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

Abstract

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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Figure 1: Affinity purification of SSU intermediates.
Figure 2: SSU intermediates purified with all three tags contain 17S rRNA as a major pre-16S rRNA species.
Figure 3: Distinct architecture of the three purified SSU assembly intermediates.
Figure 4: Multiple pathways for ribosomal-protein addition to the three intermediates.
Figure 5: Maturation of 17S rRNA can initiate either at the 5′ or 3′ end in vivo and in vitro.

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References

  1. 1

    Kaczanowska, M. & Ryden-Aulin, M. Ribosome biogenesis and the translation process in Escherichia coli. Microbiol. Mol. Biol. Rev. 71, 477–494 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2

    Kressler, D., Hurt, E. & Bassler, J. Driving ribosome assembly. Biochim. Biophys. Acta 1803, 673–683 (2010).

    CAS  PubMed  Article  Google Scholar 

  3. 3

    Borg, S. et al. Novel Salmonella typhimurium properties in host-parasite interactions. Immunol. Lett. 68, 247–249 (1999).

    CAS  PubMed  Article  Google Scholar 

  4. 4

    Björkman, J., Samuelsson, P., Andersson, D.I. & Hughes, D. Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium. Mol. Microbiol. 31, 53–58 (1999).

    PubMed  Article  Google Scholar 

  5. 5

    Phunpruch, S. et al. A role for 16S rRNA dimethyltransferase (ksgA) in intrinsic clarithromycin resistance in Mycobacterium tuberculosis. Int. J. Antimicrob. Agents 41, 548–551 (2013).

    CAS  PubMed  Article  Google Scholar 

  6. 6

    Ilina, E.N. et al. Mutation in ribosomal protein S5 leads to spectinomycin resistance in Neisseria gonorrhoeae. Front. Microbiol. 4, 186 (2013).

    PubMed  PubMed Central  Article  Google Scholar 

  7. 7

    Champney, W.S. The other target for ribosomal antibiotics: inhibition of bacterial ribosomal subunit formation. Infect. Disord. Drug Targets 6, 377–390 (2006).

    CAS  PubMed  Article  Google Scholar 

  8. 8

    Falaleeva, M. & Stamm, S. Processing of snoRNAs as a new source of regulatory non-coding RNAs: snoRNA fragments form a new class of functional RNAs. BioEssays 35, 46–54 (2013).

    CAS  PubMed  Article  Google Scholar 

  9. 9

    Srivastava, A.K. & Schlessinger, D. Mechanism and regulation of bacterial ribosomal RNA processing. Annu. Rev. Microbiol. 44, 105–129 (1990).

    CAS  PubMed  Article  Google Scholar 

  10. 10

    Kindler, P., Keil, T.U. & Hofschneider, P.H. Isolation and characterization of a ribonuclease III deficient mutant of Escherichia coli. Mol. Gen. Genet. 126, 53–59 (1973).

    CAS  PubMed  Article  Google Scholar 

  11. 11

    Young, R.A. & Steitz, J.A. Complementary sequences 1700 nucleotides apart form a ribonuclease III cleavage site in Escherichia coli ribosomal precursor RNA. Proc. Natl. Acad. Sci. USA 75, 3593–3597 (1978).

    CAS  PubMed  Article  Google Scholar 

  12. 12

    Deutscher, M.P. Maturation and degradation of ribosomal RNA in bacteria. Prog. Mol. Biol. Transl. Sci. 85, 369–391 (2009).

    CAS  PubMed  Article  Google Scholar 

  13. 13

    Li, Z., Pandit, S. & Deutscher, M.P. RNase G (CafA protein) and RNase E are both required for the 5′ maturation of 16S ribosomal RNA. EMBO J. 18, 2878–2885 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14

    Wachi, M., Umitsuki, G., Shimizu, M., Takada, A. & Nagai, K. Escherichia coli cafA gene encodes a novel RNase, designated as RNase G, involved in processing of the 5′ end of 16S rRNA. Biochem. Biophys. Res. Commun. 259, 483–488 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15

    Sulthana, S. & Deutscher, M.P. Multiple exoribonucleases catalyze maturation of the 3′ terminus of 16S ribosomal RNA (rRNA). J. Biol. Chem. 288, 12574–12579 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16

    Jacob, A.I., Kohrer, C., Davies, B.W., RajBhandary, U.L. & Walker, G.C. Conserved bacterial RNase YbeY plays key roles in 70S ribosome quality control and 16S rRNA maturation. Mol. Cell 49, 427–438 (2013).

    CAS  PubMed  Article  Google Scholar 

  17. 17

    Connolly, K. & Culver, G. Deconstructing ribosome construction. Trends Biochem. Sci. 34, 256–263 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18

    Shajani, Z., Sykes, M.T. & Williamson, J.R. Assembly of bacterial ribosomes. Annu. Rev. Biochem. 80, 501–526 (2011).

    CAS  PubMed  Article  Google Scholar 

  19. 19

    Connolly, K. & Culver, G. Overexpression of RbfA in the absence of the KsgA checkpoint results in impaired translation initiation. Mol. Microbiol. 87, 968–981 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20

    Lindahl, L. Intermediates and time kinetics of the in vivo assembly of Escherichia coli ribosomes. J. Mol. Biol. 92, 15–37 (1975).

    CAS  PubMed  Article  Google Scholar 

  21. 21

    Youngman, E.M. & Green, R. Affinity purification of in vivo–assembled ribosomes for in vitro biochemical analysis. Methods 36, 305–312 (2005).

    CAS  PubMed  Article  Google Scholar 

  22. 22

    Youngman, E.M., Brunelle, J.L., Kochaniak, A.B. & Green, R. The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release. Cell 117, 589–599 (2004).

    CAS  PubMed  Article  Google Scholar 

  23. 23

    Persaud, C. et al. Mutagenesis of the modified bases, m(5)U1939 and psi2504, in Escherichia coli 23S rRNA. Biochem. Biophys. Res. Commun. 392, 223–227 (2010).

    CAS  PubMed  Article  Google Scholar 

  24. 24

    Asai, T. et al. Construction and initial characterization of Escherichia coli strains with few or no intact chromosomal rRNA operons. J. Bacteriol. 181, 3803–3809 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Moazed, D., Stern, S. & Noller, H.F. Rapid chemical probing of conformation in 16S ribosomal RNA and 30S ribosomal subunits using primer extension. J. Mol. Biol. 187, 399–416 (1986).

    CAS  PubMed  Article  Google Scholar 

  26. 26

    Dammel, C.S. & Noller, H.F. A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly. Genes Dev. 7, 660–670 (1993).

    CAS  PubMed  Article  Google Scholar 

  27. 27

    Roy-Chaudhuri, B., Kirthi, N. & Culver, G.M. Appropriate maturation and folding of 16S rRNA during 30S subunit biogenesis are critical for translational fidelity. Proc. Natl. Acad. Sci. USA 107, 4567–4572 (2010).

    CAS  PubMed  Article  Google Scholar 

  28. 28

    Powers, T. & Noller, H.F. A functional pseudoknot in 16S ribosomal RNA. EMBO J. 10, 2203–2214 (1991).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29

    Vila, A., Viril-Farley, J. & Tapprich, W.E. Pseudoknot in the central domain of small subunit ribosomal RNA is essential for translation. Proc. Natl. Acad. Sci. USA 91, 11148–11152 (1994).

    CAS  PubMed  Article  Google Scholar 

  30. 30

    Poot, R.A., van den Worm, S.H., Pleij, C.W. & van Duin, J. Base complementarity in helix 2 of the central pseudoknot in 16S rRNA is essential for ribosome functioning. Nucleic Acids Res. 26, 549–553 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31

    Moazed, D. & Noller, H.F. Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16S rRNA. J. Mol. Biol. 211, 135–145 (1990).

    CAS  PubMed  Article  Google Scholar 

  32. 32

    Yusupova, G.Z., Yusupov, M.M., Cate, J.H. & Noller, H.F. The path of messenger RNA through the ribosome. Cell 106, 233–241 (2001).

    CAS  Article  PubMed  Google Scholar 

  33. 33

    Noller, H.F. et al. Structure of the ribosome at 5.5Å resolution and its interactions with functional ligands. Cold Spring Harb. Symp. Quant. Biol. 66, 57–66 (2001).

    CAS  PubMed  Article  Google Scholar 

  34. 34

    Schuwirth, B.S. et al. Structures of the bacterial ribosome at 3.5Å resolution. Science 310, 827–834 (2005).

    CAS  Article  PubMed  Google Scholar 

  35. 35

    Nguyenle, T., Laurberg, M., Brenowitz, M. & Noller, H.F. Following the dynamics of changes in solvent accessibility of 16S and 23S rRNA during ribosomal subunit association using synchrotron-generated hydroxyl radicals. J. Mol. Biol. 359, 1235–1248 (2006).

    CAS  PubMed  Article  Google Scholar 

  36. 36

    Culver, G.M. Assembly of the 30S ribosomal subunit. Biopolymers 68, 234–249 (2003).

    CAS  PubMed  Article  Google Scholar 

  37. 37

    Chen, S.S., Sperling, E., Silverman, J.M., Davis, J.H. & Williamson, J.R. Measuring the dynamics of E. coli ribosome biogenesis using pulse-labeling and quantitative mass spectrometry. Mol. Biosyst. 8, 3325–3334 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38

    Mulder, A.M. et al. Visualizing ribosome biogenesis: parallel assembly pathways for the 30S subunit. Science 330, 673–677 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39

    Ogle, J.M., Carter, A.P. & Ramakrishnan, V. Insights into the decoding mechanism from recent ribosome structures. Trends Biochem. Sci. 28, 259–266 (2003).

    CAS  PubMed  Article  Google Scholar 

  40. 40

    Kirthi, N., Roy-Chaudhuri, B., Kelley, T. & Culver, G.M. A novel single amino acid change in small subunit ribosomal protein S5 has profound effects on translational fidelity. RNA 12, 2080–2091 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41

    Calidas, D., Lyon, H. & Culver, G.M. The N-terminal extension of S12 influences small ribosomal subunit assembly in Escherichia coli. RNA 20, 321–330 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42

    Held, W.A., Mizushima, S. & Nomura, M. Reconstitution of Escherichia coli 30S ribosomal subunits from purified molecular components. J. Biol. Chem. 248, 5720–5730 (1973).

    CAS  PubMed  Google Scholar 

  43. 43

    Chen, S.S. & Williamson, J.R. Characterization of the ribosome biogenesis landscape in E. coli using quantitative mass spectrometry. J. Mol. Biol. 425, 767–779 (2013).

    CAS  PubMed  Article  Google Scholar 

  44. 44

    Adilakshmi, T., Bellur, D.L. & Woodson, S.A. Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly. Nature 455, 1268–1272 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45

    Yates, L.A., Norbury, C.J. & Gilbert, R.J. The long and short of microRNA. Cell 153, 516–519 (2013).

    CAS  PubMed  Article  Google Scholar 

  46. 46

    Mori, H., Dammel, C., Becker, E., Triman, K. & Noller, H.F. Single base alterations upstream of the E. coli 16S rRNA coding region result in temperature-sensitive 16S rRNA expression. Biochim. Biophys. Acta 1050, 323–327 (1990).

    CAS  PubMed  Article  Google Scholar 

  47. 47

    Balzer, M. & Wagner, R. Mutations in the leader region of ribosomal RNA operons cause structurally defective 30S ribosomes as revealed by in vivo structural probing. J. Mol. Biol. 276, 547–557 (1998).

    CAS  PubMed  Article  Google Scholar 

  48. 48

    Besançon, W. & Wagner, R. Characterization of transient RNA-RNA interactions important for the facilitated structure formation of bacterial ribosomal 16S RNA. Nucleic Acids Res. 27, 4353–4362 (1999).

    PubMed  PubMed Central  Article  Google Scholar 

  49. 49

    Jomaa, A. et al. Understanding ribosome assembly: the structure of in vivo assembled immature 30S subunits revealed by cryo-electron microscopy. RNA 17, 697–709 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50

    Leong, V., Kent, M., Jomaa, A. & Ortega, J. Escherichia coli rimM and yjeQ null strains accumulate immature 30S subunits of similar structure and protein complement. RNA 19, 789–802 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51

    Guo, Q. et al. Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process. Nucleic Acids Res. 41, 2609–2620 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52

    Clatterbuck Soper, S.F., Dator, R.P., Limbach, P.A. & Woodson, S.A. In vivo X-ray footprinting of pre-30S ribosomes reveals chaperone-dependent remodeling of late assembly intermediates. Mol. Cell 52, 506–516 (2013).

    CAS  PubMed  Article  Google Scholar 

  53. 53

    Bubunenko, M., Baker, T. & Court, D.L. Essentiality of ribosomal and transcription antitermination proteins analyzed by systematic gene replacement in Escherichia coli. J. Bacteriol. 189, 2844–2853 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. 54

    Mizushima, S. & Nomura, M. Assembly mapping of 30S ribosomal proteins from E. coli. Nature 226, 1214 (1970).

    CAS  PubMed  Article  Google Scholar 

  55. 55

    Held, W.A., Ballou, B., Mizushima, S. & Nomura, M. Assembly mapping of 30S ribosomal proteins from Escherichia coli: further studies. J. Biol. Chem. 249, 3103–3111 (1974).

    CAS  PubMed  Google Scholar 

  56. 56

    Bubunenko, M. et al. 30S ribosomal subunits can be assembled in vivo without primary binding ribosomal protein S15. RNA 12, 1229–1239 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. 57

    Adilakshmi, T., Ramaswamy, P. & Woodson, S.A. Protein-independent folding pathway of the 16S rRNA 5′ domain. J. Mol. Biol. 351, 508–519 (2005).

    CAS  PubMed  Article  Google Scholar 

  58. 58

    Dennis, P.P., Russell, A.G. & Moniz De Sa, M. Formation of the 5′ end pseudoknot in small subunit ribosomal RNA: involvement of U3-like sequences. RNA 3, 337–343 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Talkington, M.W., Siuzdak, G. & Williamson, J.R. An assembly landscape for the 30S ribosomal subunit. Nature 438, 628–632 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  60. 60

    Van Duin, J. & Wijnands, R. The function of ribosomal protein S21 in protein synthesis. Eur. J. Biochem. 118, 615–619 (1981).

    CAS  PubMed  Article  Google Scholar 

  61. 61

    Douthwaite, S., Powers, T., Lee, J.Y. & Noller, H.F. Defining the structural requirements for a helix in 23S ribosomal RNA that confers erythromycin resistance. J. Mol. Biol. 209, 655–665 (1989).

    CAS  PubMed  Article  Google Scholar 

  62. 62

    Sigmund, C.D., Ettayebi, M. & Morgan, E.A. Antibiotic resistance mutations in 16S and 23S ribosomal RNA genes of Escherichia coli. Nucleic Acids Res. 12, 4653–4663 (1984).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  63. 63

    Makosky, P.C. & Dahlberg, A.E. Spectinomycin resistance at site 1192 in 16S ribosomal RNA of E. coli: an analysis of three mutants. Biochimie 69, 885–889 (1987).

    CAS  PubMed  Article  Google Scholar 

  64. 64

    Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  65. 65

    Sun, Q., Vila-Sanjurjo, A. & O'Connor, M. Mutations in the intersubunit bridge regions of 16S rRNA affect decoding and subunit-subunit interactions on the 70S ribosome. Nucleic Acids Res. 39, 3321–3330 (2011).

    CAS  PubMed  Article  Google Scholar 

  66. 66

    LeCuyer, K.A., Behlen, L.S. & Uhlenbeck, O.C. Mutants of the bacteriophage MS2 coat protein that alter its cooperative binding to RNA. Biochemistry 34, 10600–10606 (1995).

    CAS  PubMed  Article  Google Scholar 

  67. 67

    Xu, Z. & Culver, G.M. Differential assembly of 16S rRNA domains during 30S subunit formation. RNA 16, 1990–2001 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  68. 68

    Al Refaii, A. & Alix, J.H. Ribosome biogenesis is temperature-dependent and delayed in Escherichia coli lacking the chaperones DnaK or DnaJ. Mol. Microbiol. 71, 748–762 (2009).

    CAS  PubMed  Article  Google Scholar 

  69. 69

    Xu, Z. & Culver, G.M. Chemical probing of RNA and RNA/protein complexes. Methods Enzymol. 468, 147–165 (2009).

    CAS  PubMed  Article  Google Scholar 

  70. 70

    Stern, S., Moazed, D. & Noller, H.F. Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Methods Enzymol. 164, 481–489 (1988).

    CAS  PubMed  Article  Google Scholar 

  71. 71

    Das, R., Laederach, A., Pearlman, S.M., Herschlag, D. & Altman, R.B. SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. RNA 11, 344–354 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  72. 72

    Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. 73

    Eisen, M.B., Spellman, P.T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  74. 74

    Saldanha, A.J. Java Treeview-extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).

    CAS  Article  PubMed  Google Scholar 

  75. 75

    Maki, J.A., Schnobrich, D.J. & Culver, G.M. The DnaK chaperone system facilitates 30S ribosomal subunit assembly. Mol. Cell 10, 129–138 (2002).

    CAS  PubMed  Article  Google Scholar 

  76. 76

    Connolly, K., Rife, J.P. & Culver, G. Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA. Mol. Microbiol. 70, 1062–1075 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

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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N.G. and G.M.C. designed the study and experiments and wrote the manuscript; N.G. performed experiments and analyzed the data.

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Correspondence to Gloria M Culver.

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The authors declare no competing financial interests.

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.

Supplementary Figure 4 R-protein content of the three purified SSU intermediates is distinct.

(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.

Supplementary Figure 5 Delayed processing of 17S rRNA in the absence of RNase G and RNase E.

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 information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 2881 kb)

Supplementary Data Set 1

Chemical modification of 17S rRNA–containing RNPs using kethoxal (XLSX 50 kb)

Supplementary Data Set 2

Relative levels of R proteins in SSU intermediates (XLSX 15 kb)

Supplementary Data Set 3

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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