Biology subjects

Nadler, F.

Publications and source records attributed to Nadler, F..

3 recordsLinked to original sources

bioRxiv2022

Human mtRF1 terminates COX1 translation and its ablation induces mitochondrial ribosome-associated quality control

Translation termination requires release factors that read a STOP codon in the decoding center and subsequently facilitate the hydrolysis of the nascent peptide chain from the peptidyl tRNA within the ribosome. In human mitochondria eleven open reading frames terminate in the standard UAA or UAG STOP codon, which can be recognized by mtRF1a, the proposed major mitochondrial release factor. However, two transcripts encoding for COX1 and ND6 terminate in the non-conventional AGA or AGG codon, respectively. How translation termination is achieved in these two cases is not known. We solve this long-standing open question by showing that the non-canonical release factor mtRF1 is a specialized release factor that triggers COX1 translation termination, while mtRF1a terminates the majority of other mitochondrial translation events including the non-canonical ND6. Loss of mtRF1 leads to isolated COX deficiency and activates the mitochondrial ribosome-associated quality control accompanied by the degradation of COX1 mRNA to prevent an overload of the ribosome rescue system. Taken together, these results establish the role of mtRF1 in mitochondrial translation, which had been a mystery for almost 25 years, and lead to a comprehensive picture of translation termination in human mitochondria.

biochemistry↗

bioRxiv2021

Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

molecular biology↗

bioRxiv2018

CopySwitch - in vivo optimization of gene copy numbers for heterologous gene expression in Bacillus subtilis

1.The Gram-positive bacterium Bacillus subtilis has long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacterium Bacillus subtilis, we have developed an automatable system for the tuning of heterologous gene expression based on the hosts intrinsic natural competence and homologous recombination capabilities. By supplying our reporter strains with a linearized, low copy number plasmid containing homology regions left and right of the reporter genes and an antibiotic resistance marker, we could show an up to 3.6-fold higher gfp (green fluorescent protein) expression and up to 1.3-fold higher mPLC (mature phospholipase C) expression after successful recombination and thus circularization of our plasmid. Furthermore, the plasmid-borne gfp expression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher.

bioengineering↗