Nucleic Acids Research Advance Access originally published online on January 7, 2009
Nucleic Acids Research 2009 37(4):1182-1192; doi:10.1093/nar/gkn1035
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2009, Vol. 37, No. 4 1182-1192
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Insights into the pre-initiation events of bacteriophage 
6 RNA-dependent RNA polymerase: towards the assembly of a productive binary complex
1Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki, Biocenter 2, 00014 University of Helsinki, Finland and 2Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Oxford University, Oxford OX3 7BN, UK
*To whom correspondence should be addressed. Tel: +358 9 19159100; Email: dennis.bamford{at}helsinki.fi Present address: Alberdina A. van Dijk, Biochemistry Division, North-West University, Private Bag X6001, 2520 Potchefstroom, South Africa
Received October 17, 2008. Revised December 9, 2008. Accepted December 11, 2008.
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 
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 6 RdRP can be greatly enhanced.