Poster abstracts
Poster number 65 submitted by Brandon Iwaniec
Identifying substrates 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)
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 some biologically relevant RNA species. Here, we used a variety of in vitro biochemical assays to gain a more in-depth picture of the biological role of MxTLP in M. xanthus. Addition of an essential G-1 nucleotide to the 5'-end of tRNAHis is an important function of members of the tRNAHis guanylyltransferase (Thg1) enzyme family. This activity has also been associated with one TLP, and therefore we sought to test whether MxTLP could exhibit a similar function. However, we demonstrated that MxTLP is not apparently needed for addition of the G-1 nucleotide in vivo in M. xanthus, based on primer extension assays of RNA isolated from MXTLP deletion strains. This observation is also consistent with the RNase P-dependent pathway for obtaining G-1 in bacteria, and our in vitro histidylation assays confirmed that the form of G-1-containing tRNAHis preferred by the M. xanthus histidyl tRNA synthetase is the form that would most likely result from post-transcriptional RNase P cleavage, rather than from addition by MxTLP. We also demonstrated that MxTLP can incorporate labeled nucleotides into RNA isolated from vegetative and developing cells, but with different patterns of incorporation, indicating that MxTLP may play distinct roles in each life cycle. Efforts to identify the substrates that are acted on during each life cycle stage using various approaches 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