Bacterial Toxin RelE: a Highly Efficient Ribonuclease with Exquisite Substrate Specificity Using Atypical Catalytic Residues

Overview

Journal Biochemistry

Specialty Biochemistry

Date 2013 Nov 21

PMID 24251350

Citations 26

Authors

Meghan A Griffin

Jared H Davis

Scott A Strobel

Affiliations

Soon will be listed here.

Abstract

The toxin RelE is a ribosome-dependent endoribonuclease implicated in diverse cellular processes, including persistence. During amino acid starvation, RelE inhibits translation by cleaving ribosomal A-site mRNA. Although RelE is structurally similar to other microbial endoribonucleases, the active-site amino acid composition differs substantially and lacks obvious candidates for general acid-base functionality. Highly conserved RelE residues (Lys52, Lys54, Arg61, Arg81, and Tyr87) surround the mRNA scissile phosphate, and specific 16S rRNA contacts further contribute to substrate positioning. We used a single-turnover kinetic assay to evaluate the catalytic importance of individual residues in the RelE active site. Within the context of the ribosome, RelE rapidly cleaves A-site mRNA at a rate similar to those of traditional ribonucleases. Single-turnover rate constants decreased between 10(2)- and 10(6)-fold for the RelE active-site mutants of Lys52, Lys54, Arg61, and Arg81. RelE may principally promote catalysis via transition-state charge stabilization and leaving-group protonation, in addition to achieving in-line substrate positioning in cooperation with the ribosome. This kinetic analysis complements structural information to provide a foundation for understanding the molecular mechanism of this atypical endoribonuclease.

Citing Articles

Computational analysis of genes with lethal knockout phenotype and prediction of essential genes in archaea.

Makarova K, Zhang C, Wolf Y, Karamycheva S, Whitaker R, Koonin E mBio. 2024; 15(2):e0309223.

PMID: 38189270 PMC: 10865827. DOI: 10.1128/mbio.03092-23.

A trailing ribosome speeds up RNA polymerase at the expense of transcript fidelity via force and allostery.

Wee L, Tong A, Ariza A, Canari-Chumpitaz C, Grob P, Nogales E Cell. 2023; 186(6):1244-1262.e34.

PMID: 36931247 PMC: 10135430. DOI: 10.1016/j.cell.2023.02.008.

Enzymatic properties of CARF-domain proteins in sp. PCC 6803.

Ding J, Schuergers N, Baehre H, Wilde A Front Microbiol. 2022; 13:1046388.

PMID: 36419420 PMC: 9676260. DOI: 10.3389/fmicb.2022.1046388.

Efficient quantitative monitoring of translational initiation by RelE cleavage.

Focht C, Strobel S Nucleic Acids Res. 2022; 50(18):e105.

PMID: 35871288 PMC: 9561414. DOI: 10.1093/nar/gkac614.

Significant Loop Motions in the SsoPTP Protein Tyrosine Phosphatase Allow for Dual General Acid Functionality.

Pinkston J, Jo J, Olsen K, Comer D, Glaittli C, Loria J Biochemistry. 2021; 60(38):2888-2901.

PMID: 34496202 PMC: 8561395. DOI: 10.1021/acs.biochem.1c00365.

References

Song H, Ismagilov R . Millisecond kinetics on a microfluidic chip using nanoliters of reagents. J Am Chem Soc. 2003; 125(47):14613-9. PMC: 1769313. DOI: 10.1021/ja0354566. View

Gotfredsen M, Gerdes K . The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family. Mol Microbiol. 1998; 29(4):1065-76. DOI: 10.1046/j.1365-2958.1998.00993.x. View

Nakano S, Chadalavada D, Bevilacqua P . General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme. Science. 2000; 287(5457):1493-7. DOI: 10.1126/science.287.5457.1493. View

Harris T, Turner G . Structural basis of perturbed pKa values of catalytic groups in enzyme active sites. IUBMB Life. 2002; 53(2):85-98. DOI: 10.1080/15216540211468. View

Shapiro R, Fox E, Riordan J . Role of lysines in human angiogenin: chemical modification and site-directed mutagenesis. Biochemistry. 1989; 28(4):1726-32. DOI: 10.1021/bi00430a045. View

Kamada K, Hanaoka F . Conformational change in the catalytic site of the ribonuclease YoeB toxin by YefM antitoxin. Mol Cell. 2005; 19(4):497-509. DOI: 10.1016/j.molcel.2005.07.004. View

Yamaguchi Y, Inouye M . Regulation of growth and death in Escherichia coli by toxin-antitoxin systems. Nat Rev Microbiol. 2011; 9(11):779-90. DOI: 10.1038/nrmicro2651. View

Bevilacqua P . Mechanistic considerations for general acid-base catalysis by RNA: revisiting the mechanism of the hairpin ribozyme. Biochemistry. 2003; 42(8):2259-65. DOI: 10.1021/bi027273m. View

Yang W . Nucleases: diversity of structure, function and mechanism. Q Rev Biophys. 2010; 44(1):1-93. PMC: 6320257. DOI: 10.1017/S0033583510000181. View

10.

Doherty M, Pealing S, Miles C, Moysey R, Taylor P, Walkinshaw M . Identification of the active site acid/base catalyst in a bacterial fumarate reductase: a kinetic and crystallographic study. Biochemistry. 2000; 39(35):10695-701. DOI: 10.1021/bi000871l. View

11.

Rodnina M, Fricke R, Kuhn L, Wintermeyer W . Codon-dependent conformational change of elongation factor Tu preceding GTP hydrolysis on the ribosome. EMBO J. 1995; 14(11):2613-9. PMC: 398375. DOI: 10.1002/j.1460-2075.1995.tb07259.x. View

12.

Loverix S, Winqvist A, Stromberg R, Steyaert J . Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond. Chem Biol. 2000; 7(8):651-8. DOI: 10.1016/s1074-5521(00)00005-3. View

13.

Inoue-Ito S, Yajima S, Fushinobu S, Nakamura S, Ogawa T, Hidaka M . Identification of the catalytic residues of sequence-specific and histidine-free ribonuclease colicin E5. J Biochem. 2012; 152(4):365-72. DOI: 10.1093/jb/mvs077. View

14.

Guglielmini J, Van Melderen L . Bacterial toxin-antitoxin systems: Translation inhibitors everywhere. Mob Genet Elements. 2012; 1(4):283-290. PMC: 3337138. DOI: 10.4161/mge.18477. View

15.

Frederiksen J, Piccirilli J . Separation of RNA phosphorothioate oligonucleotides by HPLC. Methods Enzymol. 2010; 468:289-309. DOI: 10.1016/S0076-6879(09)68014-9. View

16.

Meiering E, Serrano L, Fersht A . Effect of active site residues in barnase on activity and stability. J Mol Biol. 1992; 225(3):585-9. DOI: 10.1016/0022-2836(92)90387-y. View

17.

Sekimoto T, Matsuyama T, Fukui T, Tanizawa K . Evidence for lysine 80 as general base catalyst of leucine dehydrogenase. J Biol Chem. 1993; 268(36):27039-45. View

18.

Wang X, Wood T . Toxin-antitoxin systems influence biofilm and persister cell formation and the general stress response. Appl Environ Microbiol. 2011; 77(16):5577-83. PMC: 3165247. DOI: 10.1128/AEM.05068-11. View

19.

Ruben E, Schwans J, Sonnett M, Natarajan A, Gonzalez A, Tsai Y . Ground state destabilization from a positioned general base in the ketosteroid isomerase active site. Biochemistry. 2013; 52(6):1074-81. PMC: 3651043. DOI: 10.1021/bi301348x. View

20.

Stivers J, Nagarajan R . Probing enzyme phosphoester interactions by combining mutagenesis and chemical modification of phosphate ester oxygens. Chem Rev. 2006; 106(8):3443-67. PMC: 2729714. DOI: 10.1021/cr050317n. View