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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) Representative fits to Equation (2) for Ile340 and Ile500 are shown for P2 in the apo state (state 1). The scaling factor used in the y-axis is the ratio (4) of the number of scans used in experiments A and B. (b) η values for the Ile residues in P2 are plotted against residue number. η values for states 1, 2, 3 and 4 are represented by black, red, green and purple circles respectively. The regions corresponding to the fingers, thumb, palm and C-terminal domains are shaded red, blue, green and yellow respectively. Data corresponding to Ile211, Ile283, Ile617 and Ile641 were not analyzed.
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Figure 3: (a) Representative fits to Equation (2) for Ile340 and Ile500 are shown for P2 in the apo state (state 1). The scaling factor used in the y-axis is the ratio (4) of the number of scans used in experiments A and B. (b) η values for the Ile residues in P2 are plotted against residue number. η values for states 1, 2, 3 and 4 are represented by black, red, green and purple circles respectively. The regions corresponding to the fingers, thumb, palm and C-terminal domains are shaded red, blue, green and yellow respectively. Data corresponding to Ile211, Ile283, Ile617 and Ile641 were not analyzed.

Mentions: η values for 21 of 25 Ile [1H,13C]-δ1 resonances were analyzed in detail for states 1 through 4. Signals corresponding to Ile617 and Ile641 from the C-terminal domain were weak and excluded from the analysis as were those belonging to Ile211 and Ile283. Representative fits to Equation (2) (see ‘Materials and Methods’ section) and the η values for the 21 Ile resonances for the four states analyzed are shown in Figure 3.Figure 3.


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) Representative fits to Equation (2) for Ile340 and Ile500 are shown for P2 in the apo state (state 1). The scaling factor used in the y-axis is the ratio (4) of the number of scans used in experiments A and B. (b) η values for the Ile residues in P2 are plotted against residue number. η values for states 1, 2, 3 and 4 are represented by black, red, green and purple circles respectively. The regions corresponding to the fingers, thumb, palm and C-terminal domains are shaded red, blue, green and yellow respectively. Data corresponding to Ile211, Ile283, Ile617 and Ile641 were not analyzed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926596&req=5

Figure 3: (a) Representative fits to Equation (2) for Ile340 and Ile500 are shown for P2 in the apo state (state 1). The scaling factor used in the y-axis is the ratio (4) of the number of scans used in experiments A and B. (b) η values for the Ile residues in P2 are plotted against residue number. η values for states 1, 2, 3 and 4 are represented by black, red, green and purple circles respectively. The regions corresponding to the fingers, thumb, palm and C-terminal domains are shaded red, blue, green and yellow respectively. Data corresponding to Ile211, Ile283, Ile617 and Ile641 were not analyzed.
Mentions: η values for 21 of 25 Ile [1H,13C]-δ1 resonances were analyzed in detail for states 1 through 4. Signals corresponding to Ile617 and Ile641 from the C-terminal domain were weak and excluded from the analysis as were those belonging to Ile211 and Ile283. Representative fits to Equation (2) (see ‘Materials and Methods’ section) and the η values for the 21 Ile resonances for the four states analyzed are shown in Figure 3.Figure 3.

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