Fluorescence-based approach to study mRNA mutations in genetic disorders
Fluorescence-based approach to study mRNA mutations in genetic disorders
luorescence spectroscopy methods for investigating the process of termination-codon recognition in genetic disorders: Many genetic disorders are caused by mutations which introduce premature termination codons (so-called nonsense mutations) into a protein-encoding gene, causing premature termination of translation of an mRNA template containing the premature termination codon and resulting in the production of truncated, non-functional ...
New York, NY, United States
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Background

Fluorescence spectroscopy methods for investigating the process of termination-codon recognition in genetic disorders: Many genetic disorders are caused by mutations which introduce premature termination codons (so-called nonsense mutations) into a protein-encoding gene, causing premature termination of translation of an mRNA template containing the premature termination codon and resulting in the production of truncated, non-functional proteins. In addition, detection of premature termination codons often triggers an mRNA quality control pathway, known as nonsense-mediated mRNA decay, that rapidly and specifically degrades mRNAs harboring premature termination codons. The overall result of these cellular responses to premature termination codons is a severe downregulation in the expression of specific proteins, some of which may be essential to the viability of the cell and/or the organism. Human diseases in which nonsense mutations are known to be among the causes include cystic fibrosis, Duchenne muscular dystrophy (DMD), beta thalassaemia, Hurler syndrome and numerous cancers including a widespread form of breast cancer. 

Class I release factors (RFs) are the cellular proteins that specifically bind to the ribosome in response to a termination codon on the mRNA template and trigger the hydrolysis, or release, of the nascent polypeptide from the ribosome. Despite decades of study, the mechanism through which the ribosome decodes termination codons using RFs and couples termination-codon recognition to hydrolysis of the nascent polypeptide remains elusive. Even less is known about the mechanism through which the ribosome uses RFs and additional accessory factors to specifically detect premature termination codons and trigger the non-sense mediated mRNA decay pathway. Although numerous other ribosomal substrates have been previously labeled with fluorescent probes, RFs have not been labeled with fluorescent probes; and thus a convenient, fluorescence-based approach for investigating translation termination has not been previously established. 

Escherichia coli-based model to study ribosome-catalyzed protein synthesis in genetic disorders: Using a highly-purified, in vitro, Escherichia coli-based model system for studies of ribosome-catalyzed protein synthesis, the researchers have developed a number of complementary strategies that report on the substrate selection and activity of RFs. Using fluorescently-labeled tRNAs and RFs, ensemble-based or single-molecule fluorescence spectroscopy methods are employed for investigating the processes of termination-codon recognition and nascent-polypeptide release from the ribosome. This is the first example in which RFs are labeled with fluorescent probes and a fluorescence-based approach is used to study translation termination. Thus, this technology provides a number of unique observables to probe the termination reaction. 

Applications: • This technology offers a novel system for sensitively probing the translation termination reaction • This technology can be extended and co-opted for high-throughput approaches to screen novel antibiotic drugs designed to specifically inhibit translation termination in prokaryotic cells (i.e. by specifically targeting the interaction between prokaryotic RFs and the prokaryotic ribosome) and the associated nascent-polypeptide hydrolysis and/or nonsense-mediated mRNA decay either • This technology can be extended and co-opted for high-throughput approaches to screen drugs designed to specifically inhibit translation termination at a premature termination codon (i.e. by promoting a termination-codon read-through event that suppresses the termination reaction specifically at the premature stop codon, allowing the ribosome to continue translation of the premature termination codon-containing mRNA). • High-throughput approaches with high sensitivity can be used for characterizing the efficacy of pharmacological drugs/agents designed to inhibit and/or perturb the termination reaction either specifically within the prokaryotic translational machinery (i.e. for antibiotic drug discovery efforts) or specifically at premature termination codons (i.e. fordrug discovery efforts aimed at identifying drugs that promote termination-codon read-through events specifically at premature termination codons), as monitored by fluorescently-labeled RF reporters

Advantages: • Using a fluorescence signal as the observable, this technology offers extremely high sensitivity; thus, only small amount of material is required in commercial applications • The surface-based translation system used allows these experiments to be carried out in large arrays, using either ensemble-based or single molecule-based fluorescence detection, and thus can be easily co-opted for high throughput screens • Single molecule-based fluorescence detection can elucidate mechanistic details that cannot be easily accessed using ensemble methods, and thus provide powerful insight into the mechanism of translation termination 

Publications: Translation factors direct intrinsic ribosome dynamics during translation termination and ribosome recycling. Sternberg, S.H., Fei, J., Prywes, N., McGrath, K.A., and Gonzalez Jr, R.L. Nature Structural & Molecular Biology, advanced online publication (2009)”

Lead Inventor: Ruben L. Gonzalez, Jr. Ph.D.


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