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Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation
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- Kim, Changil (author)
- Uppsala universitet,Institutionen för cell- och molekylärbiologi
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- Holm, Mikael, 1984- (author)
- Uppsala universitet,Institutionen för cell- och molekylärbiologi
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- Mandava, Chandra Sekhar, 1978- (author)
- Uppsala universitet,Molekylärbiologi
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- (creator_code:org_t)
- 2021-05-03
- 2021
- English.
- In: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 18:12, s. 2363-2375
- Journal article (peer-reviewed)
Abstract
Subject headings
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- 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.
Subject headings
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Keyword
- Ribosome
- protein synthesis
- translocation
- pyrene mRNA
- N-acetyl Phe-tRNA
- EF-G
- GTP hydrolysis
Publication and Content Type
- ref (subject category)
- art (subject category)
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