Poster abstracts
Poster number 70 submitted by Brandon Iwaniec
Investigating functions outside of tRNA for a bacterial 3'-5' reverse polymerase from the Thg1/TLP superfamily
Brandon Iwaniec (Chemistry and Biochemistry, The Ohio State University), Susanne Mueller (Department of Microbiology and Immunology, Medical College of Wisconsin), John. R. Kirby (Department of Microbiology and Immunology, Medical College of Wisconsin), Jane Jackman (Chemistry and Biochemistry, The Ohio State University)
Abstract:
tRNAHis guanylyltransferase (Thg1)-like proteins (TLPs) catalyze nucleotide addition to RNA substrates in the opposite direction (3'-5') of canonical RNA polymerases. This biochemical activity is seen in vitro with bacterial TLPs, which can act on the 5'-ends of tRNA substrates, similar to the known biological functions of the two characterized eukaryotic TLPs. However, no physiologically relevant substrates for any bacterial TLPs have been identified to date and there is no known requirement to repair tRNA in these species that would make use of the observed in vitro activity. Here we report the first investigation of the biological function of the 3'-5' RNA polymerase in the bacterium Myxococcus xanthus. When the M. xanthus TLP (MxTLP) gene is deleted, the cells exhibit defects in starvation-induced fruiting body formation and sporulation, suggesting that this enzyme plays a role in maturation or maintenance of one or more biologically relevant RNAs. We used the Δmxtlp strain to rule out a function for MxTLP in adding of an essential G-1 nucleotide to the 5'-end of tRNAHis, which is an important function of some eukaryotic members of the Thg1 family. These results are consistent with the existence of an alternative RNase P-dependent pathway for incorporation of G-1 into tRNAHis in bacteria like M. xanthus, whereby this residue is encoded in the tRNA gene and not necessary to be added post-transcriptionally. Instead, biochemical studies with purified MxTLP demonstrate that the enzyme can incorporate labeled nucleotides into several larger RNA species isolated from vegetative and developing cells, consistent with a function for the enzyme involving substrates other than tRNA. Efforts are ongoing to identify and test RNA targets for MxTLP activity using enhanced CLIP (eCLIP) that would be leading candidates as substrates of MxTLP. These results will be used to help elucidate the first biological function of a TLP in any bacterial system.
Keywords: Reverse polymerization , tRNA, Myxococcus xanthus