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Dynamics on multiple timescales in the RNA-directed RNA polymerase from the cystovirus phi6.

Ren Z, Wang H, Ghose R - Nucleic Acids Res. (2010)

Bottom Line: By measurement and quantitative analysis of multiple-quantum spin-relaxation data for the delta1 positions of Ile residues that are distributed over the 3D-fold of P2, we find that the enzyme is dynamic both on the fast (ps-ns) and slow (micros-ms) timescales.The characteristics of several motional modes including those that coincide with the catalytic timescale (500-800/s) are altered in the presence of substrate analogs and single-stranded RNA templates.These studies reveal the plasticity of this finely tuned molecular machine and represent a first step towards linking structural information available from a host of crystal structures to catalytic mechanisms and timescales obtained from the measurements of kinetics for homologous systems in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The City College of New York, New York, NY 10031, USA.

ABSTRACT
The de novo initiating RNA-directed RNA polymerase (RdRP), P2, forms the central machinery in the infection cycle of the bacteriophage phi6 by performing the dual tasks of replication and transcription of the double-stranded RNA genome in the host cell. By measurement and quantitative analysis of multiple-quantum spin-relaxation data for the delta1 positions of Ile residues that are distributed over the 3D-fold of P2, we find that the enzyme is dynamic both on the fast (ps-ns) and slow (micros-ms) timescales. The characteristics of several motional modes including those that coincide with the catalytic timescale (500-800/s) are altered in the presence of substrate analogs and single-stranded RNA templates. These studies reveal the plasticity of this finely tuned molecular machine and represent a first step towards linking structural information available from a host of crystal structures to catalytic mechanisms and timescales obtained from the measurements of kinetics for homologous systems in solution.

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(a) Formation of a stable initiation complex results from the proper base pairing of the template bases T1 (C) and T2 (C) (numbered 3′→5′) with the substrate (GTP analog) bases D1 (G) and D2 (G). Nucleophilic attack by the 3′-OH of D1 and cleavage between the α and β−phosphate groups of D2 (represented by the dotted line), results in the formation of the first 3′–5′ phosphodiester bond of the daughter chain between D1 and D2 (numbered 5′→3′), releasing pyrophosphate. The requirements for the reaction to proceed are thus, (1) proper T1–D1 and T2–D2 base pairing (indicated by solid lines) and (2) cleavability of the substrate (indicated by dashed lines) between the α,β−phosphates. (b) Schematic representation of the three ternary complexes (states 4, 5 and 6) between P2, substrate GTP analogs and 5-nt ssRNA templates. State 4: T1 (C)-D1 (G) and T2 (C)-D2 (G) are correctly base-paired, the substrate GMPPNP is α−β cleavable and the reaction proceeds for one cycle generating 5′-PPP-G-P-3′. The reaction subsequently stalls since the base (D3, A) complementary to T3 (U) from ATP (or an analog) is not available. State 5: The substrate GMPPNP is α−β cleavable but proper base paring between T2 (U) and D2 (G) cannot occur. State 6: T1–D1 and T2–D2 properly base pair, however the substrate, GMPCPP, is not is α−β cleavable and the reaction cannot proceed.
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Figure 2: (a) Formation of a stable initiation complex results from the proper base pairing of the template bases T1 (C) and T2 (C) (numbered 3′→5′) with the substrate (GTP analog) bases D1 (G) and D2 (G). Nucleophilic attack by the 3′-OH of D1 and cleavage between the α and β−phosphate groups of D2 (represented by the dotted line), results in the formation of the first 3′–5′ phosphodiester bond of the daughter chain between D1 and D2 (numbered 5′→3′), releasing pyrophosphate. The requirements for the reaction to proceed are thus, (1) proper T1–D1 and T2–D2 base pairing (indicated by solid lines) and (2) cleavability of the substrate (indicated by dashed lines) between the α,β−phosphates. (b) Schematic representation of the three ternary complexes (states 4, 5 and 6) between P2, substrate GTP analogs and 5-nt ssRNA templates. State 4: T1 (C)-D1 (G) and T2 (C)-D2 (G) are correctly base-paired, the substrate GMPPNP is α−β cleavable and the reaction proceeds for one cycle generating 5′-PPP-G-P-3′. The reaction subsequently stalls since the base (D3, A) complementary to T3 (U) from ATP (or an analog) is not available. State 5: The substrate GMPPNP is α−β cleavable but proper base paring between T2 (U) and D2 (G) cannot occur. State 6: T1–D1 and T2–D2 properly base pair, however the substrate, GMPCPP, is not is α−β cleavable and the reaction cannot proceed.

Mentions: The choice of the ternary complexes of P2 with substrate (Mg2+/GTP analogs) and ssRNA warrants some discussion (Figure 2). For RdRPs that initiate RNA synthesis de novo i.e. without the use of a primer, the initiation NTP acts as a 1-nt primer and base-pairs with the extreme 3′-nt (T1) of the template forming the first nucleobase (D1) from the 5′-end of the daughter strand. The second substrate NTP (D2) base pairs with the penultimate template nucleotide from the 3′-end (T2). Formation of a stable initiation complex requires both T1–D1 and T2–D2 base-pairing to occur. The first phosphodiester bond is thus formed between D1 and D2 with the cleavage of the latter between the α and β−phosphates releasing pyrophosphate (shown schematically in Figure 2a, also see Supplementary Figure S3). The process continues with each incoming substrate nucleotide (CTP, UTP, ATP or GTP) complementary to the corresponding template nucleobase (36). Thus, among the ternary complexes studied here, state 4 (Table 1, Figure 2b) represents a dead-end or stalled complex where the first phosphodiester bond between D1 and D2 can form since GMPPNP is a viable substrate for P2 (Supplementary Figure S3) given the fact that it can be cleaved between the α and β phosphates (37). However, additional polymerization beyond the one-nucleotide addition cycle and formation of a 2-nt daughter chain (5′-PPP-G-P-G-3′) becomes extremely inefficient, as the substrate nucleotide (A from ATP or an ATP-analog) complementary to T3 (U) is unavailable. Examples of stalling through nucleotide deprivation in ϕ6 P2 has been described in the literature (38) and characterized structurally (10). In state 5 (Table 1) a complex capable of forming a stable initiation platform cannot form since the complementary nucleotide D2 (A from ATP or analog) for T2 (U) is unavailable (Figure 2b). State 6 (Table 1), on the other hand, represents a stable initiation complex, stabilized by proper base-pairing of D1 with T1 and D2 with T2 (D1,2 = GMPCPP and T1,2 = C). However the phosphodiester linkage between D1 and D2 cannot form since in GMPCPP the bond between the α and β phosphates cannot be cleaved (Figure 2b and Supplementary Figure S3).Table 2.


Dynamics on multiple timescales in the RNA-directed RNA polymerase from the cystovirus phi6.

Ren Z, Wang H, Ghose R - Nucleic Acids Res. (2010)

(a) Formation of a stable initiation complex results from the proper base pairing of the template bases T1 (C) and T2 (C) (numbered 3′→5′) with the substrate (GTP analog) bases D1 (G) and D2 (G). Nucleophilic attack by the 3′-OH of D1 and cleavage between the α and β−phosphate groups of D2 (represented by the dotted line), results in the formation of the first 3′–5′ phosphodiester bond of the daughter chain between D1 and D2 (numbered 5′→3′), releasing pyrophosphate. The requirements for the reaction to proceed are thus, (1) proper T1–D1 and T2–D2 base pairing (indicated by solid lines) and (2) cleavability of the substrate (indicated by dashed lines) between the α,β−phosphates. (b) Schematic representation of the three ternary complexes (states 4, 5 and 6) between P2, substrate GTP analogs and 5-nt ssRNA templates. State 4: T1 (C)-D1 (G) and T2 (C)-D2 (G) are correctly base-paired, the substrate GMPPNP is α−β cleavable and the reaction proceeds for one cycle generating 5′-PPP-G-P-3′. The reaction subsequently stalls since the base (D3, A) complementary to T3 (U) from ATP (or an analog) is not available. State 5: The substrate GMPPNP is α−β cleavable but proper base paring between T2 (U) and D2 (G) cannot occur. State 6: T1–D1 and T2–D2 properly base pair, however the substrate, GMPCPP, is not is α−β cleavable and the reaction cannot proceed.
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Figure 2: (a) Formation of a stable initiation complex results from the proper base pairing of the template bases T1 (C) and T2 (C) (numbered 3′→5′) with the substrate (GTP analog) bases D1 (G) and D2 (G). Nucleophilic attack by the 3′-OH of D1 and cleavage between the α and β−phosphate groups of D2 (represented by the dotted line), results in the formation of the first 3′–5′ phosphodiester bond of the daughter chain between D1 and D2 (numbered 5′→3′), releasing pyrophosphate. The requirements for the reaction to proceed are thus, (1) proper T1–D1 and T2–D2 base pairing (indicated by solid lines) and (2) cleavability of the substrate (indicated by dashed lines) between the α,β−phosphates. (b) Schematic representation of the three ternary complexes (states 4, 5 and 6) between P2, substrate GTP analogs and 5-nt ssRNA templates. State 4: T1 (C)-D1 (G) and T2 (C)-D2 (G) are correctly base-paired, the substrate GMPPNP is α−β cleavable and the reaction proceeds for one cycle generating 5′-PPP-G-P-3′. The reaction subsequently stalls since the base (D3, A) complementary to T3 (U) from ATP (or an analog) is not available. State 5: The substrate GMPPNP is α−β cleavable but proper base paring between T2 (U) and D2 (G) cannot occur. State 6: T1–D1 and T2–D2 properly base pair, however the substrate, GMPCPP, is not is α−β cleavable and the reaction cannot proceed.
Mentions: The choice of the ternary complexes of P2 with substrate (Mg2+/GTP analogs) and ssRNA warrants some discussion (Figure 2). For RdRPs that initiate RNA synthesis de novo i.e. without the use of a primer, the initiation NTP acts as a 1-nt primer and base-pairs with the extreme 3′-nt (T1) of the template forming the first nucleobase (D1) from the 5′-end of the daughter strand. The second substrate NTP (D2) base pairs with the penultimate template nucleotide from the 3′-end (T2). Formation of a stable initiation complex requires both T1–D1 and T2–D2 base-pairing to occur. The first phosphodiester bond is thus formed between D1 and D2 with the cleavage of the latter between the α and β−phosphates releasing pyrophosphate (shown schematically in Figure 2a, also see Supplementary Figure S3). The process continues with each incoming substrate nucleotide (CTP, UTP, ATP or GTP) complementary to the corresponding template nucleobase (36). Thus, among the ternary complexes studied here, state 4 (Table 1, Figure 2b) represents a dead-end or stalled complex where the first phosphodiester bond between D1 and D2 can form since GMPPNP is a viable substrate for P2 (Supplementary Figure S3) given the fact that it can be cleaved between the α and β phosphates (37). However, additional polymerization beyond the one-nucleotide addition cycle and formation of a 2-nt daughter chain (5′-PPP-G-P-G-3′) becomes extremely inefficient, as the substrate nucleotide (A from ATP or an ATP-analog) complementary to T3 (U) is unavailable. Examples of stalling through nucleotide deprivation in ϕ6 P2 has been described in the literature (38) and characterized structurally (10). In state 5 (Table 1) a complex capable of forming a stable initiation platform cannot form since the complementary nucleotide D2 (A from ATP or analog) for T2 (U) is unavailable (Figure 2b). State 6 (Table 1), on the other hand, represents a stable initiation complex, stabilized by proper base-pairing of D1 with T1 and D2 with T2 (D1,2 = GMPCPP and T1,2 = C). However the phosphodiester linkage between D1 and D2 cannot form since in GMPCPP the bond between the α and β phosphates cannot be cleaved (Figure 2b and Supplementary Figure S3).Table 2.

Bottom Line: By measurement and quantitative analysis of multiple-quantum spin-relaxation data for the delta1 positions of Ile residues that are distributed over the 3D-fold of P2, we find that the enzyme is dynamic both on the fast (ps-ns) and slow (micros-ms) timescales.The characteristics of several motional modes including those that coincide with the catalytic timescale (500-800/s) are altered in the presence of substrate analogs and single-stranded RNA templates.These studies reveal the plasticity of this finely tuned molecular machine and represent a first step towards linking structural information available from a host of crystal structures to catalytic mechanisms and timescales obtained from the measurements of kinetics for homologous systems in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The City College of New York, New York, NY 10031, USA.

ABSTRACT
The de novo initiating RNA-directed RNA polymerase (RdRP), P2, forms the central machinery in the infection cycle of the bacteriophage phi6 by performing the dual tasks of replication and transcription of the double-stranded RNA genome in the host cell. By measurement and quantitative analysis of multiple-quantum spin-relaxation data for the delta1 positions of Ile residues that are distributed over the 3D-fold of P2, we find that the enzyme is dynamic both on the fast (ps-ns) and slow (micros-ms) timescales. The characteristics of several motional modes including those that coincide with the catalytic timescale (500-800/s) are altered in the presence of substrate analogs and single-stranded RNA templates. These studies reveal the plasticity of this finely tuned molecular machine and represent a first step towards linking structural information available from a host of crystal structures to catalytic mechanisms and timescales obtained from the measurements of kinetics for homologous systems in solution.

Show MeSH
Related in: MedlinePlus