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Nucleic-acid-binding properties of the C2-L1Tc nucleic acid chaperone encoded by L1Tc retrotransposon.

Heras SR, Thomas MC, Macias F, Patarroyo ME, Alonso C, López MC - Biochem. J. (2009)

Bottom Line: These domains also contribute to bind single- and double-stranded DNA and have a duplex-stabilizing effect.However, the peptide containing the zinc finger situated towards the C-terminal end of C2-L1Tc protein has a slight destabilization effect on a mismatched DNA duplex and shows a strong preference for single-stranded nucleic acids, such as C2-L1Tc.These results provide further insight into the essential properties of the C2-L1Tc protein as a NAC.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Instituto de Parasitología y Biomedicina López Neyra, CSIC, 18001 Granada, Spain.

ABSTRACT
It has been reported previously that the C2-L1Tc protein located in the Trypanosoma cruzi LINE (long interspersed nuclear element) L1Tc 3' terminal end has NAC (nucleic acid chaperone) activity, an essential activity for retrotransposition of LINE-1. The C2-L1Tc protein contains two cysteine motifs of a C2H2 type, similar to those present in TFIIIA (transcription factor IIIA). The cysteine motifs are flanked by positively charged amino acid regions. The results of the present study show that the C2-L1Tc recombinant protein has at least a 16-fold higher affinity for single-stranded than for double-stranded nucleic acids, and that it exhibits a clear preference for RNA binding over DNA. The C2-L1Tc binding profile (to RNA and DNA) corresponds to a non-co-operative-binding model. The zinc fingers present in C2-L1Tc have a different binding affinity to nucleic acid molecules and also different NAC activity. The RRR and RRRKEK [NLS (nuclear localization sequence)] sequences, as well as the C2H2 zinc finger located immediately downstream of these basic stretches are the main motifs responsible for the strong affinity of C2-L1Tc to RNA. These domains also contribute to bind single- and double-stranded DNA and have a duplex-stabilizing effect. However, the peptide containing the zinc finger situated towards the C-terminal end of C2-L1Tc protein has a slight destabilization effect on a mismatched DNA duplex and shows a strong preference for single-stranded nucleic acids, such as C2-L1Tc. These results provide further insight into the essential properties of the C2-L1Tc protein as a NAC.

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Related in: MedlinePlus

Binding analysis of C2-L1Tc-derived peptides to RNA by EMSAA 0.72 nM concentration of in-vitro-transcribed 32P-labelled 130nt-RNA was pre-incubated with increasing peptide concentrations at 37 °C for 30 min. (a) Peptides 5015, 5016 and 5031, from 1 to 14 μM; (b) peptide 5033 from 1 to 9 μM, and peptide 10987 from 3 to 24 μM; (c) peptides 5020, 5030 and 5032, from 9 to 30 μM (see Figure 1 and Table 1 for the peptide sequence details). Control reactions were performed without peptide (no peptide). The samples were run on 5% native polyacrylamide gels. (d) Curves representing the binding of peptides 5015, 5033, 10987, 5020, 5016 and 5031 to the 130nt-RNA. The data were obtained from three independent experiments as those shown in (a)–(c). The average values corresponding to the bound RNA fraction were plotted against the peptide concentration. The curves correspond to the best fit of the Hill equation to the experimental data [R2 (coefficient of determination) ≥0.99]. The equation used was as follows\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\def\rm#1{{\textrm #1}}\def\it#1{{\textit #1}}	\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{array}{l} y=\displaystyle\frac{B_{\mathrm {max}}\cdot x^{{\alpha}_{\scriptsize H}}} {K_{\mathrm d}\vspace*{6pt}^{{\alpha}_{\scriptsize H}}+ x^{{ {{\alpha}_{\scriptsize H}}}}}\\ \end{array}\end{document}where x is the peptide concentration, y is the radiolabelled RNA-bound fraction and Bmax is the maximum binding. Kd, defined as the peptide concentration at which 50% of the RNA is bound, is indicated. The position of the ‘complex’ and ‘free’ nucleic acids is indicated on the left-hand side of the panels as an arrowhead and F respectively.
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Figure 5: Binding analysis of C2-L1Tc-derived peptides to RNA by EMSAA 0.72 nM concentration of in-vitro-transcribed 32P-labelled 130nt-RNA was pre-incubated with increasing peptide concentrations at 37 °C for 30 min. (a) Peptides 5015, 5016 and 5031, from 1 to 14 μM; (b) peptide 5033 from 1 to 9 μM, and peptide 10987 from 3 to 24 μM; (c) peptides 5020, 5030 and 5032, from 9 to 30 μM (see Figure 1 and Table 1 for the peptide sequence details). Control reactions were performed without peptide (no peptide). The samples were run on 5% native polyacrylamide gels. (d) Curves representing the binding of peptides 5015, 5033, 10987, 5020, 5016 and 5031 to the 130nt-RNA. The data were obtained from three independent experiments as those shown in (a)–(c). The average values corresponding to the bound RNA fraction were plotted against the peptide concentration. The curves correspond to the best fit of the Hill equation to the experimental data [R2 (coefficient of determination) ≥0.99]. The equation used was as follows\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\def\rm#1{{\textrm #1}}\def\it#1{{\textit #1}} \setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{array}{l} y=\displaystyle\frac{B_{\mathrm {max}}\cdot x^{{\alpha}_{\scriptsize H}}} {K_{\mathrm d}\vspace*{6pt}^{{\alpha}_{\scriptsize H}}+ x^{{ {{\alpha}_{\scriptsize H}}}}}\\ \end{array}\end{document}where x is the peptide concentration, y is the radiolabelled RNA-bound fraction and Bmax is the maximum binding. Kd, defined as the peptide concentration at which 50% of the RNA is bound, is indicated. The position of the ‘complex’ and ‘free’ nucleic acids is indicated on the left-hand side of the panels as an arrowhead and F respectively.

Mentions: The number and the sequence of the peptides employed are indicated. The basic stretches of the C2-L1Tc protein are in bold and the residues forming the zinc fingers are underlined. The peptides derived from peptides 5015 and 5016 containing point mutations and deletions are labelled with *. Dissociation constants, Kd values, were obtained by fitting the experimental data to the Hill equation\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\def\rm#1{{\textrm #1}}\def\it#1{{\textit #1}} \setlength{\oddsidemargin}{-69pt}\begin{document}}{}$\begin{array}{l} y=\displaystyle\frac{B_{\mathrm {max}}\cdot x^{{\alpha}_{\scriptsize H}}}{K_{\mathrm d}\vspace*{6pt}^{{\alpha}_{\scriptsize H}}+ x^{{ {{\alpha}_{\scriptsize H}}}}}\\ \end{array}\end{document}where Kd is the ligand concentration at which 50% of the nucleic acid is bound and Bmax is the maximum binding (Figures 5, 6 and 7). The Hill formalism used has been described by Henriet et al. [44]. (a) The Kd of C2-L1Tc protein and of peptide 10987 and 5020 for dsDNA were obtained from the x intercept in the Hill transformation equation (Figures 2d and 7f). The dependent variable ‘y’ value is 0 when Y/(1−Y)=1. (b) Increase (+) or decrease (−) in temperature relative to the Tm of the 29/mm29c DNA duplex (40 °C) in the presence of 0.1 μM C2-L1Tc protein or 1 μM of each peptide. In the presence of peptide 5015 the duplex was not melted even at 55 °C. Conc50 represents the peptide concentration required to reach the formation of 50% of stable duplexes as a measure of NAC activity [22]. Values are means±S.D. NO, no observed activity.


Nucleic-acid-binding properties of the C2-L1Tc nucleic acid chaperone encoded by L1Tc retrotransposon.

Heras SR, Thomas MC, Macias F, Patarroyo ME, Alonso C, López MC - Biochem. J. (2009)

Binding analysis of C2-L1Tc-derived peptides to RNA by EMSAA 0.72 nM concentration of in-vitro-transcribed 32P-labelled 130nt-RNA was pre-incubated with increasing peptide concentrations at 37 °C for 30 min. (a) Peptides 5015, 5016 and 5031, from 1 to 14 μM; (b) peptide 5033 from 1 to 9 μM, and peptide 10987 from 3 to 24 μM; (c) peptides 5020, 5030 and 5032, from 9 to 30 μM (see Figure 1 and Table 1 for the peptide sequence details). Control reactions were performed without peptide (no peptide). The samples were run on 5% native polyacrylamide gels. (d) Curves representing the binding of peptides 5015, 5033, 10987, 5020, 5016 and 5031 to the 130nt-RNA. The data were obtained from three independent experiments as those shown in (a)–(c). The average values corresponding to the bound RNA fraction were plotted against the peptide concentration. The curves correspond to the best fit of the Hill equation to the experimental data [R2 (coefficient of determination) ≥0.99]. The equation used was as follows\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\def\rm#1{{\textrm #1}}\def\it#1{{\textit #1}}	\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{array}{l} y=\displaystyle\frac{B_{\mathrm {max}}\cdot x^{{\alpha}_{\scriptsize H}}} {K_{\mathrm d}\vspace*{6pt}^{{\alpha}_{\scriptsize H}}+ x^{{ {{\alpha}_{\scriptsize H}}}}}\\ \end{array}\end{document}where x is the peptide concentration, y is the radiolabelled RNA-bound fraction and Bmax is the maximum binding. Kd, defined as the peptide concentration at which 50% of the RNA is bound, is indicated. The position of the ‘complex’ and ‘free’ nucleic acids is indicated on the left-hand side of the panels as an arrowhead and F respectively.
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Figure 5: Binding analysis of C2-L1Tc-derived peptides to RNA by EMSAA 0.72 nM concentration of in-vitro-transcribed 32P-labelled 130nt-RNA was pre-incubated with increasing peptide concentrations at 37 °C for 30 min. (a) Peptides 5015, 5016 and 5031, from 1 to 14 μM; (b) peptide 5033 from 1 to 9 μM, and peptide 10987 from 3 to 24 μM; (c) peptides 5020, 5030 and 5032, from 9 to 30 μM (see Figure 1 and Table 1 for the peptide sequence details). Control reactions were performed without peptide (no peptide). The samples were run on 5% native polyacrylamide gels. (d) Curves representing the binding of peptides 5015, 5033, 10987, 5020, 5016 and 5031 to the 130nt-RNA. The data were obtained from three independent experiments as those shown in (a)–(c). The average values corresponding to the bound RNA fraction were plotted against the peptide concentration. The curves correspond to the best fit of the Hill equation to the experimental data [R2 (coefficient of determination) ≥0.99]. The equation used was as follows\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\def\rm#1{{\textrm #1}}\def\it#1{{\textit #1}} \setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{array}{l} y=\displaystyle\frac{B_{\mathrm {max}}\cdot x^{{\alpha}_{\scriptsize H}}} {K_{\mathrm d}\vspace*{6pt}^{{\alpha}_{\scriptsize H}}+ x^{{ {{\alpha}_{\scriptsize H}}}}}\\ \end{array}\end{document}where x is the peptide concentration, y is the radiolabelled RNA-bound fraction and Bmax is the maximum binding. Kd, defined as the peptide concentration at which 50% of the RNA is bound, is indicated. The position of the ‘complex’ and ‘free’ nucleic acids is indicated on the left-hand side of the panels as an arrowhead and F respectively.
Mentions: The number and the sequence of the peptides employed are indicated. The basic stretches of the C2-L1Tc protein are in bold and the residues forming the zinc fingers are underlined. The peptides derived from peptides 5015 and 5016 containing point mutations and deletions are labelled with *. Dissociation constants, Kd values, were obtained by fitting the experimental data to the Hill equation\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\def\rm#1{{\textrm #1}}\def\it#1{{\textit #1}} \setlength{\oddsidemargin}{-69pt}\begin{document}}{}$\begin{array}{l} y=\displaystyle\frac{B_{\mathrm {max}}\cdot x^{{\alpha}_{\scriptsize H}}}{K_{\mathrm d}\vspace*{6pt}^{{\alpha}_{\scriptsize H}}+ x^{{ {{\alpha}_{\scriptsize H}}}}}\\ \end{array}\end{document}where Kd is the ligand concentration at which 50% of the nucleic acid is bound and Bmax is the maximum binding (Figures 5, 6 and 7). The Hill formalism used has been described by Henriet et al. [44]. (a) The Kd of C2-L1Tc protein and of peptide 10987 and 5020 for dsDNA were obtained from the x intercept in the Hill transformation equation (Figures 2d and 7f). The dependent variable ‘y’ value is 0 when Y/(1−Y)=1. (b) Increase (+) or decrease (−) in temperature relative to the Tm of the 29/mm29c DNA duplex (40 °C) in the presence of 0.1 μM C2-L1Tc protein or 1 μM of each peptide. In the presence of peptide 5015 the duplex was not melted even at 55 °C. Conc50 represents the peptide concentration required to reach the formation of 50% of stable duplexes as a measure of NAC activity [22]. Values are means±S.D. NO, no observed activity.

Bottom Line: These domains also contribute to bind single- and double-stranded DNA and have a duplex-stabilizing effect.However, the peptide containing the zinc finger situated towards the C-terminal end of C2-L1Tc protein has a slight destabilization effect on a mismatched DNA duplex and shows a strong preference for single-stranded nucleic acids, such as C2-L1Tc.These results provide further insight into the essential properties of the C2-L1Tc protein as a NAC.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Instituto de Parasitología y Biomedicina López Neyra, CSIC, 18001 Granada, Spain.

ABSTRACT
It has been reported previously that the C2-L1Tc protein located in the Trypanosoma cruzi LINE (long interspersed nuclear element) L1Tc 3' terminal end has NAC (nucleic acid chaperone) activity, an essential activity for retrotransposition of LINE-1. The C2-L1Tc protein contains two cysteine motifs of a C2H2 type, similar to those present in TFIIIA (transcription factor IIIA). The cysteine motifs are flanked by positively charged amino acid regions. The results of the present study show that the C2-L1Tc recombinant protein has at least a 16-fold higher affinity for single-stranded than for double-stranded nucleic acids, and that it exhibits a clear preference for RNA binding over DNA. The C2-L1Tc binding profile (to RNA and DNA) corresponds to a non-co-operative-binding model. The zinc fingers present in C2-L1Tc have a different binding affinity to nucleic acid molecules and also different NAC activity. The RRR and RRRKEK [NLS (nuclear localization sequence)] sequences, as well as the C2H2 zinc finger located immediately downstream of these basic stretches are the main motifs responsible for the strong affinity of C2-L1Tc to RNA. These domains also contribute to bind single- and double-stranded DNA and have a duplex-stabilizing effect. However, the peptide containing the zinc finger situated towards the C-terminal end of C2-L1Tc protein has a slight destabilization effect on a mismatched DNA duplex and shows a strong preference for single-stranded nucleic acids, such as C2-L1Tc. These results provide further insight into the essential properties of the C2-L1Tc protein as a NAC.

Show MeSH
Related in: MedlinePlus