Complex-I-ty in aging

The role of mitochondrial complex I in aging has been studied in both C. elegans and Drosophila, where RNAi knock down of specific complex I subunits has been shown to extend lifespan. More recently, studies in Drosophila have shown that an increase in mitochondrial activity, including complex I-like activity, can also slow aging. In this review, we discuss this apparent paradox. Improved maintenance of mitochondrial activity, mitochondrial homeostasis, may be responsible for lifespan extension in both cases. Decreased electron transport chain activity caused by reducing complex I subunit expression prompts an increase in stress response signaling that leads to enhanced mitochondrial homeostasis during aging. Increased complex I activity, as well as mitochondrial biogenesis, is expected to both directly counteract the decline in mitochondrial health that occurs during aging and may also increase cellular NAD(+) levels, which have been linked to mitochondrial homeostatic mechanisms through activation of sirtuins. We suggest that manipulations that increase or decrease complex I activity both converge on improved mitochondrial homeostasis during aging, resulting in prolonged lifespan

Engineering posttranslational proofreading to discriminate nonstandard amino acids

Incorporation of nonstandard amino acids (nsAAs) leads to chemical diversification of proteins, which is an important tool for the investigation and engineering of biological processes. However, the aminoacyl-tRNA synthetases crucial for this process are polyspecific in regard to nsAAs and standard amino acids. Here, we develop a quality control system called “posttranslational proofreading” to more accurately and rapidly evaluate nsAA incorporation. We achieve this proofreading by hijacking a natural pathway of protein degradation known as the N-end rule, which regulates the lifespan of a protein based on its amino-terminal residue. We find that proteins containing certain desired N-terminal nsAAs have much longer half-lives compared with those proteins containing undesired amino acids. We use the posttranslational proofreading system to further evolve a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) variant and a tRNATyr species for improved specificity of the nsAA biphenylalanine in vitro and in vivo. Our newly evolved biphenylalanine incorporation machinery enhances the biocontainment and growth of genetically engineered Escherichia coli strains that depend on biphenylalanine incorporation. Finally, we show that our posttranslational proofreading system can be designed for incorporation of other nsAAs by rational engineering of the ClpS protein, which mediates the N-end rule. Taken together, our posttranslational proofreading system for in vivo protein sequence verification presents an alternative paradigm for molecular recognition of amino acids and is a major advance in our ability to accurately expand the genetic code

Characterizing the portability of phage-encoded homologous recombination proteins

© 2021, The Author(s), under exclusive licence to Springer Nature America, Inc. Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host’s single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions. [Figure not available: see fulltext.