We have recently validated Ribonuclease HI (RNase HI) as a new drug target for M. tuberculosis1. RNase HI carries out metal-aided hydrolysis of the RNA moiety of an RNA:DNA hybrid. These hybrids occur during lagging strand replication as Okazaki fragments and as R-loops when nascent RNA invades the genome and hybridizes to its template DNA. These hybrids block polymerase activity, and so resolution of RNA:DNA hybrids is a crucial activity in all cells. Although active as a single-domain enzyme in many bacteria, RNases HI can also be associated with domains that help to localise it to substrate eg hybrid-binding domain in human RNase HI, or with catalytic domains that are associated with nucleotide metabolism eg polymerase domain in HIV reverse transcriptase.
In M. tuberculosis the RNase HI domain is associated with an acid phosphatase domain which catalyses the last step in vitamin B12 biosynthesis. Although the catalytic link between B12 biosynthesis and RNA hydrolysis is cryptic, this acid phosphatase domain is necessary for full activity of RNase HI, prompting an investigation into the mechanism for this. We show through SAXS analysis that despite a 20 amino acid linker region, this domain is closely associated with the RNase HI domain. We also show, using surface plasmon resonance and electrophoretic mobility shifts assays, that, in addition to its catalytic activity, the acid phosphatase domain promotes association of RNase HI to substrate, revealing an unexpected moonlighting activity.