A general RNA-templated RNA extension activity of E. coli RNA polymerase.

Abstract

Multi-subunit "DNA-dependent" RNA polymerases (RNAPs) have noncanonical RNA-directed RNA synthesis activity; this allows synthesis of complementary RNA from RNA templates. Such noncanonical RNAP activities are biologically significant, serving RNA pathogens such as hepatitis delta virus (HDV) and contributing to cellular gene regulation. Despite the broad biological implications of these processes, our understanding of the underlying RNAP mechanisms remains incomplete. Using E. coli RNAP, a multi-subunit RNAP, as a model, we describe here the general RNA-templated RNA extension activity of that enzyme. Our data argue that the 3' end of an added RNA template can fold back and pair with upstream bases in the template, creating an intramolecular primer-template duplex as short as 1-2 base pairs. The RNAP then extends this intramolecular duplex, incorporating nucleotides complementary to the template. RNA-templated RNA extension occurred in minutes and did not appear to be suppressed by the presence of a promoter-containing DNA template. Excepting oligonucleotides implicitly designed to prevent any possibility of 3' end self-priming, every RNA template we tested could be extended by the enzyme, highlighting the general nature of this reaction. These data define a general activity of a cellular RNAP. Unrestricted, this activity could contribute to emergence and replication of RNA-based agents such as HDV and viroids; if highly regulated, the activity could limit these same elements.