Proteins form the structural and functional backbone of the cell, and any perturbation in their synthesis can disrupt normal cellular functions. The DNA blueprint is carefully read, transcribed, and translated into functional proteins through a tightly regulated process. The 'ribosome' plays a crucial role in orchestrating the translation of the messenger RNA transcript by assembling amino acids into the corresponding polypeptide sequence. Ribosomal functions beyond protein synthesis have been uncovered over the years, revealing its role not only in the synthesis of proteins but also in the regulation of the complex process through interactions with several regulatory factors and the nascent (newly synthesized) peptide itself.
Translation initiation begins with the ribosome recognizing the initiation site and catalyzing the transfer of amino acids to the growing peptide chain through elongation. However, some nascent peptides interact with the ribosomal tunnel and rearrange the internal structure, resulting in elongation stalling—known as "translation arrest."
Interestingly, translation arrest in bacterial cells is often triggered by environmental factors such as the presence/absence of specific nutrients and growth factors or inhibitory agents such as antibiotics as a mechanism to regulate the expression of downstream genes. However, ribosome arrest peptides (RAPs), which are encoded by upstream small open reading frames (sORFs) and induce translation arrest, remain largely elusive.
To bridge this knowledge gap, Dr. Yuhei Chadani, an Associate Professor at the Faculty of Environmental, Life, Natural Science and Technology, Okayama University, Japan, together with Yushin Ando (a master's student), Associate Professor Yuzuru Itoh from the University of Tokyo, and Akinao Kobo (a doctoral student) from the Institute of Science Tokyo, sought to identify and characterize RAPs from Escherichia coli (E. coli) and examine the mechanisms underlying translation arrest.
Giving further insight into their work published in Volume 16 of Nature Communications on 08 March 2025 , Dr. Chadani says, "Understanding the structural diversity of nascent peptides formed in the ribosomal tunnel and their role in translational regulation can aid the elimination of bottlenecks in protein synthesis and the development of biosensors utilizing regulatory nascent peptides."
Overexpression of TnaC, a tryptophan-dependent RAP, is known to impede cell growth and induce cytotoxicity, thus reflecting RAP activity. The researchers screened and analyzed 38 sORFs: 26 annotated and 12 putative sequences. Upon overexpression, 18 sORFs induced growth inhibition. Notably, their cytotoxic effects were not associated with the regulation of downstream genes.
In bacterial cells, cold shock proteins (CSPs) are expressed in response to the inhibition of translation elongation induced by environmental and intrinsic stressors. The researchers conducted a comparative proteomic analysis to elucidate the effects of RAP activity and stress response. TnaC and antibiotic-mediated translation arrest are associated with the expression of CSPs. Similarly, overexpression of 12 sORFs was associated with an increased expression of CSPs.
Ribosome profiling and analysis of the peptidyl-tRNA intermediates that accumulate due to translation arrest revealed that the arrest peptides 'PepNL' and 'NanCL' induced translation arrest in E. coli. The researchers further analyzed the structure of the ribosome arrested by the PepNL nascent peptide. Their findings revealed that the PepNL nascent peptide adopts a stable mini-hairpin conformation in the exit tunnel of the ribosome.
Normally, on subsequently encountering a stop codon in the transcript, peptide release factors (RF) trigger the dissociation of the peptide chain from the transfer RNA. Structural comparisons between the arrested ribosome and canonical translation termination revealed steric clashes between the nascent peptide and amino acid residues in the ribosomal RNA, leading to a rearrangement in RF2, shifting it to an inactive conformation. Notably, folding of the PepNL nascent peptide within the ribosomal tunnel does not require an arrest inducer, unlike other sensory RAPs like TnaC, and functions by recognizing the stop codon read-through as an arrest cue.
Overall, these findings reveal two previously unknown RAPs in E. coli and shed light on novel structural mechanisms underlying their regulatory roles in gene regulation and environmental adaptation.
"Our approaches to identifying PepNL and NanCL, as well as the distinct molecular mechanism of translation stalling and regulation, provide valuable insights into deciphering the hidden genetic codes within polypeptide sequences," Dr. Chadani concludes.
About Okayama University, Japan
As one of the leading universities in Japan, Okayama University aims to create and establish a new paradigm for the sustainable development of the world. Okayama University offers a wide range of academic fields, which become the basis of the integrated graduate schools. This not only allows us to conduct the most advanced and up-to-date research, but also provides an enriching educational experience.
Website: https://www.okayama-u.ac.jp/index_e.html
About Dr. Yuhei Chadani from Okayama University, Japan
Dr. Yuhei Chadani is currently an Associate Professor at the Faculty of Environmental, Life, Natural Science and Technology, Okayama University, Japan. His research focuses on decoding gene regulatory elements in peptide sequences and understanding the mechanisms of gene regulation and translation dynamics, and ribosome stabilization using bacteria and yeast model systems. He is a recipient of several awards from the Genetics Society of Japan and is affiliated with various academic societies and research groups. He has authored several research articles in his research domain.
