Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Elsevier Science full text link Elsevier Science Free PMC article
Full text links

Actions

Share

.2009 Aug 1;391(1):45-55.
doi: 10.1016/j.ab.2009.04.035. Epub 2009 May 3.

A quantitative stopped-flow fluorescence assay for measuring polymerase elongation rates

Affiliations

A quantitative stopped-flow fluorescence assay for measuring polymerase elongation rates

Peng Gong et al. Anal Biochem..

Abstract

The measurement of nucleic acid polymerase elongation rates is often done via a lengthy experimental process involving radiolabeled substrates, quenched elongation experiments, electrophoretic product separation, and band quantitation. In this work, we describe an alternative real-time stopped-flow assay for obtaining kinetic parameters for elongation of extended sequences. The assay builds on our earlier PETE (polymerase elongation template element) assay designed for high-throughput screening purposes [S.P. Mestas, A.J. Sholders, O.B. Peersen, A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity, Anal. Biochem. 365 (2007) 194-200] and relies on measuring how long it takes a polymerase to reach the end of a defined length template. Using poliovirus polymerase and self-priming hairpin RNA substrates with 6- to 26-nt-long templating regions, we demonstrate that the assay can be used to determine V(max) rates for elongation and apparent K(m) values for nucleotide triphosphate (NTP) use. Modeling the reaction kinetics as a series of irreversible steps allows us to numerically fit the entire time-based dataset by properly accounting for the temporal distribution of intermediate species. This enables us to determine average elongation rates over heterogeneous templating regions that mimic viral genome substrates. The assay is easily extendable to other RNA and DNA polymerases, can accommodate secondary structures in the template, and can in principle be used for any enzyme traversing along an extended substrate.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Schematic illustration of the hairpin RNA molecules used in this study. The PETE (polymerase elongation template element) oligonucleotides are named with anx-y convention wherex is the length of the duplex stem andy is the length of the templating single stranded 5′ extension. All RNA molecules are labeled at their 5′ ends with fluorescein (F) attached to the terminal phosphate via a six carbon linker. Prior to the stopped-flow experiments, stalled elongation complexes are pre-assembled by incubating the RNA-polymerase complex with the first nucleotide tri-phosphate (boxed), i.e. the original RNA molecules have a 3′ cytosine. The 10-26 PETE is predicted to have a weak secondary structure due to a fold-back of the 5′ end that is stabilized by three canonical (●) and one G-U (○) base pair. Some experiments utilize 10-26 PETE synthesized with an amino linker modified deoxythymidine (*) base in the tetraloop that is then labeled with an infrared fluorescent dye.
Fig. 2
Fig. 2
Stopped flow elongation data for the 8-6, 8-8, and 8-10 PETE constructs showing the template length dependent lag phase prior to the signal increase associated with terminal nucleotide additions. Data were obtained at pH 7.0 and 20 μM each NTP. (A) Total fluorescence (TF) data and (B) fluorescence anisotropy (FA) data that both show the effect but with different curve shapes. (C) Direct comparison of the TF and FA data for the 8-6 and 8-8 PETEs showing how the TF signal change runs slightly ahead of the change in the FA signal. For all panels, the data for each trace were normalized to a total amplitude change of 1.0 and the individual traces were offset along the Y-axis for clarity.
Fig. 3
Fig. 3
Verification that the observed fluorescence increase is associated with terminal nucleotide addition. Samples are from the following four reactions:1:RNA only2:RNA + polymerase3: RNA + polymerase + ATP/UTP/GTP4: RNA + polymerase + all four NTPs. (A) Stopped flow data from 10-26 PETE in the absence (3) and presence (4) of CTP, which is required to elongate to the end of the template (see Figure 1). (B) Electrophoresis analysis of the RNA in reactions1-4 showing that the fully elongated RNA (4) migrates faster than the RNA missing the terminal five bases (3). The image of the same gel treated with Stains-all dye shows two bands reflecting the ≈40% IRdye labeling efficiency. (C) Mass spectrometry data verifying the molecular weights of the RNAs in reactions2-4. The doublet in the starting material reflects the added mass of the IRdye and we consistently failed to see significant MALDI-TOF signals from the elongated form of the IRdye labeled RNA.
Fig. 4
Fig. 4
Stopped flow data showing relative amplitude changes of terminal nucleotide additions. (A) The 8-6_v2 PETE was elongated stepwise by mixing it with the appropriate nucleotide triphosphates as indicated in the figure. The relative values of the endpoint amplitudes were calculated for both total fluorescence and fluorescence anisotropy traces (not shown) and are listed in Table 1. The left panel is an expansion of the first five seconds of the data showing the similar initial curve behavior for all the reactions. Data were obtained with 10 μM each NTP at pH 7.0 in dual timebase mode with a higher sampling frequency during the first ten seconds of acquisition, and baseline corrected to a starting value of zero by subtracting the initial signal value. (B) Comparison of 8-6 PETE elongation data obtained in the presence of 100 μM and 400 μM each NTP showing the signal decrease observed at higher NTP concentrations. (C) Extended timecourse data showing the slow reduction in both total fluorescence and fluorescence anisotropy signals observed at high NTP concentrations.
Fig. 5
Fig. 5
Analysis of stopped flow data to determine elongation rates. (A) Simple analysis showing two independent single exponential curve fits to the initial signal decrease, attributed to secondary structure in 10-26 PETE, and the final signal increase due to terminal nucleotide addition. Elongation rate is determined by dividing the time of the curve intercept (circled) by the 21 nucleotides incorporated during the lag phase. (B) Full kinetic analysis by curve fitting an entire stopped flow dataset using the Berkeley Madonna software package. Experimental data is shown by the grey line and the curve fit to Eq. [1] is shown by the solid line. (C) Full kinetic analysis of data obtained at 37°C showing the loss of the initial signal decrease at higher temperature. The lower curves show the calculated temporal distribution of select elongation intermediates during the reaction, demonstrating the broadening of the distribution during the lag phase. The reaction is initiated with the stalled +1 complex and increase in the observed fluorescence signal corresponds to the accumulation of the +22 species, i.e. addition of the 5th nucleotide from the end of the template.
Fig. 6
Fig. 6
Determination of Michaelis-Menten parameters for polymerase elongation. (A) Stopped flow data traces from 10-26 PETE obtained at 22.5°C, pH 6.5, with NTP concentrations of 5, 10, 15, 30, 50, 80, and 120 μM each. (B) Michaelis-Menten plot of the elongation rates obtained from the Berkeley Madonna analysis vs. NTP concentration. The lag phase data is of the single average elongation rate obtained from the fit and the terminal nucleotide rates are the average of the 5th, 4th, and 3rd nucleotide additions from the end of the template. Vmax and apparent Km values from fitting data obtained at different temperatures are listed in the text and Table 2.
Fig. 7
Fig. 7
pH and temperature dependencies observed in the kinetic PETE assay with 10-26 RNA and 120 μM each NTP. (A) Total fluorescence data showing a ≈2-fold reduction in signal amplitude at pH 7.5 as compared to pH 6.5 while the fluorescence anisotropy data (panel B) is not greatly affected. In the time dimension the shorter lag phase indicates that the elongation rate almost triples as a function of pH. (C,D) Steady state fluorescence excitation and emission spectra of stalled (solid), partially (grey), and fully (dashed) elongated complexes at pH 6.5 and 7.5. Data are from identical sample concentrations and intensities can be directly compared, demonstrating that while the absolute signal is higher at pH 7.5, there is a larger elongation-dependent change in the signal at pH 6.5. (E) Elongation rates as a function of incubation time of the stalled elongation complex at temperatures of 22.5, 30, and 37 °C. These data are summarized in Table 2. (F) Amplitudes of signal changes associated with terminal nucleotide additions for the same data as in panel E. The grey lines in panels E and F are simple linear curve fits to the data and the slopes (δ) shown in panel F corresponding to the loss of elongation signal as a function of incubation time. Panel F datasets were normalized to a Y-intercept of 1.0 and offset by 0.25 units each along the Y-axis for clarity.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Johnson KA. Rapid quench kinetic analysis of polymerases, adenosinetriphosphatases, and enzyme intermediates. Methods Enzymol. 1995;249:38–61. - PubMed
    1. Patel SS, Bandwar RP. Fluorescence methods for studying the kinetics and thermodynamics of transcription initiation. Methods Enzymol. 2003;370:668–86. - PubMed
    1. Datta K, Johnson NP, von Hippel PH. Mapping the conformation of the nucleic acid framework of the T7 RNA polymerase elongation complex in solution using low-energy CD and fluorescence spectroscopy. J Mol Biol. 2006;360:800–13. - PubMed
    1. Johnson RS, Strausbauch M, Cooper R, Register JK. Rapid kinetic analysis of transcription elongation by Escherichia coli RNA polymerase. J Mol Biol. 2008;381:1106–13. - PubMed
    1. Upson RH, Haugland RP, Malekzadeh MN, Haugland RP. A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate. Anal Biochem. 1996;243:41–5. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full text links
Elsevier Science full text link Elsevier Science Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp