PRJNA503322
High-throughput profiling of TnaC sensory peptide
Translation is a fundamental biological process that makes proteins with mRNA as template. During the elongation process of translation, the nascent peptide moves through the exit tunnel within the bacterial ribosome 50S subunit. Growing body of evidences suggest that this tunnel is not only a simple passive pathway for the nascent peptide. In contrast, the interaction between the nascent peptide and the ribosome enables the regulation of many important biological processes, such as protein translation dynamics, downstream gene expression, protein folding, protein location in the cell, etc. However, the detailed molecular mechanism of this category of regulation, especially the kinetics issue during the process, remains elusive, largely due to the difficult to develop in vivo approach to monitor such process in real time.As a classical model of nascent peptide stalling, TnaC controls the downstream gene expression via a tryptophan induced ribosome-arrest mechanism. Tryptophan induced ribosome stalling can prevent the premature transcription termination of the downstream tnaA gene, thus resulting in a tryptophan dependent expression of tnaA. In this project, we presented a deep mutational scanning approach to study the kinetics of this gene expression regulation process. Briefly, we constructed a tnaC library consisting of 1,450 mutants, covering all possible single nucleotide mutations of this genetic element (codon 2~24 of 24aa TnaC peptide). Via fusing a GFP reporter downstream this tnaC variant library, we profiled the responses of all these 1,450 tnaC variants at three tryptophan concentrations (0, 100 and 500 uM). FACS is subsequently used to sort cells carrying different tnaC variants into different bins according to the GFP reporter expression. For each bin, we recovered ~100,000 cells and used NGS to dissect the abundance of each tnaC variant. Via this strategy, we constructed a comprehensive tnaC genetic map, shedding light on the key components for TnaC nascent peptide to conduct such gene regulation function. This map provides rich resources to understand the dynamics of this gene expression regulation process.Together with previous biochemical as well as structural studies, we proposed a kinetic model for this molecular mechanism consisted of two dynamic steps: the synchronization of transcription and translation, and subsequent nascent-peptide-ribosome-interaction derived translation stalling. This model demonstrates an interesting kinetics process at molecular level that coordinating the processing of biological macromolecules (RNA polymerase and ribosome) by a gene composed of only 75 nucleotides and one inducer small molecule. We think such method should also catalyze studies focusing on other nascent peptide (uORF in mammalian genome) based dynamic gene regulation process, which is actually widely adopted by organisms from bacteria to mammals to cope with signaling tasks.