-like sequence in the leader has been proposed to act as a chaperone for central pseudoknot formation 58 . Specific conditions that allow 17S rRNA or intermediate pre-16S rRNA to be incorporated into 70S-like ribosomes, which has detrimental effects on translation 19,27 . Thus, the presence of 17S rRNA in RNPs with "immature" functional sites in wild-type bacteria suggests a mechanism that allows maturation and function of SSUs in the same cellular environment without deleterious consequences.
While there are many obvious differences between these purified intermediates, there are also commonalities between them. The length of the purified pre-rRNA is the most obvious, 17S rRNA (Fig. 2a). Our chemical probing data suggests that the SSU body (5′ domain of 16S rRNA), is most similar between all intermediates and mature SSUs (Fig. 3b). A common theme was revealed where most functional sites are not formed in any of these pre-SSUs. Moreover, r-protein S4 is equally represented in all three intermediates and S4 is generally regarded as an early binding protein 36,55 . In contrast, r-proteins S2, S3 and S21, which have all been shown to bind late and with slow kinetics 38,55,59 are depleted in these intermediates. The relative lack of r-protein S21 is of particular note as it is important for proper translational initiation 19,60 . Therefore, while it appears that there are multiple pathways for SSU formation in vivo, a common theme to limit functional site formation until later in biogenesis and thus likely preclude immature subunits from entering the translational cycle has emerged.
Our data indicates that ribosome biogenesis occurs via multiple pathways in vivo. Multiple in vivo pathways for r-protein addition and 17S rRNA maturation in wild-type E. coli can be observed (see Fig. 5, Supplementary Fig. 5). Additionally, there are multiple pathways for auxiliary factor action on 17S rRNA containing RNPs in vivo (N.G., G.M.C., unpublished results). Thus several SSU biogenesis processes occur simultaneously in multiple pathways on 17S rRNPs to ultimately form mature, functional subunits. This work highlights the potential of E. coli to adapt under different circumstances and challenges in antimicrobial development. This is the first work that allows concurrent analysis of rRNA processing, rprotein binding and rRNA conformational changes, yielding comprehensive analysis of SSU intermediates and assembly pathways. This work is a major advance in our understanding of in vivo formation of ribosomal subunits. Perhaps as importantly, this system provides the potential to study most other dynamic functional RNPs in vivo.

Insertion of MS2 tags in transcribed spacers of 16S rRNA
pLK35 plasmid containing an rDNA operon under the control of lambda P L promoter and ampicillin resistance marker was used for MS2 tag insertion 61

Growth analysis of rDNA plasmids containing MS2 tags
Plasmids containing the MS2 tag and a non-tagged plasmid were transformed and shuffled in a Δ7rrn strain, MC338, lacking chromosomal rDNA operons and carrying a wild-type rDNA on a plasmid, pCsacB7(ref. 24,65). LB media containing ampicillin (100µg/ml) was inoculated with overnight saturated cultures at an optical density of 0.01 at 600 nm (OD 600 ) and cells were then grown at 37°C. The growth supported by the tagged ribosomes in the absence of wild-type ribosomes was monitored by measuring OD 600 . The experiment was done in triplicates and the average OD 600 was plotted on a semi-log scale. The difference in growth rate among the strains with different tagged plasmids was calculated during the exponential phase of growth.
Plasmids containing MS2 tagged rDNA operons and pLK35 were transformed in E. coli strain MRE600 (carrying all seven chromosomal rDNA operons). Growth of these strains was monitored and analyzed at 25°C as described above.

Purification of the SSU intermediates
MRE600, carrying plasmids with MS2 tagged rDNA operon, were grown at 37°C in LB media with ampicillin (100 µg/ml) and 0.2% glucose until OD 600 reached 0.6. Cells were cooled, pelleted, resuspended in MBP binding buffer (20mM Tris-HCl [pH=7.4], 100mM KCl, 10mM MgCl 2 , 1mM EDTA) and lysed using a high pressure homogenizer. 1/100U of amplification grade DNase I was added to the lysate and incubated on ice for 10 minutes. The clarified lysate was incubated with amylose beads (New England Biolabs) which were pre-bound with MS2 fusion protein, for 2 hours on a rotating platform at 4°C. The beads were washed with 40 bead volumes of binding buffer, 20 bead volumes of washing buffer (20mM Tris-HCl [pH=7.4], 200mM KCl, 10mM MgCl 2 , 1mM EDTA) and then packed into a column. The beads were washed again with a 15 column volume of washing buffer. The tagged assembly intermediates were isolated with elution buffer (20mM Tris-HCl [pH=7.4], 500mM KCl, 10mM MgCl 2 , 1mM EDTA). Allele specific primer extension for the C1192U change in plasmid borne 16S rRNA was performed to check the purity of the isolated intermediates as described previously 28,67 .

RNA isolation and analysis
The RNA was isolated from the purified MS2 tagged RNPs as described 25 . 1µg of the RNA isolated from each elution fraction of the purified rRNA containing assembly intermediates was resolved on formaldehyde containing 2% denaturing agarose gel. The gel was visualized using a BioRad Versa Doc. Northern analysis was performed as described with the following oligos 19 : mature 16S -5′-CGCATTTCACCGCTACA-3′, leader -5′-CGTGTTCACTCTTGAGACTTGG-3′ and trailer 5′-CAAAGTACGCTTCTTTAAG-3′ Modified 3′5′ RACE was performed as outlined with the following modifications 68 . 200ng of purified RNA was used for initial circularization with T4 RNA ligase (New England Biolabs) overnight at 16°C. This step was done with and without a heat denaturation step. For heat denaturation, RNA was heated at 74°C and cooled quickly before ligation. Reverse transcription was performed with AMV reverse transcriptase (New England Biolabs) for 60 minutes followed by PCR amplification. The PCR amplified products were resolved on a 2% agarose gel and visualized using the Bio-Rad Versa Doc system. To sequence the different 3′5′ RACE products, the products were gel purified with Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare) and PCR amplified using the same primers as for initial PCR reaction. The PCR products were resolved on a 2% agarose gel to check the purity and re-purified as described above. The purified products were sequenced by GENEWIZ.

Chemical probing
Chemical probing by kethoxal was performed as detailed 25,69 . The purified rRNA containing RNPs were concentrated using Spin-X UF 500, MWCO 5000 (Corning) and buffer exchanged in RA + buffer (80mM HEPES [pH = 7.6], 20mM MgCl 2 , 330mM KCl, 0.01% Nikkol). 25 µg of mature small subunits, naked 16S rRNA and purified tagged RNPs were incubated with 0.4% kethoxal on ice for 60 minutes. Primer extensions were performed and the products were resolved on a 6% sequencing urea-polyacrylamide gel to visualize the modifications 70 . The intensity of nucleotide modifications in the mature 16S rRNA region was quantified using SAFA 71 and ImageJ 72 . The change in nucleotide intensity of purified intermediates (I I ) and naked 16S rRNA (I N ) was compared with mature small subunits (I M ).
Nucleotides showing consistent changes in at least two experiments were averaged. The relative change (RC) in reactivity was calculated as RC= log 2 (I I / I M ) or RC= log 2 (I N / I M ) and plotted as a histogram ( Supplementary Fig.3, Supplementary Data Set 1). Only values of −0.55< RC > 0.55 (or change in reactivity of greater than 50% as compared to mature SSU) for nucleotide reactivity change were considered as protected or exposed. For plotting these changes on the 3D structure, changes in the nucleotide reactivity were binned as follow: 0.55<|RC|≤1 were considered as a weak enhancement or protection and represented by small circles while |RC|>1 was considered as a strong enhancement or protection, represented by large circles. The changes were plotted on the 3D structure of SSU showing only 16S rRNA (PDB: 2AVY). The nucleotide reactivity changes in the leader were measured with ImageJ 72 and plotted as weak (small circles) and strong (large circles) changes.

Ribosomal protein analysis
The purified RNPs were concentrated and buffer exchanged into 6M Urea, 10mM DTT solution as described above. They were normalized to equal RNA concentration by measuring OD 260 with Nanodrop (Thermo Scientific). All quantitative results were obtained from three biological replicates and two technical replicates performed for each sample. LC-MS/MS analysis was performed by the URMC Proteomics Center at University of Rochester. The protein search was done with Mascot software (Matrix Science Inc) and spectral counts were calculated with ProteoIQ (Premier Biosoft). The spectral count of each protein in a sample was normalized according to the total spectral count of the sample. The spectral counts were averaged for each protein in biological and technical replicates in each intermediate and 86M to improve protein quantification (Supplementary Data Set 2). Relative levels of r-proteins associated with the intermediates were compared to those associated with 86M and the ratios were expressed as percentages. The relative levels were binned into three groups and plotted on the 3D structure of SSU as a heat map (Fig. 4a,  Supplementary Fig 4a). To calculate Relative Protein Abundance (RPA) for individual rprotein present in three intermediates, the relative levels of each r-protein (ratio of intermediate and mature SSU) among the three intermediates were median centered and log 2 transformed and followed by hierarchical clustering analysis by Cluster3.0(ref. 73) and visualized using Java TreeView 74 .

In vitro processing analysis
The purified intermediates were concentrated and buffer exchanged (20mM Tris-HCl [pH7.4], 100mM KCl, 10mM MgCl 2 , 1mM EDTA, and 6mM BME). S100 extracts 75 were prepared from MRE600 strain (wild-type) along with Δrng and rne ts strains of E. coli 13,14 . Purified intermediates were incubated with different S100 extracts (containing 100 µM ATP and GTP) at 37°C. Aliquots were taken at 0 and 60 minutes time points and the length and identity of pre-16S RNA were analyzed by the methods described above.

Ribosome sedimentation profile analysis
Wild-type strain carrying non-tagged and tagged plasmids were grown to OD 600 of 0.5 at 37°C. The ribosome profiles were obtained as described previously 76 . Various fractions were collected and ethanol precipitated for RNA extraction and analysis by allele specific primer extension as described above. Percentages of plasmid-borne rRNA were calculated using ImageJ software as described above.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Figure 1. Affinity Purification of SSU intermediates
(a) Schematic of 16S rRNA maturation. Precursor 16S rRNA is cleaved by several RNases to form mature 16S rRNA. Cleavage sites and RNases involved are as indicated. * indicate PNPase, RNase PH, RNase R, RNase II and YbeY, the RNases implicated in 3′end maturation of 16S rRNA [13][14][15][16] . During transcription, leader and trailer base pairs to form double helicalstructure encompassing entire 16S rRNA and the helix contains the site for RNase III cleavage. The numbers represent the nucleotides away from the mature ends. (b) Sites of MS2 tag (marked as MS2) insertion for tag 105L, 11L, 20T and 86M. Tag 105L is 105nts (nucleotides) into the Leader and between the RNase III and RNase E cleavage sites. Tag 11L is 11nts into the Leader and placed between RNase E and RNase G cleavage sites. 20T tag is 20nts into the Trailer. Tag 86M is 86nts into the Mature 16S rRNA and has been previously used to isolate mature, mutant ribosomes (named Spur) 21 and is used as a positive control. (c) Growth measurements of the Δ7rrn strain containing rDNA plasmid with MS2 tags at different positions and a non-tagged plasmid (WT, wild-type) as a sole source of rRNA. Average values are from three independent experiments (n = 3). Change in the growth rate of tagged plasmids as compared to non-tagged plasmid is ±5%. PCR (Reverse Transcription -Polymerase Chain Reaction) products of the ligated junctions of pre-16S rRNA purified with the three tags (modified 3′5′RACE; see Methods) resolved on a 2% agarose gel and stained with ethidium bromide. Only one fraction from the above analysis is shown; similar results are obtained for all elution fractions. Ŧ indicates heat treatment of RNA before ligation. The experiments in panel a, b and c were carried out at least four times.(d) Modified 3′5′ RACE products of pre-16S RNA purified with tag 11L from the Δrng strain of E. coli, which has been previously shown to accumulate 16.3S rRNA species, processed by RNase E at the 5′ end 13 . 16.3S rRNA is marked. Uncropped image is shown in Supplementary Data Set 3. The identity of the products is confirmed by sequencing (data not shown). The experiments were carried out two times. (a) Altered nucleotide reactivity of leader in the three purified pre-SSU intermediates, as revealed by kethoxal probing, is classified as strong (large circle) and weak (small circle). Hairpin indicates the position of the MS2 tag. (b) Nucleotides with the altered reactivity in three pre-SSU 17S rRNA containing complexes purified with different tags and mature, naked 16S rRNA (r-proteins are removed) are shown on the three-dimensional crystal structure of the SSU (all r-proteins are omitted for clarity) (PDB: 2AVY) 34 . The relative changes in the nucleotide reactivity intensity of the intermediates and naked 16S rRNA (referred as the starting point in biogenesis process) are calculated relative to mature SSUs reactivity (as baseline; see Methods, Supplementary Fig. 3, Supplementary Data Set 1) and plotted as circles. Three independent biological replicates were performed; the nucleotides showing consistent changes in at least two experiments are shown. Residues with > 50% changes relative to mature SSUs are shown. The figures are generated using PyMol (http:// www.pymol.org/).  (a) Modified 3′5′ RACE products from the three purified intermediates separated on a 2% agarose gel. Intermediate pre-16S rRNA species are indicated by an arrow and is observed in all purifications (n > 10). 17S rRNA and intermediate pre-16S rRNA are gel purified and PCR amplified and products are resolved on a 2% agarose gel marked as Product 1 and Product 2. (b) Sequencing results of the various pre-16S rRNA processing species associated with the three different purified SSU intermediates products, each product is given a designation (a-d). The experiments were carried out two times. (c) Modified 3′5′ RACE products that result from incubation of the purified 105L, 11L and 20T assembly intermediates with wild-type extracts for 0 and 60 minutes (in vitro). '+' lanes indicate addition of S100 and incubation time (in mins) is shown. The experiments were carried out three times. Uncropped image is shown in Supplementary Data Set 3. Various pre-16S rRNA species are marked as in Panel b.