A minimized rRNA-binding site for ribosomal protein S4 and its implications for 30S assembly.
Primary ribosomal protein S4 is essential for 30S ribosome biogenesis in eubacteria, because it nucleates subunit assembly and helps coordinate assembly with the synthesis of its rRNA and protein components. S4 binds a five-helix junction (5WJ) that bridges the 5' and 3' ends of the 16S 5' domain. To delineate which nucleotides contribute to S4 recognition, sequential deletions of the 16S 5' domain were tested in competitive S4-binding assays based on electrophoretic mobility shifts. S4 binds the minimal 5WJ RNA containing just the five-helix junction as well or better than with affinity comparable to or better than the 5' domain or native 16S rRNA. Internal deletions and point mutations demonstrated that helices 3, 4, 16 and residues at the helix junctions are necessary for S4 binding, while the conserved helix 18 pseudoknot is dispensable. Hydroxyl radical footprinting and chemical base modification showed that S4 makes the same interactions with minimal rRNA substrates as with the native 16S rRNA, but the minimal substrates are more pre-organized for binding S4. Together, these results suggest that favorable interactions with S4 offset the energetic penalty for folding the 16S rRNA.
Bellur
Deepti L
DL
Program in Cell, Molecular and Developmental Biology and Biophysics, Johns Hopkins University, Baltimore, MD 21218-2685, USA.
Woodson
Sarah A
SA
D001665:Binding Sites; D001667:Binding, Competitive; D004926:Escherichia coli / Q000235:genetics; D011485:Protein Binding; D023302:Protein Footprinting; D017434:Protein Structure, Tertiary; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism
D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; C031798:ribosomal protein S4