Year: 2016
Type: article
Abstract: Bacteria encode multiple type II toxin-antitoxin modules that cleave ribosome-bound mRNAs in response to stress. All ribosome-dependent toxin family members structurally characterized to date adopt similar microbial RNase architectures despite possessing low sequence identities. Therefore, determining which residues are catalytically important in this specialized RNase family has been a challenge in the field. Structural studies of RelE and YoeB toxins bound to the ribosome provided significant insights but biochemical experiments with RelE were required to clearly demonstrate which residues are critical for acid-base catalysis of mRNA cleavage. Here, we solved an X-ray crystal structure of the wild-type, ribosome-dependent toxin HigB bound to the ribosome revealing potential catalytic residues proximal to the mRNA substrate. Using cell-based and biochemical assays, we further determined that HigB residues His54, Asp90, Tyr91 and His92 are critical for activity in vivo, while HigB H54A and Y91A variants have the largest effect on mRNA cleavage in vitro Comparison of X-ray crystal structures of two catalytically inactive HigB variants with 70S-HigB bound structures reveal that HigB active site residues undergo conformational rearrangements likely required for recognition of its mRNA substrate. These data support the emerging concept that ribosome-dependent toxins have diverse modes of mRNA recognition.
Source: Nucleic Acids Research
DOI: 10.1093/nar/gkw598
Institution: Emory University
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,992
Funders: National Science Foundation, U.S. Department of Energy, National Institutes of Health, Office of Science, National Institute of General Medical Sciences, National Center for Research Resources, Office of Research Infrastructure Programs, National Institutes of Health, Argonne National Laboratory
Awards: G102163711, G107546177, G1313983767, G1664757510, G2092860589, G2164036156, G2287611682, Molecular Basis of Ribosomal Frameshifting, G254657075, G262962599, G2686365521, Mechanism of HigB-mediated ribosome-dependent mRNA degradation, G3075337988, Pixel Array Detector for X-ray Crystallography, G3354192016, G3407338877, G409777406, G498139845, G513797252, G5201631574, G5607048534, G5915780612, G666778509, G6671297155, G6848031779, G6918803902, CAREER: Structure-function studies of bacterial toxin-ribosome complexes, G8059870437, TRAINING PROGRAM IN BIOCHEMISTRY, CELL AND DEVELOPMENT BIOLOGY, NE-CAT Center for Advanced Macromolecular Crystallography, G9000119994, G921583954, G969889393, G993149817
OpenAlex ID: W2464132585