title: Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly.
abstract: 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.
authors.affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218-2685, USA.
authors.forename: Tadepalli
authors.initials: T
authors.lastname: Adilakshmi
authors.forename: Deepti L
authors.initials: DL
authors.lastname: Bellur
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Woodson
mesh_terms: 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
chemical_list: D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins
references.citation: Nature. 2000 Sep 21;407(6802):327-39
references.pmid: 11014182
references.citation: Biopolymers. 2003 Feb;68(2):234-49
references.pmid: 12548626
references.citation: Biochimie. 1991 Jun;73(6):757-67
references.pmid: 1764521
references.citation: Science. 2005 Nov 4;310(5749):827-34
references.pmid: 16272117
references.citation: Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):7884-8
references.pmid: 8058729
references.citation: J Biol Chem. 1973 Aug 25;248(16):5720-30
references.pmid: 4579428
references.citation: Nature. 2005 Dec 1;438(7068):628-32
references.pmid: 16319883
references.citation: RNA. 2003 Feb;9(2):165-7
references.pmid: 12554857
references.citation: J Mol Biol. 1993 Jul 20;232(2):362-74
references.pmid: 8345517
references.citation: Science. 1986 Mar 28;231(4745):1562-4
references.pmid: 3513310
references.citation: Nat Struct Mol Biol. 2004 Feb;11(2):179-86
references.pmid: 14730351
references.citation: J Mol Biol. 2008 Feb 8;376(1):92-108
references.pmid: 18155048
references.citation: J Mol Biol. 1997 Oct 17;273(1):7-13
references.pmid: 9367740
references.citation: RNA. 1995 Apr;1(2):194-209
references.pmid: 7585249
references.citation: J Mol Biol. 2002 Feb 22;316(3):725-68
references.pmid: 11866529
references.citation: Methods Enzymol. 2000;317:353-68
references.pmid: 10829290
references.citation: Biochemistry. 1988 Sep 6;27(18):7051-5
references.pmid: 2461734
references.citation: Science. 1999 Sep 24;285(5436):2095-104
references.pmid: 10497122
references.citation: Cold Spring Harb Symp Quant Biol. 2001;66:567-74
references.pmid: 12762058
references.citation: J Mol Biol. 2006 May 12;358(4):1179-90
references.pmid: 16574145
references.citation: Biochemistry. 2000 Nov 7;39(44):13602-13
references.pmid: 11063598
references.citation: J Mol Biol. 2005 Aug 19;351(3):508-19
references.pmid: 16023137
references.citation: Science. 1989 May 19;244(4906):783-90
references.pmid: 2658053
references.citation: J Mol Biol. 1969 Mar 28;40(3):391-413
references.pmid: 4903714
references.citation: Curr Opin Chem Biol. 2005 Apr;9(2):127-34
references.pmid: 15811796
references.citation: J Biol Chem. 1995 Jan 20;270(3):1238-42
references.pmid: 7836385
references.citation: Biochimie. 1991 Jun;73(6):739-55
references.pmid: 1764520