Limits...
Insights into the pre-initiation events of bacteriophage phi 6 RNA-dependent RNA polymerase: towards the assembly of a productive binary complex.

Sarin LP, Poranen MM, Lehti NM, Ravantti JJ, Koivunen MR, Aalto AP, van Dijk AA, Stuart DI, Grimes JM, Bamford DH - Nucleic Acids Res. (2009)

Bottom Line: In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3' terminus of the template and position it, so that a productive binary complex is formed.The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site.Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi 6 RdRP can be greatly enhanced.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki, Biocenter 2, Helsinki, Finland.

ABSTRACT
The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3' terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage phi 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi 6 RdRP can be greatly enhanced.

Show MeSH

Related in: MedlinePlus

Biochemical characterization of φ6 RdRP mutants. (a) Summary of the RNA polymerization activities of the φ6 RdRP mutants in comparison to the WT or YKW(630–632)GSG mutant (initiation by back-priming) (8). (b–g) Screening of the optimal reaction conditions for the mutant polymerases in terms of temperature, pH, ammonium acetate concentration and divalent cation concentrations (Mn2+, Mg2+, Zn2+). All results have been normalized against the highest attained polymerization activity for each RdRP. (h) Electrophoretic mobility shift assay (EMSA) of the φ6 RdRP mutants. The effect of an increasing concentration (0.04, 0.08 and 0.16 mg/ml) of WT (lanes 2–4) and R30A (lanes 6–8) polymerases on the migration of the s+rep template in 8% native polyacrylamide gel. The baseline (white dotted line) determines the position of the control (s+rep without polymerase; lanes 1, 5 and 9). The numbers below lanes 2–4 and 6–8 indicate the retardation of the s+rep template in millimetres.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651803&req=5

Figure 2: Biochemical characterization of φ6 RdRP mutants. (a) Summary of the RNA polymerization activities of the φ6 RdRP mutants in comparison to the WT or YKW(630–632)GSG mutant (initiation by back-priming) (8). (b–g) Screening of the optimal reaction conditions for the mutant polymerases in terms of temperature, pH, ammonium acetate concentration and divalent cation concentrations (Mn2+, Mg2+, Zn2+). All results have been normalized against the highest attained polymerization activity for each RdRP. (h) Electrophoretic mobility shift assay (EMSA) of the φ6 RdRP mutants. The effect of an increasing concentration (0.04, 0.08 and 0.16 mg/ml) of WT (lanes 2–4) and R30A (lanes 6–8) polymerases on the migration of the s+rep template in 8% native polyacrylamide gel. The baseline (white dotted line) determines the position of the control (s+rep without polymerase; lanes 1, 5 and 9). The numbers below lanes 2–4 and 6–8 indicate the retardation of the s+rep template in millimetres.

Mentions: The polymerase reaction conditions preferred by the mutants were generally quite similar to those of the WT polymerase, showing only minor deviations in terms of temperature, pH, salt concentration and divalent cation concentrations (Figure 2a-g). Despite strong similarities between the optimal conditions, the template tunnel mutant R30A and the specificity pocket mutant E634Q retained polymerization activity at temperatures up to 55–60°C, i.e. high enough temperatures to cause RNA hydrolysis, whereas the other mutants retained WT characteristics with temperature optima of roughly 40°C (Figure 2b). The template tunnel rim mutants R30A and K541L displayed a significantly reduced level of RNA synthesis for both replication and transcription, whereas the specificity pocket mutant E634Q showed an increased overall activity (Figure 2a).Figure 2.


Insights into the pre-initiation events of bacteriophage phi 6 RNA-dependent RNA polymerase: towards the assembly of a productive binary complex.

Sarin LP, Poranen MM, Lehti NM, Ravantti JJ, Koivunen MR, Aalto AP, van Dijk AA, Stuart DI, Grimes JM, Bamford DH - Nucleic Acids Res. (2009)

Biochemical characterization of φ6 RdRP mutants. (a) Summary of the RNA polymerization activities of the φ6 RdRP mutants in comparison to the WT or YKW(630–632)GSG mutant (initiation by back-priming) (8). (b–g) Screening of the optimal reaction conditions for the mutant polymerases in terms of temperature, pH, ammonium acetate concentration and divalent cation concentrations (Mn2+, Mg2+, Zn2+). All results have been normalized against the highest attained polymerization activity for each RdRP. (h) Electrophoretic mobility shift assay (EMSA) of the φ6 RdRP mutants. The effect of an increasing concentration (0.04, 0.08 and 0.16 mg/ml) of WT (lanes 2–4) and R30A (lanes 6–8) polymerases on the migration of the s+rep template in 8% native polyacrylamide gel. The baseline (white dotted line) determines the position of the control (s+rep without polymerase; lanes 1, 5 and 9). The numbers below lanes 2–4 and 6–8 indicate the retardation of the s+rep template in millimetres.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651803&req=5

Figure 2: Biochemical characterization of φ6 RdRP mutants. (a) Summary of the RNA polymerization activities of the φ6 RdRP mutants in comparison to the WT or YKW(630–632)GSG mutant (initiation by back-priming) (8). (b–g) Screening of the optimal reaction conditions for the mutant polymerases in terms of temperature, pH, ammonium acetate concentration and divalent cation concentrations (Mn2+, Mg2+, Zn2+). All results have been normalized against the highest attained polymerization activity for each RdRP. (h) Electrophoretic mobility shift assay (EMSA) of the φ6 RdRP mutants. The effect of an increasing concentration (0.04, 0.08 and 0.16 mg/ml) of WT (lanes 2–4) and R30A (lanes 6–8) polymerases on the migration of the s+rep template in 8% native polyacrylamide gel. The baseline (white dotted line) determines the position of the control (s+rep without polymerase; lanes 1, 5 and 9). The numbers below lanes 2–4 and 6–8 indicate the retardation of the s+rep template in millimetres.
Mentions: The polymerase reaction conditions preferred by the mutants were generally quite similar to those of the WT polymerase, showing only minor deviations in terms of temperature, pH, salt concentration and divalent cation concentrations (Figure 2a-g). Despite strong similarities between the optimal conditions, the template tunnel mutant R30A and the specificity pocket mutant E634Q retained polymerization activity at temperatures up to 55–60°C, i.e. high enough temperatures to cause RNA hydrolysis, whereas the other mutants retained WT characteristics with temperature optima of roughly 40°C (Figure 2b). The template tunnel rim mutants R30A and K541L displayed a significantly reduced level of RNA synthesis for both replication and transcription, whereas the specificity pocket mutant E634Q showed an increased overall activity (Figure 2a).Figure 2.

Bottom Line: In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3' terminus of the template and position it, so that a productive binary complex is formed.The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site.Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi 6 RdRP can be greatly enhanced.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki, Biocenter 2, Helsinki, Finland.

ABSTRACT
The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3' terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage phi 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi 6 RdRP can be greatly enhanced.

Show MeSH
Related in: MedlinePlus