Protein-independent Folding Pathway of the 16 S rRNA 5 0 Domain Tadepalli Adilakshmi 1 , Priya Ramaswamy 2 and Sarah A. Woodson 1 * 1 T.C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles St. Baltimore, MD 21218-7865 USA 2 Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218-7865, USA Evolution of the ribosome from an RNA catalyst suggests that the intrinsic folding pathway of the rRNA dictates the hierarchy of ribosome assembly. To address this possibility, we probed the tertiary folding pathway of the 5 0 domain of the Escherichia coli 16 S rRNA at 20 ms intervals using X-ray- dependent hydroxyl radical footprinting. Comparison with crystallo- graphic structures and footprinting reactions on native 30 S ribosomes showed that the RNA formed all of the predicted tertiary interactions in the absence of proteins. In 20 mM MgCl 2 , many tertiary interactions appeared within 20 ms. By contrast, interactions between H6, H15 and H17 near the spur of the 30 S ribosome evolved over several minutes, likely due to mispairing of a central helix junction. The kinetic folding pathway of the RNA corresponded to the expected order of protein binding, suggesting that the RNA folding pathway forms the basis for early steps of ribosome assembly. q 2005 Published by Elsevier Ltd. Keywords: ribosome assembly; RNA folding; hydroxyl radical footprinting; RNA structure *Corresponding author Introduction The sequence conservation and functional importance of ribosomal RNA suggests that the ribosome evolved from an early RNA catalyst. 1,2 High-resolution X-ray structures confirmed that the catalytic center of the ribosome is primarily composed of RNA, and that the ribosomal proteins mostly interact with the rRNA rather than each other. 3 Therefore, the path of ribosome assembly in modern cells may reflect self-folding of the ancestral rRNA. Pioneering experiments by Nomura and co- workers showed that the 16 S rRNA and ribosomal proteins can themselves assemble into 30 S ribo- somes. 4,5 The hierarchy of assembly, which involves the sequential association of ribosomal proteins, 6 arises from protein-induced conformational changes in the 16 S rRNA. 7 For example, Williamson and co-workers showed that binding of protein S15 to the central domain of the 16 S rRNA greatly favors the correct Mg 2C -dependent helix orientation and pre-organizes the rRNA for recognition by S11 and S18. 8–10 In general, binding of ribosomal proteins is coupled to folding of the rRNA. 11–13 As 30 S ribosome assembly strongly depends on conformational changes in the 16 S rRNA, we reasoned that the initial order of protein binding may be dictated by the kinetic folding pathway of the rRNA. To investigate this, we probed the folding pathway of the 5 0 domain of the Escherichia coli 16 S rRNA. The 5 0 domain forms a separate tertiary structure that makes up the body and spur of the small subunit. 14,15 It is the earliest region of the 16 S rRNA to fold, consistent with co-transcriptional assembly. 16 A 16 S fragment containing the 5 0 domain forms a stable ribonucleoprotein (RNP) complex with ribosomal proteins S4, S17, S20 and S16. 17 As three of the six primary assembly proteins (S4, S7, S8, S15, S17, S20) bind the 16 S 5 0 domain, it is likely to play an important role in the nucleation of 30 S structure. 18 Remarkably, we find that the 16 S 5 0 domain RNA forms all of the expected tertiary interactions in the absence of ribosomal proteins, demonstrating that the 5 0 domain folds independently. Time-resolved hydroxyl radical footprinting using a synchrotron X-ray beam shows that the RNA begins to fold within 20 ms, and that RNA tertiary interactions appear in the order corresponding to the predicted hierarchy of protein association. These results suggest that the kinetic folding pathway of the 0022-2836/$ - see front matter q 2005 Published by Elsevier Ltd. Abbreviation used: RNP, ribonucleoprotein. E-mail address of the corresponding author: swoodson@jhu.edu doi:10.1016/j.jmb.2005.06.020 J. Mol. Biol. (2005) 351, 508–519