Abstract
RNA primers for DNA replication are usually synthesized by specialized enzymes, the primases1. However, some replication systems have evolved to use cellular DNA-dependent RNA polymerase for primer synthesis1,2. The main requirement for the replication primer, an exposed RNA 3′ end annealed to the DNA template, is not compatible with known conformations of the transcription elongation complex3, raising a question of how the priming is achieved. Here we show that a previously unrecognized kind of transcription complex is formed during RNA polymerase-catalysed synthesis of the M13 bacteriophage replication primer. The complex contains an overextended RNA–DNA hybrid bound in the RNA-polymerase trough that is normally occupied by downstream double-stranded DNA, thus leaving the 3′ end of the RNA available for interaction with DNA polymerase. Transcription complexes with similar topology may prime the replication of other bacterial mobile elements and may regulate transcription elongation under conditions that favour the formation of an extended RNA–DNA hybrid.
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Acknowledgements
This work is dedicated to Dmitry Salonin who inspired it. We thank A. Mustaev for crosslinking reagents. This work was supported by an NIH R01 grant and a Borroughs Wellcome Career Award to K.S.
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Supplementary Figure 1
This figure shows a schematic view of formation of the priming complex on bacteriophage M13 origin of replication during primer synthesis by DNA-dependent RNA polymerase. The unusual topology of the origin leads to conformational rearrangement during RNA polymerase transcription that extrudes the 3' end of the primer RNA, base-paired to template DNA, from the RNAP catalytic centre and makes it available for interaction with DNA polymerase. (DOC 78 kb)
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Zenkin, N., Naryshkina, T., Kuznedelov, K. et al. The mechanism of DNA replication primer synthesis by RNA polymerase. Nature 439, 617–620 (2006). https://doi.org/10.1038/nature04337
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DOI: https://doi.org/10.1038/nature04337
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