title: Slow formation of stable complexes during coincubation of minimal rRNA and ribosomal protein S4.
abstract: Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5' domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation.
authors.affiliation: Program in Cell, Molecular and Developmental Biology and Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Megan
authors.initials: M
authors.lastname: Mayerle
authors.forename: Deepti L
authors.initials: DL
authors.lastname: Bellur
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Woodson
mesh_terms: D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
chemical_list: D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C031798:ribosomal protein S4