Copper catalyzed cycloaddition for the synthesis of non isomerisable 2' and 3'-regioisomers of arg-tRNAarg.

In this report, non-isomerisable analogs of arginine tRNA (Arg-triazole-tRNA) have been synthesized as tools to study tRNA-dependent aminoacyl-transferases. The synthesis involves the incorporation of 1,4 substituted-1,2,3 triazole ring to mimic the ester bond that connects the amino acid to the terminal adenosine in the natural substrate. The synthetic procedure includes (i) a coupling between 2'- or 3'-azido-adenosine derivatives and a cytidine phosphoramidite to access dinucleotide molecules, (ii) Cu-catalyzed cycloaddition reactions between 2'- or 3'-azido dinucleotide in the presence of an alkyne molecule mimicking the arginine, providing the corresponding Arg-triazole-dinucleotides, (iii) enzymatic phosphorylation of the 5'-end extremity of the Arg-triazole-dinucleotides with a polynucleotide kinase, and (iv) enzymatic ligation of the 5'-phosphorylated dinucleotides with a 23-nt RNA micro helix that mimics the acceptor arm of arg-tRNA or with a full tRNAarg. Characterization of nucleoside and nucleotide compounds involved MS spectrometry, 1H, 13C and 31P NMR analysis. This strategy allows to obtain the pair of the two stable regioisomers of arg-tRNA analogs (2' and 3') which are instrumental to explore the regiospecificity of arginyl transferases enzyme. In our study, a first binding assay of the arg-tRNA micro helix with the Arginyl-tRNA-protein transferase 1 (ATE1) was performed by gel shift assays.

The structural basis of tRNA recognition by arginyl-tRNA-protein transferase.

Arginyl-tRNA-protein transferase 1 (ATE1) is a master regulator of protein homeostasis, stress response, cytoskeleton maintenance, and cell migration. The diverse functions of ATE1 arise from its unique enzymatic activity to covalently attach an arginine onto its protein substrates in a tRNA-dependent manner. However, how ATE1 (and other aminoacyl-tRNA transferases) hijacks tRNA from the highly efficient ribosomal protein synthesis pathways and catalyzes the arginylation reaction remains a mystery. Here, we describe the three-dimensional structures of Saccharomyces cerevisiae ATE1 with and without its tRNA cofactor. Importantly, the putative substrate binding domain of ATE1 adopts a previously uncharacterized fold that contains an atypical zinc-binding site critical for ATE1 stability and function. The unique recognition of tRNAArg by ATE1 is coordinated through interactions with the major groove of the acceptor arm of tRNA. Binding of tRNA induces conformational changes in ATE1 that helps explain the mechanism of substrate arginylation.

Insight into the AcrAB-TolC Complex Assembly Process Learned from Competition Studies.

The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called "dominant negative" effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.

Accessibility from the Cytoplasm Is Critical for ssrA Tag-Mediated Degradation of Integral Membrane Proteins by ClpXP Protease.

The AAA+ protease ClpXP has long been established as the cellular rescue system that degrades ssrA-tagged proteins resulting from stalled ribosomes. Until recently, in all of these studies soluble proteins were used as model substrates, since the ClpXP complex and the related adapter SspB are all cytosolic proteins. In a previous study, we found that the introduction of an ssrA tag can facilitate complete degradation of a large and stable trimeric integral membrane protein AcrB, which is the first reported example of a membrane protein substrate. To investigate the mechanism of degradation of a membrane protein by a soluble protein complex, we experimented with the truncation of the C-terminal tail of AcrB. We found that the C-terminal tail is important for degradation, as systematic truncation of the tail diminished degradation. Thus, we hypothesize that membrane proteins need a cytosolic tail/domain for ClpXP-SspB to latch on to initiate degradation. To test this hypothesis, we introduced the ssrA tag at the C-terminal of several membrane proteins, including AqpZ, YiiP, YajR, as well as their truncation fragments, and examined their degradation. We found that the ssrA-facilitated degradation of membrane proteins by ClpXP-SspB depends on the presence of a CT tail or domain, which is critical for accessibility of the tag by ClpXP-SspB. When the ssrA tag is not well-exposed to the cytosol, FtsH can access and degrade the tagged protein, given that the substrate protein is metastable.