Journal
BIOCHEMISTRY
Volume 53, Issue 19, Pages 3237-3247Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi500198b
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- National Science Foundation [MCB-1121024]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1121024] Funding Source: National Science Foundation
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In cells, RNAs likely adopt numerous intermediate conformations prior to formation of functional RNA protein complexes. We used single-nucleotide resolution selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to probe the structure of Escherichia coli 16S rRNA in healthy growing bacteria. SHAPE-directed modeling indicated that the predominant steady-state RNA conformational ensemble in dividing cells had a base-paired structure different from that expected on the basis of comparative sequence analysis and high-resolution studies of the 30S ribosomal subunit. We identified the major cause of these differences by stopping ongoing in-cell transcription (in essence, an in-cell RNA structure pulse chase experiment) which caused the RNA to chase into a structure that closely resembled the expected one. Most helices that formed alternate RNA conformations under growth conditions interact directly with tertiary-binding ribosomal proteins and form a C-shape that surrounds the mRNA channel and decoding site. These in-cell experiments lead to a model in which ribosome assembly factors function as molecular struts to preorganize this intermediate and emphasize that the final stages of ribonucleoprotein assembly involve extensive protein-facilitated RNA conformational changes.
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