Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity

Most bacteria use a two-step indirect pathway to aminoacylate tRNA Gln and tRNA Asn , despite the fact that the indirect pathway consumes more energy and is error prone. We have previously shown that the higher protein synthesis errors from this indirect pathway in mycobacteria allow adaptation to hostile environments such as antibiotic treatment through generation of novel alternate proteins not coded by the genome.

Clinically relevant mutations of mycobacterial GatCAB inform regulation of translational fidelity

Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error-prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampicin tolerance. However, the mechanisms by which these clinically-derived mutations in gatA impact GatCAB function was unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains, but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity.

Eukaryotic protein uS19: a component of the decoding site of ribosomes and a player in human diseases

Proteins belonging to the universal ribosomal protein (rp) uS19 family are constituents of small ribosomal subunits, and their conserved globular parts are involved in the formation of the head of these subunits. The eukaryotic rp uS19 (previously known as S15) comprises a C-terminal extension that has no homology in the bacterial counterparts. This extension is directly implicated in the formation of the ribosomal decoding site and thereby affects translational fidelity in a manner that has no analogy in bacterial ribosomes. Another eukaryote-specific feature of rp uS19 is its essential participance in the 40S subunit maturation due to the interactions with the subunit assembly factors required for the nuclear exit of pre-40S particles. Beyond properties related to the translation machinery, eukaryotic rp uS19 has an extra-ribosomal function concerned with its direct involvement in the regulation of the activity of an important tumor suppressor p53 in the Mdm2/Mdmx-p53 pathway. Mutations in the RPS15 gene encoding rp uS19 are linked to diseases (Diamond Blackfan anemia, chronic lymphocytic leukemia and Parkinson's disease) caused either by defects in the ribosome biogenesis or disturbances in the functioning of ribosomes containing mutant rp uS19, likely due to the changed translational fidelity. Here, we review currently available data on the involvement of rp uS19 in the operation of the translational machinery and in the maturation of 40S subunits, on its extra-ribosomal function, and on relationships between mutations in the RPS15 gene and certain human diseases.

Conformational communication mediates the reset step in t6A biosynthesis

The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

Altered patterns of global protein synthesis and translational fidelity in RPS15-mutated chronic lymphocytic leukemia

Genomic studies have recently identified RPS15 as a new driver gene in aggressive and chemorefractory cases of chronic lymphocytic leukemia (CLL). RPS15 encodes a ribosomal protein whose conserved C-terminal domain extends into the decoding center of the ribosome. We demonstrate that mutations in highly conserved residues of this domain affect protein stability, by increasing its ubiquitin-mediated degradation, and cell-proliferation rates. On the other hand, we show that mutated RPS15 can be loaded into the ribosomes, directly impacting on global protein synthesis and/or translational fidelity in a mutation-specific manner. Quantitative mass spectrometry analyses suggest that RPS15 variants may induce additional alterations in the translational machinery, as well as a metabolic shift at the proteome level in HEK293T and MEC-1 cells. These results indicate that CLL-related RPS15 mutations might act following patterns known for other ribosomal diseases, likely switching from a hypo- to a hyperproliferative phenotype driven by mutated ribosomes. In this scenario, loss of translational fidelity causing altered cell proteostasis can be proposed as a new molecular mechanism involved in CLL pathobiology.

Not so lost in translation: RPS15 mutations in CLL

In this issue of Blood, Bretones et al expand knowledge of the functional consequences of recurrent mutations in RPS15, a gene that encodes a ribosomal protein of the 40S subunit and is enriched in patients with clinically aggressive chronic lymphocytic leukemia (CLL).1 By transfecting RPS15 mutants and applying different technologies to assess ribosome activity and efficiency in combination with high-throughput proteome profiling, they were able to demonstrate reduced half-life of RPS15, impaired translational fidelity, and changes in the expressed proteome in mutant vs wild-type RPS15.

Studies on Aminoglycoside Susceptibility Identify a Novel Function of KsgA To Secure Translational Fidelity during Antibiotic Stress

ABSTRACTAntibiotic resistance has become a global crisis. Studies on the mechanism of bacterial tolerance to antibiotics will not only increase our conceptual understanding of bacterial death but also provide potential targets for novel inhibitors. We screened a mutant library containing a full set of in-frame deletion mutants ofEscherichia coliK-12 and identified 140 genes that possibly contribute to gentamicin tolerance. The deletion ofksgAincreased the inhibition and killing potency against mid-log-phase bacteria by aminoglycosides. Initially identified as a 16S rRNA methyltransferase, KsgA also has additional functions as a ribosomal biogenesis factor and a DNA glycosylase. We found that the methyltransferase activity of KsgA is responsible for the tolerance, as demonstrated by a site-directed mutagenesis analysis. In contrast to the mechanism for cold sensitivity, the decreased tolerance to aminoglycoside is not related to the failure of ribosomal biogenesis. Furthermore, the DNA glycosylase activity of KsgA contributes minimally to kanamycin tolerance. Importantly, we discovered that KsgA secures protein translational fidelity upon kanamycin killing, in contrast to its role during cold stress and kasugamycin treatment. The results suggest that the compromise in protein translational fidelity in the absence of KsgA is the root cause of an increased sensitivity to a bactericidal aminoglycoside. In addition, KsgA in the pathogenicAcinetobacter baumanniicontributes not only to the tolerance against aminoglycoside killing but also to virulence in the host, warranting its potential application as a target for inhibitors that potentiate aminoglycoside therapeutic killing as well as disarm bacterial virulence simultaneously.