Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA-protein cross-linking.
Understanding of eukaryotic ribosome synthesis has been slowed by a lack of structural data for the pre-ribosomal particles. We report rRNA-binding sites for six late-acting 40S ribosome synthesis factors, three of which cluster around the 3' end of the 18S rRNA in model 3D structures. Enp1 and Ltv1 were previously implicated in 'beak' structure formation during 40S maturation--and their binding sites indicate direct functions. The kinase Rio2, putative GTPase Tsr1 and dimethylase Dim1 bind sequences involved in tRNA interactions and mRNA decoding, indicating that their presence is incompatible with translation. The Dim1- and Tsr1-binding sites overlap with those of homologous Escherichia coli proteins, revealing conservation in assembly pathways. The primary binding sites for the 18S 3'-endonuclease Nob1 are distinct from its cleavage site and were unaltered by mutation of the catalytic PIN domain. Structure probing indicated that at steady state the cleavage site is likely unbound by Nob1 and flexible in the pre-rRNA. Nob1 binds before pre-rRNA cleavage, and we conclude that structural reorganization is needed to bring together the catalytic PIN domain and its target.
Granneman
Sander
S
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, Scotland, UK.
Petfalski
Elisabeth
E
Swiatkowska
Agata
A
Tollervey
David
D
D001483:Base Sequence; D001665:Binding Sites; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012337:RNA, Ribosomal, 18S / Q000737:chemistry* / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D012337:RNA, Ribosomal, 18S; D012269:Ribosomal Proteins
Mol Cell. 2005 Apr 29;18(3):319-29
15866174
Biopolymers. 2007 Sep;87(1):1-11
17514744
J Am Chem Soc. 2007 Apr 11;129(14):4144-5
17367143
Trends Biochem Sci. 2009 May;34(5):256-63
19376708
Biochemistry. 1972 Jun 20;11(13):2503-9
4339244
J Biol Chem. 2009 Dec 11;284(50):35079-91
19801658
Mol Cell. 2004 Jul 23;15(2):295-301
15260980
J Bacteriol. 2008 Apr;190(7):2537-45
18223068
Cell. 2001 Nov 2;107(3):373-86
11701127
Nat Struct Mol Biol. 2008 May;15(5):534-6
18391965
Science. 2008 Jun 13;320(5882):1465-70
18467557
Mol Cell. 2007 Apr 13;26(1):41-50
17434125
Genes Dev. 1995 Oct 15;9(20):2470-81
7590228
Mol Cell Biol. 2003 Mar;23(6):2083-95
12612080
J Cell Biol. 2009 Jun 29;185(7):1167-80
19564402
Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9613-8
19482942
RNA. 2009 Sep;15(9):1716-28
19628622
EMBO J. 2010 Jan 6;29(1):80-92
19893492
Mol Cell Biol. 2003 Mar;23(5):1798-807
12588997
Gene. 2003 Aug 14;313:17-42
12957375
J Mol Microbiol Biotechnol. 2006;11(1-2):41-52
16825789
Genes Dev. 1990 Dec;4(12A):2132-45
2269428
Nat Struct Mol Biol. 2009 May;16(5):528-33
19363482
J Mol Biol. 2005 Jan 28;345(4):681-93
15588818
Mol Cell. 2009 Nov 25;36(4):583-92
19941819
Mol Cell. 2004 Jan 30;13(2):225-39
14759368
Nature. 1999 Aug 26;400(6747):833-40
10476960
J Mol Biol. 2003 Oct 31;333(4):697-709
14568531
Nucleic Acids Res. 2003 Dec 1;31(23):6798-805
14627813
Cell Mol Life Sci. 2008 Aug;65(15):2334-59
18408888
Mol Cell. 2005 Oct 28;20(2):263-75
16246728
J Proteome Res. 2009 Jul;8(7):3390-402
19469554
Mol Cell. 2004 May 7;14(3):331-42
15125836
EMBO J. 1987 Dec 20;6(13):4169-75
3327689
EMBO J. 2004 Jun 16;23(12):2336-47
15167894
Biochemistry. 1990 Jun 26;29(25):5911-8
2116901
Genetics. 2006 Oct;174(2):679-91
16888326
Genes Dev. 2008 Aug 15;22(16):2242-55
18708582
Proc Natl Acad Sci U S A. 2009 Aug 25;106(34):14259-64
19706509
RNA. 2001 Sep;7(9):1268-83
11565749
Genes Dev. 2002 Dec 15;16(24):3142-57
12502737
Yeast. 1998 Jan 30;14(2):115-32
9483801
Cell. 2009 Sep 4;138(5):911-22
19737519
RNA. 2004 Nov;10(11):1698-701
15388878
Nature. 2006 Jun 1;441(7093):651-5
16738661
Genes Dev. 2003 Nov 15;17(22):2786-97
14600024
J Mol Biol. 1994 Aug 19;241(3):492-7
8064863
J Mol Biol. 2002 Feb 22;316(3):725-68
11866529
J Mol Biol. 2008 Aug 29;381(2):467-77
18588897
EMBO J. 2006 Nov 1;25(21):5117-25
17053788
EMBO J. 2003 Mar 17;22(6):1370-80
12628929
Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4569-74
11283351
J Struct Biol. 2007 Jan;157(1):281-7
16963278
Structure. 2001 Sep;9(9):751-8
11566123
RNA. 2004 Apr;10(4):645-56
15037774
Nat Biotechnol. 1999 Oct;17(10):1030-2
10504710