Stop Codon Readthrough: Key Factors Behind Extended Proteins

Cells rely on stop codons to signal the end of protein synthesis, ensuring proteins are produced with precise lengths and functions. However, in certain cases, the translation machinery reads through these signals, resulting in extended proteins with potential biological significance. This phenomenon, known as stop codon readthrough, can be influenced by genetic, molecular, and environmental factors.

Stop Codon Types

Stop codons serve as molecular punctuation marks in the genetic code, signaling translation termination. In most organisms, three distinct stop codons—UAA, UAG, and UGA—fulfill this role by interacting with release factors. Despite their shared function, these codons differ in efficiency and susceptibility to readthrough.

Among them, UAA, the “ochre” codon, is the most efficient at terminating translation, reducing the probability of readthrough. UAG, the “amber” codon, is slightly less effective, while UGA, the “opal” codon, is the least stringent and frequently involved in programmed translational readthrough. These differences arise from molecular interactions between stop codons and release factors, as well as the broader sequence context.

In some organisms, UGA is reassigned to encode selenocysteine when accompanied by a specific RNA structure known as the selenocysteine insertion sequence (SECIS). Certain viral genomes also exploit stop codon differences to regulate protein production, enhancing adaptability and survival.

Codon Context And Surrounding Elements

Stop codon readthrough is influenced by the nucleotide sequence flanking the stop signal. The bases immediately upstream and downstream affect ribosomal dynamics, determining whether termination proceeds or an additional amino acid is incorporated. Specific nucleotide motifs can either enhance termination fidelity or promote readthrough.

A cytosine-rich sequence preceding a stop codon has been associated with increased readthrough rates, likely due to its impact on ribosomal positioning. Similarly, a purine-rich downstream context, particularly adenine and guanine, increases readthrough likelihood by influencing termination complex stability.

Secondary mRNA structures, such as stem-loops or pseudoknots, can also contribute by delaying release factor engagement, increasing the probability of near-cognate tRNA incorporation. Certain viruses, including retroviruses, use these structural elements to regulate alternative protein isoform production.

Translation Machinery Interplay

The ribosome’s ability to recognize stop codons and terminate translation depends on interactions between molecular components. Release factors decode stop codons and promote peptide chain release. In eukaryotes, eRF1 binds to all three stop codons, while eRF3 facilitates GTP hydrolysis. In prokaryotes, RF1 and RF2 perform similar roles, with RF1 recognizing UAA and UAG, and RF2 targeting UAA and UGA.

Near-cognate tRNAs, which have a single nucleotide mismatch with stop codons, can transiently enter the ribosomal A-site and occasionally incorporate an amino acid instead of terminating translation. This likelihood increases under conditions that weaken release factor activity, such as oxidative stress, viral infection, or specific cellular signals.

Ribosomal structural dynamics also influence termination efficiency. High-resolution cryo-electron microscopy studies reveal that conformational changes affect release factor binding. Mutations, post-translational modifications, and certain antibiotics, such as aminoglycosides, can disrupt normal termination, increasing readthrough. This phenomenon has been explored as a therapeutic strategy for genetic disorders caused by premature stop codons, including cystic fibrosis and Duchenne muscular dystrophy.

Occurrences In Organisms

Stop codon readthrough occurs across diverse biological systems and is sometimes a regulated mechanism expanding proteomic diversity. Viruses frequently exploit this process to generate multiple proteins from a single transcript. The retrovirus HIV-1, for example, uses readthrough to produce the Gag-Pol polyprotein, a precursor to essential viral enzymes, ensuring a precise ratio of structural and enzymatic components.

In eukaryotes, readthrough contributes to functional protein extensions. In Drosophila melanogaster, the enzyme glutamate decarboxylase undergoes partial readthrough, generating a longer variant with distinct regulatory properties. In humans, certain selenoproteins rely on programmed readthrough to incorporate selenocysteine at UGA sites, an essential process for antioxidant defense and thyroid hormone metabolism.

Functional Relevance Of Extended Proteins

Stop codon readthrough was once considered an error, but many extended proteins serve distinct biological purposes. These variants can acquire additional functional domains, alter cellular localization, or undergo unique post-translational modifications. Some extensions modulate protein stability, influencing degradation rates and overall homeostasis.

One example is the readthrough extension of vascular endothelial growth factor A (VEGFA), a key regulator of angiogenesis. Extended VEGFA isoforms exhibit altered receptor-binding properties, impacting vascular development and tissue repair. In yeast, readthrough-generated protein variants contribute to stress adaptation, allowing cells to respond dynamically to environmental changes. These findings highlight the adaptive potential of stop codon readthrough in expanding protein function.