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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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Related in: MedlinePlus

Assignment of 1H NMR signals to double-stranded basepairs that form the 3′(−)SL RNA secondary structure.The 3′(−)SL RNA displays 5 distinct major imino protonsignals in the 1H NMR spectrum that correspond to a stem-loopRNA secondary structure comprising a 5 base-paired stem, a penta-loop,and 4 and 2 nucleotide single-stranded parts at the 3′- and5′-ends, respectively. (A) A spectrum of the wild-type 3′(−)SLRNA was compared with the spectrum of a 3′(−)SL RNAmutant G13C, which features only 4 prominent base pairs. This enabledthe assignment of the imino proton signal of G13, as indicatedby the arrow. (B) An alternative stem-loop structure with an additionalsixth base pair, U8–A12, resulting ina conformation consisting of a 6 base-paired double-stranded stemand a triple-loop, is less populated in solution. This was shown bysubstitution of the wild-type U8–A12 byC8–G12, which resulted in a new iminoproton signal in the 1H NMR spectrum at higher field, asindicated by the arrows. (C) Two-dimensional 1H–1H NOESY spectrum of 1 mM 3′(−)SL RNA was recorded,yielding intramolecular imino–imino NOE signals. Depicted arethe following NOE cross peaks: G16–G15, turquoise; G16–G3, red; G13–G14, ocher.
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fig5: Assignment of 1H NMR signals to double-stranded basepairs that form the 3′(−)SL RNA secondary structure.The 3′(−)SL RNA displays 5 distinct major imino protonsignals in the 1H NMR spectrum that correspond to a stem-loopRNA secondary structure comprising a 5 base-paired stem, a penta-loop,and 4 and 2 nucleotide single-stranded parts at the 3′- and5′-ends, respectively. (A) A spectrum of the wild-type 3′(−)SLRNA was compared with the spectrum of a 3′(−)SL RNAmutant G13C, which features only 4 prominent base pairs. This enabledthe assignment of the imino proton signal of G13, as indicatedby the arrow. (B) An alternative stem-loop structure with an additionalsixth base pair, U8–A12, resulting ina conformation consisting of a 6 base-paired double-stranded stemand a triple-loop, is less populated in solution. This was shown bysubstitution of the wild-type U8–A12 byC8–G12, which resulted in a new iminoproton signal in the 1H NMR spectrum at higher field, asindicated by the arrows. (C) Two-dimensional 1H–1H NOESY spectrum of 1 mM 3′(−)SL RNA was recorded,yielding intramolecular imino–imino NOE signals. Depicted arethe following NOE cross peaks: G16–G15, turquoise; G16–G3, red; G13–G14, ocher.

Mentions: RNA synthesis catalyzed by the HCV-polymeraseon the structured3′(−)SL RNA template. (A) The HCV-polymerase NS5B isschematically depicted with thumb (T), palm (P), and finger (F) domains.The 3′(−)SL RNA template forms a stem-loop structure;the inset provides the secondary structures that were determined byNMR (see also Figures 4 and 5 and Table 3). For the synthesis ofprogeny (+)RNA molecules, the HCV-polymerase binds in a first half-reaction(binary complex formation). The interaction with NTPs (as indicated)results in the formation of the catalysis-competent ternary complex.Our data suggest that RNA secondary structures of the template haveto be resolved to accomplish double-stranded RNA product formationand release (second half-reaction). (B) Schematic presentation ofthe primary structure of the 3′(−)SL RNA template. Twopossible structures of the stem-loop were derived by the program mfold.45


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)

Assignment of 1H NMR signals to double-stranded basepairs that form the 3′(−)SL RNA secondary structure.The 3′(−)SL RNA displays 5 distinct major imino protonsignals in the 1H NMR spectrum that correspond to a stem-loopRNA secondary structure comprising a 5 base-paired stem, a penta-loop,and 4 and 2 nucleotide single-stranded parts at the 3′- and5′-ends, respectively. (A) A spectrum of the wild-type 3′(−)SLRNA was compared with the spectrum of a 3′(−)SL RNAmutant G13C, which features only 4 prominent base pairs. This enabledthe assignment of the imino proton signal of G13, as indicatedby the arrow. (B) An alternative stem-loop structure with an additionalsixth base pair, U8–A12, resulting ina conformation consisting of a 6 base-paired double-stranded stemand a triple-loop, is less populated in solution. This was shown bysubstitution of the wild-type U8–A12 byC8–G12, which resulted in a new iminoproton signal in the 1H NMR spectrum at higher field, asindicated by the arrows. (C) Two-dimensional 1H–1H NOESY spectrum of 1 mM 3′(−)SL RNA was recorded,yielding intramolecular imino–imino NOE signals. Depicted arethe following NOE cross peaks: G16–G15, turquoise; G16–G3, red; G13–G14, ocher.
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Related In: Results  -  Collection

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

fig5: Assignment of 1H NMR signals to double-stranded basepairs that form the 3′(−)SL RNA secondary structure.The 3′(−)SL RNA displays 5 distinct major imino protonsignals in the 1H NMR spectrum that correspond to a stem-loopRNA secondary structure comprising a 5 base-paired stem, a penta-loop,and 4 and 2 nucleotide single-stranded parts at the 3′- and5′-ends, respectively. (A) A spectrum of the wild-type 3′(−)SLRNA was compared with the spectrum of a 3′(−)SL RNAmutant G13C, which features only 4 prominent base pairs. This enabledthe assignment of the imino proton signal of G13, as indicatedby the arrow. (B) An alternative stem-loop structure with an additionalsixth base pair, U8–A12, resulting ina conformation consisting of a 6 base-paired double-stranded stemand a triple-loop, is less populated in solution. This was shown bysubstitution of the wild-type U8–A12 byC8–G12, which resulted in a new iminoproton signal in the 1H NMR spectrum at higher field, asindicated by the arrows. (C) Two-dimensional 1H–1H NOESY spectrum of 1 mM 3′(−)SL RNA was recorded,yielding intramolecular imino–imino NOE signals. Depicted arethe following NOE cross peaks: G16–G15, turquoise; G16–G3, red; G13–G14, ocher.
Mentions: RNA synthesis catalyzed by the HCV-polymeraseon the structured3′(−)SL RNA template. (A) The HCV-polymerase NS5B isschematically depicted with thumb (T), palm (P), and finger (F) domains.The 3′(−)SL RNA template forms a stem-loop structure;the inset provides the secondary structures that were determined byNMR (see also Figures 4 and 5 and Table 3). For the synthesis ofprogeny (+)RNA molecules, the HCV-polymerase binds in a first half-reaction(binary complex formation). The interaction with NTPs (as indicated)results in the formation of the catalysis-competent ternary complex.Our data suggest that RNA secondary structures of the template haveto be resolved to accomplish double-stranded RNA product formationand release (second half-reaction). (B) Schematic presentation ofthe primary structure of the 3′(−)SL RNA template. Twopossible structures of the stem-loop were derived by the program mfold.45

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