The Sarcin-ricin Loop of 23S RRNA is Essential for Assembly of the Functional Core of the 50S Ribosomal Subunit

Overview

Journal RNA

Specialty Molecular Biology

Date 2008 Aug 30

PMID 18755834

Citations 34

Authors

Laura Lancaster

Nicole J Lambert

Eric J Maklan

Lucas H Horan

Harry F Noller

Affiliations

Soon will be listed here.

Abstract

The sarcin-ricin loop (SRL) of 23S rRNA in the large ribosomal subunit is a factor-binding site that is essential for GTP-catalyzed steps in translation, but its precise functional role is thus far unknown. Here, we replaced the 15-nucleotide SRL with a GAAA tetraloop and affinity purified the mutant 50S subunits for functional and structural analysis in vitro. The SRL deletion caused defects in elongation-factor-dependent steps of translation and, unexpectedly, loss of EF-Tu-independent A-site tRNA binding. Detailed chemical probing analysis showed disruption of a network of rRNA tertiary interactions that hold together the 23S rRNA elements of the functional core of the 50S subunit, accompanied by loss of ribosomal protein L16. Our results reveal an influence of the SRL on the higher-order structure of the 50S subunit, with implications for its role in translation.

Citing Articles

The proline-rich antimicrobial peptide Api137 disrupts large ribosomal subunit assembly and induces misfolding.

Lauer S, Gasse J, Krizsan A, Reepmeyer M, Sprink T, Nikolay R Nat Commun. 2025; 16(1):567.

PMID: 39794318 PMC: 11723945. DOI: 10.1038/s41467-025-55836-8.

RNAMotifProfile: a graph-based approach to build RNA structural motif profiles.

Rahaman M, Zhang S NAR Genom Bioinform. 2024; 6(3):lqae128.

PMID: 39328267 PMC: 11426329. DOI: 10.1093/nargab/lqae128.

Human mtDNA-Encoded Long ncRNAs: Knotty Molecules and Complex Functions.

Bruni F Int J Mol Sci. 2024; 25(3).

PMID: 38338781 PMC: 10855489. DOI: 10.3390/ijms25031502.

Interactions between terminal ribosomal RNA helices stabilize the large ribosomal subunit.

Nissley A, Kamal T, Cate J RNA. 2023; 29(10):1500-1508.

PMID: 37419664 PMC: 10578474. DOI: 10.1261/rna.079690.123.

Interplay between Inter-Subunit Rotation of the Ribosome and Binding of Translational GTPases.

Das A, Adiletta N, Ermolenko D Int J Mol Sci. 2023; 24(8).

PMID: 37108045 PMC: 10138997. DOI: 10.3390/ijms24086878.

References

Sergiev P, Lesnyak D, Burakovsky D, Kiparisov S, Leonov A, Bogdanov A . Alteration in location of a conserved GTPase-associated center of the ribosome induced by mutagenesis influences the structure of peptidyltransferase center and activity of elongation factor G. J Biol Chem. 2005; 280(36):31882-9. DOI: 10.1074/jbc.M505670200. View

Wintermeyer W, Peske F, Beringer M, Gromadski K, Savelsbergh A, Rodnina M . Mechanisms of elongation on the ribosome: dynamics of a macromolecular machine. Biochem Soc Trans. 2004; 32(Pt 5):733-7. DOI: 10.1042/BST0320733. View

Spahn C, Blaha G, Agrawal R, Penczek P, Grassucci R, Trieber C . Localization of the ribosomal protection protein Tet(O) on the ribosome and the mechanism of tetracycline resistance. Mol Cell. 2001; 7(5):1037-45. DOI: 10.1016/s1097-2765(01)00238-6. View

Myasnikov A, Marzi S, Simonetti A, Giuliodori A, Gualerzi C, Yusupova G . Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome. Nat Struct Mol Biol. 2005; 12(12):1145-9. DOI: 10.1038/nsmb1012. View

Gao H, Zhou Z, Rawat U, Huang C, Bouakaz L, Wang C . RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors. Cell. 2007; 129(5):929-41. DOI: 10.1016/j.cell.2007.03.050. View

Chan Y, Dresios J, Wool I . A pathway for the transmission of allosteric signals in the ribosome through a network of RNA tertiary interactions. J Mol Biol. 2005; 355(5):1014-25. DOI: 10.1016/j.jmb.2005.11.037. View

Trieber C, Burkhardt N, Nierhaus K, Taylor D . Ribosomal protection from tetracycline mediated by Tet(O): Tet(O) interaction with ribosomes is GTP-dependent. Biol Chem. 1998; 379(7):847-55. DOI: 10.1515/bchm.1998.379.7.847. View

MONRO R . Catalysis of peptide bond formation by 50 S ribosomal subunits from Escherichia coli. J Mol Biol. 1967; 26(1):147-51. DOI: 10.1016/0022-2836(67)90271-9. View

Mazumder R, Chae Y, Ochoa S . Polypeptide chain initiation in E. coli: sulfhydryl groups and the function of initiation factor F2. Proc Natl Acad Sci U S A. 1969; 63(1):98-103. PMC: 534041. DOI: 10.1073/pnas.63.1.98. View

10.

Munishkin A, Wool I . The ribosome-in-pieces: binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA. Proc Natl Acad Sci U S A. 1997; 94(23):12280-4. PMC: 24907. DOI: 10.1073/pnas.94.23.12280. View

11.

Kunkel T . Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985; 82(2):488-92. PMC: 397064. DOI: 10.1073/pnas.82.2.488. View

12.

Geyl D, Bock A, Isono K . An improved method for two-dimensional gel-electrophoresis: analysis of mutationally altered ribosomal proteins of Escherichia coli. Mol Gen Genet. 1981; 181(3):309-12. DOI: 10.1007/BF00425603. View

13.

Noller H . RNA structure: reading the ribosome. Science. 2005; 309(5740):1508-14. DOI: 10.1126/science.1111771. View

14.

Selmer M, Dunham C, Murphy 4th F, Weixlbaumer A, Petry S, Kelley A . Structure of the 70S ribosome complexed with mRNA and tRNA. Science. 2006; 313(5795):1935-42. DOI: 10.1126/science.1131127. View

15.

LeCuyer K, Behlen L, Uhlenbeck O . Mutants of the bacteriophage MS2 coat protein that alter its cooperative binding to RNA. Biochemistry. 1995; 34(33):10600-6. DOI: 10.1021/bi00033a035. View

16.

Sanbonmatsu K, Joseph S, Tung C . Simulating movement of tRNA into the ribosome during decoding. Proc Natl Acad Sci U S A. 2005; 102(44):15854-9. PMC: 1266076. DOI: 10.1073/pnas.0503456102. View

17.

Karbstein K . Role of GTPases in ribosome assembly. Biopolymers. 2007; 87(1):1-11. DOI: 10.1002/bip.20762. View

18.

Korostelev A, Trakhanov S, Laurberg M, Noller H . Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements. Cell. 2006; 126(6):1065-77. DOI: 10.1016/j.cell.2006.08.032. View

19.

Moore V, Atchison R, Thomas G, Moran M, Noller H . Identification of a ribosomal protein essential for peptidyl transferase activity. Proc Natl Acad Sci U S A. 1975; 72(3):844-8. PMC: 432417. DOI: 10.1073/pnas.72.3.844. View

20.

Agrawal R, Linde J, Sengupta J, Nierhaus K, Frank J . Localization of L11 protein on the ribosome and elucidation of its involvement in EF-G-dependent translocation. J Mol Biol. 2001; 311(4):777-87. DOI: 10.1006/jmbi.2001.4907. View