Ultrasensitive Fluorescence-Based Detection of Nascent Proteins in Gels

Gite, Sadanand; Mamaev, Sergey; Olejnik, Judith; Rothschild, Kenneth J.

doi:10.1006/abio.1999.4472

Cited by 62 publications

(41 citation statements)

References 38 publications

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“…Excess dye was removed by four additional ethanol precipitation steps 33,34 . The degree of labelling was 497% according to HPLC analysis on Biosuite 250 HR 30 cm SEC column (Waters).…”

Section: Methodsmentioning

confidence: 99%

High-efficiency translational bypassing of non-coding nucleotides specified by mRNA structure and nascent peptide

et al. 2014

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The gene product 60 (gp60) of bacteriophage T4 is synthesized as a single polypeptide chain from a discontinuous reading frame as a result of bypassing of a non-coding mRNA region of 50 nucleotides by the ribosome. To identify the minimum set of signals required for bypassing, we recapitulated efficient translational bypassing in an in vitro reconstituted translation system from Escherichia coli. We find that the signals, which promote efficient and accurate bypassing, are specified by the gene 60 mRNA sequence. Systematic analysis of the mRNA suggests unexpected contributions of sequences upstream and downstream of the non-coding gap region as well as of the nascent peptide. During bypassing, ribosomes glide forward on the mRNA track in a processive way. Gliding may have a role not only for gp60 synthesis, but also during regular mRNA translation for reading frame selection during initiation or tRNA translocation during elongation.

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“…Excess dye was removed by four additional ethanol precipitation steps 33,34 . The degree of labelling was 497% according to HPLC analysis on Biosuite 250 HR 30 cm SEC column (Waters).…”

Section: Methodsmentioning

confidence: 99%

High-efficiency translational bypassing of non-coding nucleotides specified by mRNA structure and nascent peptide

et al. 2014

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“…19 RNCs were labeled with BOD-IPY-FL (Invitrogen, denoted as BODIPY) by the addition of BODIPY-labeled Met-tRNA fMet , prepared as described, 41,42 to produce N-terminally fluorescently labeled nascent polypeptides and proteins. Stalled ribosomes bearing nascent chains of different length were generated in E. coli cell-free systems as described.…”

Section: Sample Preparation and Biochemical Characterizationmentioning

confidence: 99%

Confined dynamics of a ribosome‐bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions

Ellis¹,

Culviner²,

Cavagnero³

2009

Protein Science

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We still know very little about how proteins achieve their native three-dimensional structure in vitro and in the cell. Folding studies as proteins emerge from the mega Dalton-sized ribosome pose special challenges due to the large size and complicated nature of the ribosomenascent chain complex. This work introduces a combination of three-component analysis of fluorescence depolarization decays (including the presence of two local motions) and in-cone analysis of diffusive local dynamics to investigate the spatial constraints experienced by a protein emerging from the ribosomal tunnel. We focus on E. coli ribosomes and an all-a-helical nascent globin in the presence and absence of the cotranslationally active chaperones DnaK and trigger factor. The data provide insights on the dynamic nature and structural plasticity of ribosome-nascent chain complexes. We find that the sub-ns motions of the N-terminal fluorophore, reporting on the globin dynamics in the vicinity of the N terminus, are highly constrained both inside and outside the ribosomal tunnel, resulting in high-order parameters (>0.85) and small cone semiangles (<30°). The shorter globin chains buried inside the tunnel are less spatially constrained than those of a reference sequence from a natively unfolded protein, suggesting either that the two nascent chain sequences have a different secondary structure and therefore sample different regions of the tunnel or that the tunnel undergoes local structural adjustments to accommodate the globin sequence. Longer globins emerging out of the ribosomal tunnel are also found to have highly spatially constrained slow (ns) motions. There are no observable spectroscopic changes in the absence of bound chaperones.

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“…The 2-nitrobenzyl moiety has been shown to have a high photocleavage efficiency under UV irradiation ( Ϸ 340 nm) (24,(27)(28)(29)(30) and thus was chosen as the PC linker in our study. BODIPY, which has been used in electrophoresis-based DNA sequencing and protein analysis (31,32), was chosen as the fluorophore to couple with dUTP-NH 2 .…”

Section: Resultsmentioning

confidence: 99%

A photocleavable fluorescent nucleotide for DNA sequencing and analysis

Bai

Ruparel

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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DNA sequencing by synthesis during a polymerase reaction using laser-induced fluorescence detection is an approach that has a great potential to increase the throughput and data quality of DNA sequencing. We report the design and synthesis of a photocleavable fluorescent nucleoside triphosphate, one of the essential molecules required for the sequencing-by-synthesis approach. We synthesized this nucleoside triphosphate by attaching a fluorophore, 4,4-difluoro-5,7-dimethyl-4-bora-3␣,4␣-diaza-s-indacene propionic acid (BODIPY), to the 5 position of 2 -deoxyuridine triphosphate via a photocleavable 2-nitrobenzyl linker. We demonstrate that the nucleotide analogue can be faithfully incorporated by a DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA-sequencing reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. These results indicate that the nucleotide analogue is an excellent substrate for Thermo Sequenase. We also systematically studied the photocleavage of the fluorescent dye from a DNA molecule that contained the nucleotide analogue. UV irradiation at 340 nm of the DNA molecule led to the efficient release of the fluorescent dye, ensuring that a previous fluorescence signal did not leave any residue that could interfere with the detection of the next nucleotide. Thus, our results indicate that it should be feasible to use four different fluorescent dyes with distinct fluorescence emissions as unique tags to label the four nucleotides (A, C, G, and T) through the photocleavable 2-nitrobenzyl linker. These fluorescent tags can be removed easily by photocleavage after the identification of each nucleotide in the DNA sequencing-by-synthesis approach.nucleoside triphosphate ͉ 2-nitrobenzyl linker ͉ photocleavage ͉ fluorescence ͉ DNA sequencing by synthesis T he ability to sequence DNA accurately and rapidly has revolutionized biology and medicine. The development of high-throughput genetic-analysis technologies involving chemistry, engineering, biology, and computer science has made it possible to move from studying single genes at a time to analyzing and comparing entire genomes. The current state-of-the-art technology for high-throughput DNA sequencing utilizes capillary array DNA sequencers with laser-induced fluorescence detection (1-4). Although capillary array DNA-sequencing technology addresses the throughput and read length requirements of large-scale DNA-sequencing projects to some extent, the sequencing throughput and accuracy required for disease-gene discovery and personalized medicine needs to be improved significantly. For example, electrophoresis-based DNAsequencing methods have difficulty detecting heterozygotes unambiguously and are not 100% accurate in regions rich in nucleotides comprising guanine or cytosine because of compression (5, 6). In addition, the first few bases after the priming site are often masked by the high fluorescence signal from excess dye-labeled primers or dye-labeled terminators and are difficult to identify. Thus, ...

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Ultrasensitive Fluorescence-Based Detection of Nascent Proteins in Gels

Cited by 62 publications

References 38 publications

High-efficiency translational bypassing of non-coding nucleotides specified by mRNA structure and nascent peptide

High-efficiency translational bypassing of non-coding nucleotides specified by mRNA structure and nascent peptide

Confined dynamics of a ribosome‐bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions

A photocleavable fluorescent nucleotide for DNA sequencing and analysis

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