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Initiation of RNA synthesis by the hepatitis C virus RNA-dependent RNA polymerase is affected by the structure of the RNA template.

Reich S, Kovermann M, Lilie H, Knick P, Geissler R, Golbik RP, Balbach J, Behrens SE - Biochemistry (2014)

Bottom Line: NS5B was found to bind to a nonstructured and a structured RNA template in different modes.Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template.Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biotechnology, Section of Microbial Biotechnology, ‡Institute of Physics, Section of Biophysics, §Institute of Biochemistry and Biotechnology, Section of Technical Biochemistry, Martin Luther University Halle-Wittenberg , D-06120 Halle/Saale, Germany.

ABSTRACT
The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D (1)H NMR spectroscopy. NS5B was found to bind to a nonstructured and a structured RNA template in different modes. Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template. By monitoring the folding/unfolding of 3'(-)SL by (1)H NMR, the base pair at the stem's edge was identified as the most stable component of the structure. (1)H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3'(-)SL showed that a pronounced lag phase preceded the processive polymerization reaction. The presence of the double-stranded stem with the edge base pair acting as the main energy barrier impaired RNA synthesis catalyzed by NS5B. Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process.

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Binding kinetics of the HCV-polymerase to short RNAs (binarycomplexformation). The kinetics of binary complex formation was measuredusing fluorescently labeled RNAs. The binding process of single-strandedRNA (16 nucleotides) (A) was recorded at template concentrations of21 nM (dark blue), 124 nM (light blue), 164 nM (dark green), 185 nM(lime), 329 nM (yellow), 410 nM (orange), 492 nM (red), and 817 nM(pink). Binding the native 3′(−)SL RNA (21 nucleotides)(C) was recorded at template concentrations of 12.5 nM (dark blue),100 nM (dark green), 200 nM (lime), 300 nM (orange), and 500 nM (red).The protein concentration was 270 nM. Complex formation proceededat least via three intermediates; accordingly, the kinetics was fittedaccording to quadruple-exponential first-order reactions (B, D). Theobserved rate constants were plotted against the respective RNA concentrationand yielded a second-order rate constant and three intramolecularfirst-order rate constants that are summarized in Table 2.
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fig3: Binding kinetics of the HCV-polymerase to short RNAs (binarycomplexformation). The kinetics of binary complex formation was measuredusing fluorescently labeled RNAs. The binding process of single-strandedRNA (16 nucleotides) (A) was recorded at template concentrations of21 nM (dark blue), 124 nM (light blue), 164 nM (dark green), 185 nM(lime), 329 nM (yellow), 410 nM (orange), 492 nM (red), and 817 nM(pink). Binding the native 3′(−)SL RNA (21 nucleotides)(C) was recorded at template concentrations of 12.5 nM (dark blue),100 nM (dark green), 200 nM (lime), 300 nM (orange), and 500 nM (red).The protein concentration was 270 nM. Complex formation proceededat least via three intermediates; accordingly, the kinetics was fittedaccording to quadruple-exponential first-order reactions (B, D). Theobserved rate constants were plotted against the respective RNA concentrationand yielded a second-order rate constant and three intramolecularfirst-order rate constants that are summarized in Table 2.

Mentions: Kinetically,the RNA binding by the HCV-polymerase proceeded alongdefined intermediates. Both RNA templates were shown to interact withthe HCV-polymerase via initial Michaelis-complex formation and somesubsequent intramolecular reactions (Figure 3 and Table 2). In analogy to earlier findings,28 four phases of template binding were resolved,which could be differentiated by their rate constant and by the amplitudeof quenching of the fluorescence emission of the labeled RNA. Thefastest process depended on the RNA concentration; accordingly, weassigned this phase to the initial complex formation as a second-orderreaction. The three slower processes did not depend on the RNA concentrationsand were attributed to intramolecular rearrangements.28 The kinetics of the interaction between the HCV-polymeraseand the two RNA templates significantly differed only in the fastestprocess, corresponding to the second-order rate constant kon of association. Here, the native 3′(−)SLRNA template was observed to bind 6-fold slower than the random ssRNA.No significant differences were observed for the three slower monomolecularreactions and the first-order dissociation rate constant koff. This indicated that the intramolecular substratepositioning and release by the polymerase mostly occurred independentlyof the RNA moiety.


Initiation of RNA synthesis by the hepatitis C virus RNA-dependent RNA polymerase is affected by the structure of the RNA template.

Reich S, Kovermann M, Lilie H, Knick P, Geissler R, Golbik RP, Balbach J, Behrens SE - Biochemistry (2014)

Binding kinetics of the HCV-polymerase to short RNAs (binarycomplexformation). The kinetics of binary complex formation was measuredusing fluorescently labeled RNAs. The binding process of single-strandedRNA (16 nucleotides) (A) was recorded at template concentrations of21 nM (dark blue), 124 nM (light blue), 164 nM (dark green), 185 nM(lime), 329 nM (yellow), 410 nM (orange), 492 nM (red), and 817 nM(pink). Binding the native 3′(−)SL RNA (21 nucleotides)(C) was recorded at template concentrations of 12.5 nM (dark blue),100 nM (dark green), 200 nM (lime), 300 nM (orange), and 500 nM (red).The protein concentration was 270 nM. Complex formation proceededat least via three intermediates; accordingly, the kinetics was fittedaccording to quadruple-exponential first-order reactions (B, D). Theobserved rate constants were plotted against the respective RNA concentrationand yielded a second-order rate constant and three intramolecularfirst-order rate constants that are summarized in Table 2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230328&req=5

fig3: Binding kinetics of the HCV-polymerase to short RNAs (binarycomplexformation). The kinetics of binary complex formation was measuredusing fluorescently labeled RNAs. The binding process of single-strandedRNA (16 nucleotides) (A) was recorded at template concentrations of21 nM (dark blue), 124 nM (light blue), 164 nM (dark green), 185 nM(lime), 329 nM (yellow), 410 nM (orange), 492 nM (red), and 817 nM(pink). Binding the native 3′(−)SL RNA (21 nucleotides)(C) was recorded at template concentrations of 12.5 nM (dark blue),100 nM (dark green), 200 nM (lime), 300 nM (orange), and 500 nM (red).The protein concentration was 270 nM. Complex formation proceededat least via three intermediates; accordingly, the kinetics was fittedaccording to quadruple-exponential first-order reactions (B, D). Theobserved rate constants were plotted against the respective RNA concentrationand yielded a second-order rate constant and three intramolecularfirst-order rate constants that are summarized in Table 2.
Mentions: Kinetically,the RNA binding by the HCV-polymerase proceeded alongdefined intermediates. Both RNA templates were shown to interact withthe HCV-polymerase via initial Michaelis-complex formation and somesubsequent intramolecular reactions (Figure 3 and Table 2). In analogy to earlier findings,28 four phases of template binding were resolved,which could be differentiated by their rate constant and by the amplitudeof quenching of the fluorescence emission of the labeled RNA. Thefastest process depended on the RNA concentration; accordingly, weassigned this phase to the initial complex formation as a second-orderreaction. The three slower processes did not depend on the RNA concentrationsand were attributed to intramolecular rearrangements.28 The kinetics of the interaction between the HCV-polymeraseand the two RNA templates significantly differed only in the fastestprocess, corresponding to the second-order rate constant kon of association. Here, the native 3′(−)SLRNA template was observed to bind 6-fold slower than the random ssRNA.No significant differences were observed for the three slower monomolecularreactions and the first-order dissociation rate constant koff. This indicated that the intramolecular substratepositioning and release by the polymerase mostly occurred independentlyof the RNA moiety.

Bottom Line: NS5B was found to bind to a nonstructured and a structured RNA template in different modes.Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template.Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biotechnology, Section of Microbial Biotechnology, ‡Institute of Physics, Section of Biophysics, §Institute of Biochemistry and Biotechnology, Section of Technical Biochemistry, Martin Luther University Halle-Wittenberg , D-06120 Halle/Saale, Germany.

ABSTRACT
The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D (1)H NMR spectroscopy. NS5B was found to bind to a nonstructured and a structured RNA template in different modes. Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template. By monitoring the folding/unfolding of 3'(-)SL by (1)H NMR, the base pair at the stem's edge was identified as the most stable component of the structure. (1)H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3'(-)SL showed that a pronounced lag phase preceded the processive polymerization reaction. The presence of the double-stranded stem with the edge base pair acting as the main energy barrier impaired RNA synthesis catalyzed by NS5B. Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process.

Show MeSH
Related in: MedlinePlus