مورد إلكتروني
Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation
| العنوان: | Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation |
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| بيانات النشر: | Uppsala universitet, Institutionen för cell- och molekylärbiologi Uppsala universitet, Molekylärbiologi 2021 |
| تفاصيل مُضافة: | Kim, Changil Holm, Mikael Mandava, Chandra Sekhar Sanyal, Suparna |
| نوع الوثيقة: | Electronic Resource |
| مستخلص: | Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNAPhe, with the natural dipeptidyl fMet-Phe-tRNAPhe in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg2+ concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA. Authors in thesis list of papers: Kim, C., Holm, M., Sanyal, S. |
| مصطلحات الفهرس: | Ribosome, protein synthesis, translocation, pyrene mRNA, N-acetyl Phe-tRNA, EF-G, GTP hydrolysis, Biochemistry and Molecular Biology, Biokemi och molekylärbiologi, Article in journal, info:eu-repo/semantics/article, text |
| DOI: | 10.1080.15476286.2021.1913312 |
| URL: | RNA Biology, 1547-6286, 2021, 18:12, s. 2363-2375 |
| الإتاحة: | Open access content. Open access content info:eu-repo/semantics/openAccess |
| ملاحظة: | application/pdf English |
| أرقام أخرى: | UPE oai:DiVA.org:uu-396099 0000-0003-4977-4384 0000-0002-4361-9554 0000-0002-3028-3270 0000-0002-7124-792X doi:10.1080/15476286.2021.1913312 PMID 33938388 ISI:000646419600001 1235232999 |
| المصدر المساهم: | UPPSALA UNIV LIBR From OAIster®, provided by the OCLC Cooperative. |
| رقم الأكسشن: | edsoai.on1235232999 |
| قاعدة البيانات: | OAIster |
| DOI: | 10.1080.15476286.2021.1913312 |
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