title: The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis.
abstract: During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.
authors.affiliation: a Department of Chemistry and Biochemistry; University of Bern ; Bern , Switzerland.
authors.forename: Miriam
authors.initials: M
authors.lastname: Koch
authors.forename: Nina
authors.initials: N
authors.lastname: Clementi
authors.forename: Nicola
authors.initials: N
authors.lastname: Rusca
authors.forename: Paul
authors.initials: P
authors.lastname: Vögele
authors.forename: Matthias
authors.initials: M
authors.lastname: Erlacher
authors.forename: Norbert
authors.initials: N
authors.lastname: Polacek
mesh_terms: D020029:Base Pairing; D016296:Mutagenesis; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013824:Thermus / Q000737:chemistry* / Q000166:cytology*
chemical_list: D012333:RNA, Messenger; D012343:RNA, Transfer
keywords: 23S rRNA; E-site; E-tRNA; atomic mutagenesis; ribosome; translation
references.citation: Nat Chem Biol. 2010 May;6(5):344-51
references.pmid: 20348921
references.citation: Nucleic Acids Res. 2010 Aug;38(14):4844-55
references.pmid: 20375101
references.citation: Cell. 1989 May 19;57(4):585-97
references.pmid: 2470511
references.citation: Biochim Biophys Acta. 1981 Nov 27;656(1):103-10
references.pmid: 6796121
references.citation: Science. 2000 Aug 11;289(5481):920-30
references.pmid: 10937990
references.citation: Nat Struct Mol Biol. 2013 May;20(5):582-8
references.pmid: 23542154
references.citation: J Mol Biol. 1988 Oct 5;203(3):699-705
references.pmid: 2463367
references.citation: J Biol Chem. 1986 Nov 5;261(31):14506-14
references.pmid: 3533922
references.citation: EMBO J. 1983;2(8):1351-6
references.pmid: 10872330
references.citation: RNA. 2004 May;10(5):805-12
references.pmid: 15100436
references.citation: J Biol Chem. 1995 Sep 1;270(35):20473-8
references.pmid: 7657623
references.citation: Eur J Biochem. 1982 Apr;123(3):643-6
references.pmid: 6176448
references.citation: Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11120-5
references.pmid: 11562497
references.citation: Nat Struct Mol Biol. 2010 May;17(5):555-60
references.pmid: 20400952
references.citation: Biochemistry. 1986 Jun 3;25(11):3245-55
references.pmid: 3524675
references.citation: RNA. 1998 Oct;4(10):1282-94
references.pmid: 9769102
references.citation: Cell. 2004 Jul 9;118(1):45-55
references.pmid: 15242643
references.citation: J Am Chem Soc. 2006 Apr 5;128(13):4453-9
references.pmid: 16569023
references.citation: FEBS Lett. 1983 Jun 27;157(1):91-4
references.pmid: 6345196
references.citation: Nature. 1989 Nov 9;342(6246):142-8
references.pmid: 2682263
references.citation: Eur J Biochem. 1991 Jun 15;198(3):713-22
references.pmid: 1904819
references.citation: J Biol Chem. 1986 Jul 15;261(20):9133-9
references.pmid: 2424904
references.citation: Science. 2003 May 16;300(5622):1159-62
references.pmid: 12750524
references.citation: Nucleic Acids Res. 2005;33(18):6048-56
references.pmid: 16243787
references.citation: Science. 1999 Sep 24;285(5436):2095-104
references.pmid: 10497122
references.citation: Nat Protoc. 2011 May;6(5):580-92
references.pmid: 21527916
references.citation: J Biol Chem. 2012 Aug 3;287(32):27049
references.pmid: 22865895
references.citation: Antimicrob Agents Chemother. 2009 Dec;53(12):5010-4
references.pmid: 19738021
references.citation: Nat Rev Microbiol. 2014 Feb;12(2):89-100
references.pmid: 24362468
references.citation: Nat Rev Microbiol. 2005 Nov;3(11):870-81
references.pmid: 16261170
references.citation: Nucleic Acids Res. 2005;33(5):1618-27
references.pmid: 15767286
references.citation: RNA. 2003 Nov;9(11):1345-52
references.pmid: 14561884
references.citation: Proc Natl Acad Sci U S A. 1981 Sep;78(9):5310-4
references.pmid: 7029532
references.citation: Crit Rev Biochem Mol Biol. 2005 Sep-Oct;40(5):285-311
references.pmid: 16257828
references.citation: Annu Rev Biochem. 2013;82:203-36
references.pmid: 23746255
references.citation: Science. 2001 May 4;292(5518):883-96
references.pmid: 11283358
references.citation: J Mol Biol. 2007 Apr 13;367(5):1471-9
references.pmid: 17321546
references.citation: J Biol Chem. 1969 Mar 25;244(6):1533-9
references.pmid: 4886309
references.citation: BMC Bioinformatics. 2002;3:2
references.pmid: 11869452
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10715-20
references.pmid: 18667704
references.citation: Curr Protein Pept Sci. 2002 Feb;3(1):1-53
references.pmid: 12370010
references.citation: RNA Biol. 2008 Jan-Mar;5(1):5-12
references.pmid: 18388484
references.citation: Chem Biol. 2008 May;15(5):485-92
references.pmid: 18439847
references.citation: Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12183-8
references.pmid: 8901554
references.citation: Nucleic Acids Res. 1994 Feb 11;22(3):279-84
references.pmid: 8127663
references.citation: J Mol Biol. 2008 Jun 27;380(1):193-205
references.pmid: 18508080
references.citation: Cell. 2006 Sep 22;126(6):1065-77
references.pmid: 16962654
references.citation: RNA. 2007 Sep;13(9):1483-91
references.pmid: 17660276
references.citation: Methods Mol Biol. 2012;848:215-26
references.pmid: 22315072
references.citation: Science. 2006 Sep 29;313(5795):1935-42
references.pmid: 16959973
references.citation: J Biol Chem. 1986 Jul 15;261(20):9140-3
references.pmid: 2424905
references.citation: RNA. 2009 Feb;15(2):255-65
references.pmid: 19095617
references.citation: FEBS Lett. 1971 Oct 1;17(2):324-326
references.pmid: 11946059
references.citation: EMBO J. 1990 Dec;9(13):4527-33
references.pmid: 2265616
references.citation: PLoS One. 2013;8(3):e58829
references.pmid: 23527033
references.citation: J Biol Chem. 2012 Apr 6;287(15):11642-8
references.pmid: 22378789
references.citation: RNA. 2001 Jan;7(1):54-63
references.pmid: 11214181
references.citation: Proc Natl Acad Sci U S A. 2008 Jul 8;105(27):9192-7
references.pmid: 18591673
references.citation: Cell Mol Life Sci. 2006 Dec;63(23):2725-37
references.pmid: 17013564