Poster abstracts
Poster number 55 submitted by Brandon Iwaniec
Determining the role of the 3’-5’ reverse polymerase in Myxococcus xanthus
Brandon Iwaniec (Chemistry and Biochemistry, The Ohio State University), Ashanti Matlock (Chemistry and Biochemistry, The Ohio State University), Jane Jackman (Department of Chemistry and Biochemistry )
Abstract:
Abstract tRNAHis guanylyltransferase-like proteins (TLPs) can catalyze nucleotide addition to RNA substrates in the opposite direction (3'-5') of all known canonical RNA polymerases. This biochemical activity is seen in vitro with bacterial TLPs, which can add nucleotides to the 5'-ends of tRNA substrates. However, no physiological relevant substrates have been identified to date in any bacterial species that has a TLP. Myxococcus xanthus, a bacterium, exhibits defects in starvation-induced fruiting body formation and sporulation when its MXTLP gene is deleted, suggesting that this enzyme plays a role in maturation or maintenance of one or more biologically relevant RNAs. We sought to test whether MxTLP catalyzes the addition of an essential G-1 nucleotide to the 5'-end of tRNAHis, which is an important function of some eukaryotic members of the tRNAHis guanylyltransferase (Thg1) family. This role for MxTLP was subsequently ruled out in M. xanthus based on the results of primer extension and histidylation assays, both of which gave results that are consistent with the RNase P-dependent pathway for maturation of tRNAHis in bacteria that does not require the activity of MxTLP. Therefore, we performed several biochemical studies to identify substrate and activity preferences of MxTLP, to identify patterns that may help suggest the enzyme's biological role in M. xanthus. We demonstrated that in vitro MxTLP can incorporate labeled nucleotides into specific RNA species isolated from vegetative and developing cells, consistent with a function for the enzyme involving other substrates than tRNA. We also recently uncovered a unique requirement of MxTLP for Mg2+ concentrations that are higher than those of other members of the Thg1/TLP family, suggesting possible mechanistic differences for the enzyme that may be relevant to its physiological role. Efforts are ongoing to identify RNAs that are acted on by MxTLP and to kinetically characterize the unique Mg2+-dependence exhibited by MxTLP, and 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