Minihairpin peptide structures found to stall protein synthesis in Escherichia coli

Proteins form the structural and functional backbone of cells, and perturbations in their synthesis can disrupt normal cellular functions. DNA blueprints are carefully read, transcribed and translated into functional proteins through a tightly regulated process.

Ribosomes play an important role in regulating the translation of messenger RNA transcripts by assembling amino acids into corresponding polypeptide sequences. Ribosomal functions beyond protein synthesis have been discovered for many years, revealing not only protein synthesis, but also its role in interactions with several regulatory factors and in regulating complex processes with the new (newly synthesized) peptide itself.

Translation initiation begins when the ribosome recognizes the starting site and catalyzes the transfer of amino acids to the peptide chain that grows through extension. However, some nascent peptides interact with ribosomal tunnels, rearrange their internal structures, and stall the elongation known as “translation halt.”

Interestingly, translation arrest in bacterial cells is often caused by environmental factors such as the presence or absence of specific nutrients or growth factors, or inhibitors such as antibiotics as mechanisms that regulate the expression of downstream genes. However, ribosomal termination peptides (RAPs), which are encoded by small upstream open reading frames (SORFs), and induce translation arrest, remain largely elusive.

To bridge this knowledge gap, Dr. Yohai Chadani, Faculty of Environment, Life, Natural Science and Technology, Okayama University, Japan, Yoshin Ando (master’s student), Professor Yuzuru Ito at the University of Tokyo, and Akinao Kobo (docs) are science and science science scientists in the science and science sciences, and examines the mechanisms underlying the arrest of translation (Escherichia coli).

“Understanding the structural diversity of neogenetic peptides formed in ribosomal tunnels and their role in translation regulation can help eliminate bottlenecks in protein synthesis and develop biosensors that utilize regulatory neogenetic peptides,” Dr. Chadani said.

Overexpression of TNAC, a tryptophan-dependent RAP, is known to interfere with cell growth and induce cytotoxicity, thus reflecting RAP activity. The researchers screened and analyzed 38 Saufes: 26 annotated sequences and 12 estimated sequences. Upon overexpression, 18 sofs induced growth stunts. In particular, their cytotoxic effects were not associated with regulation of downstream genes.

In bacterial cells, cold shock proteins (CSPs) are expressed in response to inhibition of translational elongation induced by the environment and intrinsic stressors. The researchers conducted comparative proteomic analyses to elucidate the effects of RAP activity and stress responses. TNAC and antibiotic-mediated translation arrest is associated with CSP expression. Similarly, overexpression of 12 sof was associated with increased expression of CSP.

Ribosomal profiling and analysis of peptidyl-TRNA intermediates accumulated through translation arrest revealed that the stopped peptides “PEPNL” and “NANCL” led to translation arrest in E. coli.

The researchers further analyzed the structure of ribosomes arrested by the PEPNL nascent peptide. Their findings revealed that PEPNL nascent peptides employ stable minihairpin conformations in the exit tunnel of the ribosome. This study has been published in the journal Nature Communications.

Normally, subsequent encounters with a stop codon in the transcript cause the peptide release factor (RF) to dissociate the peptide chain from the transcription RNA. Structural comparisons between stopped ribosomes and standard translation termination revealed a stereocollision between the nascent peptide and amino acid residues of ribosomal RNA, leading to RF2 rearrangement and transitioned to inactive stereolithography.

In particular, folding of PEPNL nascent peptides within ribosomal tunnels does not require that they function by recognizing stop codon read-through as stop cues, unlike other sensory wraps such as TNACs.

Overall, these findings reveal two previously unknown raps in E. coli, shedding light on the novel structural mechanisms underlying the role of regulation in gene regulation and environmental adaptation.

“The approaches to identify PEPNL and NANCL, as well as the clear molecular mechanisms of translation stalling and regulation, provide valuable insights for deciphering hidden genetic codes within polypeptide sequences,” concludes Dr. Chadani.