Poster abstracts

Poster number 16 submitted by Brandon Iwaniec

Determining the role of the 3’-5’ reverse polymerase in Myxococcus xanthus

Brandon Iwaniec (OSBP), Ashanti Matlock (OSBP), Jane Jackman (Department of Chemistry and Biochemistry )

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 the 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 in M. xanthus, based on primer extension assays of RNA isolated from MXTLP deletion vs. wild-type strains. This observation is consistent with the RNase P-dependent pathway for obtaining G-1 in bacteria, and the results of in vitro histidylation assays, in which we confirmed that M. xanthus histidyl tRNA synthetase prefers the form of G-1-containing tRNAHis that would most likely result from post-transcriptional RNase P cleavage, rather than from addition by MxTLP. Using a variety of in vitro biochemical assays, we sought to gain a more in-depth picture of the biological role of MxTLP in M. xanthus. We demonstrated that MxTLP can incorporate labeled nucleotides into specific RNA species isolated from vegetative and developing cells. Efforts to identify RNAs that are acted on by MxTLP are underway, and these results will be used to provide the first look at the potential biological function of a TLP in any bacterial system.

Keywords: Reverse polymerization , tRNA, Myxococcus xanthus