Uniformity of Peptide Release Is Maintained by Methylation of Release Factors.
Termination of protein synthesis on the ribosome is catalyzed by release factors (RFs), which share a conserved glycine-glycine-glutamine (GGQ) motif. The glutamine residue is methylated in vivo, but a mechanistic understanding of its contribution to hydrolysis is lacking. Here, we show that the modification, apart from increasing the overall rate of termination on all dipeptides, substantially increases the rate of peptide release on a subset of amino acids. In the presence of unmethylated RFs, we measure rates of hydrolysis that are exceptionally slow on proline and glycine residues and approximately two orders of magnitude faster in the presence of the methylated factors. Structures of 70S ribosomes bound to methylated RF1 and RF2 reveal that the glutamine side-chain methylation packs against 23S rRNA nucleotide 2451, stabilizing the GGQ motif and placing the side-chain amide of the glutamine toward tRNA. These data provide a framework for understanding how release factor modifications impact termination.
Department of Biology, Washington University in St. Louis, Campus Box 1137, 1 Brookings Drive, St. Louis, MO 63130, USA.
William E
WE
Pierson
Biochemistry, Cell and Developmental Biology Graduate Program, Emory University School of Medicine, 1510 Clifton Road NE, Room G223, Atlanta, GA 30322, USA; Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Room G223, Atlanta, GA 30322, USA.
Eric D
ED
Hoffer
Department of Biology, Washington University in St. Louis, Campus Box 1137, 1 Brookings Drive, St. Louis, MO 63130, USA.
Hannah E
HE
Keedy
Department of Biology, Washington University in St. Louis, Campus Box 1137, 1 Brookings Drive, St. Louis, MO 63130, USA.
Carrie L
CL
Simms
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Room G223, Atlanta, GA 30322, USA. Electronic address: cmdunha@emory.edu.
Christine M
CM
Dunham
Department of Biology, Washington University in St. Louis, Campus Box 1137, 1 Brookings Drive, St. Louis, MO 63130, USA. Electronic address: hzaher@wustl.edu.
Hani S
HS
Zaher
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018388:Codon, Terminator / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008745:Methylation; D008958:Models, Molecular; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D018388:Codon, Terminator; D010454:Peptide Termination Factors; D012338:RNA, Ribosomal, 23S; D012343:RNA, Transfer
methylation; peptide release; release factor; ribosome; termination; translation
J Biol Chem. 2002 Sep 13;277(37):33825-32
12105207
Science. 2013 Jan 4;339(6115):82-5
23239623
Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
20383002
Cell. 2005 Dec 29;123(7):1255-66
16377566
J Mol Biol. 2015 May 8;427(9):1848-60
25619162
Biochemistry. 1969 Dec;8(12):5161-8
4904043
Science. 2008 Nov 7;322(5903):953-6
18988853
EMBO J. 2010 Aug 4;29(15):2577-85
20588254
J Biol Chem. 1977 Jul 10;252(13):4514-20
873902
EMBO J. 2002 Feb 15;21(4):769-78
11847124
Structure. 2013 Jul 2;21(7):1258-63
23769667
J Biol Chem. 2007 Dec 7;282(49):35638-45
17932046
Nature. 1994 Dec 15;372(6507):701-3
7990965
Proc Natl Acad Sci U S A. 2009 Jan 6;106(1):50-4
19104062
Mol Cell. 2007 Nov 9;28(3):458-67
17996709
Biochemistry. 2009 Dec 1;48(47):11296-303
19883125
Cell. 2004 May 28;117(5):589-99
15163407
J Biol Chem. 2014 Jun 20;289(25):17589-96
24798339
EMBO J. 1996 Apr 1;15(7):1696-704
8612594
Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
20124702
Nature. 2008 Aug 14;454(7206):852-7
18596689
Proc Natl Acad Sci U S A. 2010 May 11;107(19):8593-8
20421507
Proc Natl Acad Sci U S A. 2008 Dec 16;105(50):19684-9
19064930
EMBO J. 1994 Jan 1;13(1):249-57
8306967
Nat Chem Biol. 2012 Aug;8(8):695-7
22706199
Mol Cell. 2003 Jan;11(1):103-12
12535525
Cell Rep. 2014 Nov 20;9(4):1256-64
25456128
RNA. 1999 Aug;5(8):1014-20
10445876
Curr Opin Struct Biol. 2012 Dec;22(6):733-42
22999888
Proc Natl Acad Sci U S A. 1968 Oct;61(2):768-74
4879404
Science. 2006 Sep 29;313(5795):1935-42
16959973
Proc Natl Acad Sci U S A. 2000 Feb 29;97(5):2046-51
10681447
Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32
20124692
Science. 2013 Jan 4;339(6115):85-8
23239624
Nature. 2003 Jan 2;421(6918):87-90
12511960
EMBO J. 2000 Dec 15;19(24):6900-7
11118225
Mol Cell. 2007 Sep 7;27(5):758-66
17803940
EMBO J. 2011 May 06;30(12):2445-53
21552203