Abstract
Little is known about how archaeal viruses perturb the transcription machinery of their hosts. Here we provide the first example of an archaeo-viral transcription factor that directly targets the host RNA polymerase (RNAP) and efficiently represses its activity. ORF145 from the temperate Acidianus two-tailed virus (ATV) forms a high-affinity complex with RNAP by binding inside the DNA-binding channel where it locks the flexible RNAP clamp in one position. This counteracts the formation of transcription pre-initiation complexes in vitro and represses abortive and productive transcription initiation, as well as elongation. Both host and viral promoters are subjected to ORF145 repression. Thus, ORF145 has the properties of a global transcription repressor and its overexpression is toxic for Sulfolobus. On the basis of its properties, we have re-named ORF145 RNAP Inhibitory Protein (RIP).
Highlights
In order to quantify the binding affinity, we produced a 32P-labelled variant of ORF145/RNAP Inhibitory Protein (RIP) and tested its interaction with RNA polymerase (RNAP) in electrophoretic mobility shift assays (EMSA). 32P-ORF145/RIP is resolved as a single distinct high-mobility band, while incubation with increasing amounts of RNAP led to the upshift of the signal to a new low-mobility band in a dose–response dependent manner (Fig. 1b)
In the context of the pre-initiation complexes (PICs), ORF145/RIP could interfere with the binding of transcription factor TFB core domain or DNA within the DNA-binding channel of RNAP, both would interfere with the PIC
EMSA experiments mimicking the transcription elongation complex demonstrate that ORF145/RIP can bind to RNAP in the context of the RNAP–DNA–RNA complex without disrupting it, while transcription elongation assays show that ORF145/RIP efficiently inhibits elongation
Summary
To assess how binding of ORF145/ RIP within the DNA-binding channel may affect the formation of DNA–TBP–TFB–TFE–RNAP transcription PIC, we performed EMSA experiments using 32P-labelled SSV1 T6 promoter a 32P ORF145/RIP inhibits transcription from host as well as virus promoters in agreement with its ability to directly target the RNAP.
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