Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly.
Rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. How the Escherichia coli 16S ribosomal RNA and the 20 proteins that make up the 30S ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. By providing snapshots of individual RNA and protein interactions as they emerge in real time, here we show that 30S assembly nucleates concurrently from different points along the rRNA. Time-resolved hydroxyl radical footprinting was used to map changes in the structure of the rRNA within 20 milliseconds after the addition of total 30S proteins. Helical junctions in each domain fold within 100 ms. In contrast, interactions surrounding the decoding site and between the 5', the central and the 3' domains require 2-200 seconds to form. Unexpectedly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. Although early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the rRNA.
Adilakshmi
Tadepalli
T
T.C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218-2685, USA.
Bellur
Deepti L
DL
Woodson
Sarah A
SA
D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D012336:RNA, Ribosomal, 16S / Q000235:genetics* / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017356:Synchrotrons; D014965:X-Rays
D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins
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