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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.

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RNA polymerization activity with RNA hybrids. RNA polymerization activity of WT φ6 RdRP (a) and R30A mutant (b), using different RNA hybrids. The results have been normalized against an internal control (polymerization activity with the s+ ssRNA template).
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Figure 4: RNA polymerization activity with RNA hybrids. RNA polymerization activity of WT φ6 RdRP (a) and R30A mutant (b), using different RNA hybrids. The results have been normalized against an internal control (polymerization activity with the s+ ssRNA template).

Mentions: To biochemically evaluate the process of binary complex formation, polymerase assays were performed with the above-described RNA hybrids in standard φ6 RdRP reaction mixtures. Assaying the RNA hybrids of the biologically relevant endogenous template (s+) with the WT polymerase revealed that almost no dsRNA was formed with the +0 to +2 hybrids. The +3 hybrid gave rise to a dsRNA synthesis of ∼50% compared to the control (the dsRNA yield for each polymerase mutant using the genomic s+ segment as a template), and moving the oligonucleotides further downstream (+4 to +6 hybrids) resulted in a product yield equalling that of the control (Figure 4a). The same observations also apply to mutants E165A and E634Q (Supplementary Figure 2a and c). Similar characteristics were observed for polymerase mutants R30A (Figure 4b) and K541L (Supplementary Figure 2b), although their dsRNA yield slightly exceeded that of the control with the +4 to +6 hybrids.Figure 4.


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)

RNA polymerization activity with RNA hybrids. RNA polymerization activity of WT φ6 RdRP (a) and R30A mutant (b), using different RNA hybrids. The results have been normalized against an internal control (polymerization activity with the s+ ssRNA template).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: RNA polymerization activity with RNA hybrids. RNA polymerization activity of WT φ6 RdRP (a) and R30A mutant (b), using different RNA hybrids. The results have been normalized against an internal control (polymerization activity with the s+ ssRNA template).
Mentions: To biochemically evaluate the process of binary complex formation, polymerase assays were performed with the above-described RNA hybrids in standard φ6 RdRP reaction mixtures. Assaying the RNA hybrids of the biologically relevant endogenous template (s+) with the WT polymerase revealed that almost no dsRNA was formed with the +0 to +2 hybrids. The +3 hybrid gave rise to a dsRNA synthesis of ∼50% compared to the control (the dsRNA yield for each polymerase mutant using the genomic s+ segment as a template), and moving the oligonucleotides further downstream (+4 to +6 hybrids) resulted in a product yield equalling that of the control (Figure 4a). The same observations also apply to mutants E165A and E634Q (Supplementary Figure 2a and c). Similar characteristics were observed for polymerase mutants R30A (Figure 4b) and K541L (Supplementary Figure 2b), although their dsRNA yield slightly exceeded that of the control with the +4 to +6 hybrids.Figure 4.

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