Abstract
Ribosome synthesis in bacteria is coupled with transcription of the pre-ribosomal RNA (pre-rRNA), which must fold and assemble with 20 or more ribosomal proteins. In vitro, the Escherichia coli pre-16S rRNA misfolds during transcription, delaying stable binding of ribosomal protein uS4 that nucleates assembly of the 16S 5' domain. Using single-molecule fluorescence microscopy, we show that the DEAD-box protein CsdA (DeaD) strongly accelerates uS4 binding by facilitating proper folding of the nascent rRNA. Unstable RNA structures are unfolded by CsdA, whereas stable RNA structures resist unwinding. We show that CsdA unfolding becomes less frequent as more ribosomal proteins add to the complex. The results demonstrate that disassembly of unstable, nascent RNA-protein complexes by chaperones fuels the search for native structure. We propose that general chaperones create a gradient of disassembly that steepens the hierarchy of proper protein addition until late assembly intermediates escape unwinding and commit to 30S maturation.