Biology subjects

Petrychenko, V.

Publications and source records attributed to Petrychenko, V..

4 recordsLinked to original sources

bioRxiv2026

Decoding the Sequence Requirements for Translation Initiation

Accurate selection of start codons by ribosomes is a fundamental determinant of proteome composition. Although the Kozak sequence--an 8-nucleotide sequence flanking the start codon--has long been viewed as the primary determinant of initiation in eukaryotes, it fails to explain the large diversity of start codon usage across transcripts. Here we combine massively parallel reporter assays, bioinformatics, machine learning, single-molecule imaging and cryo-electron microscopy to define the extended translation initiation sequence (eTIS), an [~]80-nucleotide sequence surrounding the start codon that governs initiation efficiency. A deep-learning model trained on eTIS features accurately predicts translation initiation across transcripts. Unexpectedly, we find that the Kozak sequence is not optimal for initiation as is widely presumed, and we identify the origin of this discrepancy. eTIS nucleotides that promote efficient initiation are enriched in the human transcriptome and are evolutionarily conserved, underscoring their functional importance. Biophysical and structural analyses reveal that specific eTIS residues--including the key +6 position and residues in the mRNA entry and exit channel--engage ribosomal proteins, rRNA and initiation factors to promote start codon recognition by stabilizing the ribosome at the start codon and facilitating the structural transitions required for initiation. Finally, optimization of the eTIS markedly enhances translational fidelity and protein output from therapeutic mRNAs, highlighting its practical utility. Together, these findings redefine the sequence logic of translation initiation and establish a framework for precise control of protein expression.

molecular biology↗

bioRxiv2025

Selective silencing of antibiotic-tethered ribosomes as a resistance mechanism against aminoglycosides

Antibiotic resistance is a growing threat, underscoring the need to understand the underlying mechanisms. Aminoglycosides kill bacteria by disrupting translation fidelity, leading to the synthesis of aberrant proteins. Surprisingly, mutations in fusA, a gene encoding translation elongation factor G (EF-G), frequently confer resistance, even though EF-G neither participates in mRNA decoding nor blocks aminoglycoside binding. Here, we show that EF-G resistance variants selectively slow ribosome movement along mRNA when aminoglycosides are bound. This delay increases the chance that the drug dissociates before misreading occurs. Over several elongation cycles, this selective silencing of drug-bound ribosomes prevents error cluster formation, preserving proteome and membrane integrity. As a result, fusA mutations confer resistance early in treatment by preventing self-promoted aminoglycoside uptake. Translation on drug-free ribosomes remains sufficiently rapid to sustain near-normal bacterial growth. This previously unrecognized resistance mechanism-- selective silencing of corrupted targets--reveals a novel antibiotic resistance strategy with potential therapeutic implications.

molecular biology↗

bioRxiv2021

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2-GTP-Met-tRNAiMet, and eIF3. The open 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The closed form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

molecular biology↗

bioRxiv2021

Structural mechanism of GTPase-powered ribosome-tRNA movement

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP-Pi form stabilizes ribosomal subunit rotation and induces twisting of the sarcin-ricin loop of the 23S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a cascade of rearrangements and a large-scale rotation of EF-G that exerts force on the ribosome and ultimately drives tRNA movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

molecular biology↗