Poster abstracts

Poster number 17 submitted by Malithi Jayasinghe

Reverse (3'-5') RNA polymerases: Applications beyond tRNA(His) maturation

Malithi I. Jayasinghe (Department of Chemistry and Biochemistry, The Ohio State University), Krishna Patel (Department of Chemistry and Biochemistry, The Ohio State University), Jane E. Jackman (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
The discovery of the reverse (3'-5') RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs) that incorporate nucleotides in the reverse (3'-5') direction as opposed to all other known RNA and DNA polymerases, opened many new possibilities, with potential applications for incorporating templated nucleotides to the 5'-end of nucleic acids that did not previously exist.1, 2 Previous studies on TLPs from a variety of organisms have demonstrated a generally greater flexibility in accommodating diverse RNA substrates than their Thg1 enzyme counterparts, consistent with the varied physiological roles for these enzymes.3, 4 However, studies to date have mainly focused on nucleotide addition to highly structured RNAs such as tRNAs. Consequently, general principles governing how well specific substrate/template sequences can be accommodated by different TLPs or the optimal nucleotide preferences that are required for efficient multiple nucleotide incorporation by TLPs have not been defined. Here, we investigated the feasibility of using TLPs for multiple nucleotide incorporation to the 5'-end of a model RNA duplex substrate designed to elicit these general features of RNA recognition by TLPs. Using optimized assay conditions, we demonstrated a remarkable capacity of several TLP enzymes to accommodate short RNA substrate-template duplexes of varying lengths and varying nucleotide combinations with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to 8 bases to 5'-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5'-end labeling tool.

References:
1. Gu, W., Jackman, J. E., Lohan, A. J., Gray, M. W., and Phizicky, E. M. (2003) tRNAHis maturation: An essential yeast protein catalyzes addition of a guanine nucleotide to the 5' end of tRNAHis, Genes & Development 17, 2889-2901.
2. Jackman, J. E., and Phizicky, E. M. (2006) tRNAHis guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G-1 addition, PNAS 103, 8640-8645.
3. Rao, B. S., Maris, E. L., and Jackman, J. E. (2011) tRNA 5'-end repair activities of tRNAHis guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea, NAR 39, 1833-1842.
4. Long, Y. C., Abad, M. G., Olson, E. D., Carrillo, E. Y., and Jackman, J. E. (2016) Identification of distinct biological functions for four 3'-5' RNA polymerases, NAR 44, 8395-8406

Keywords: Reverse RNA polymerase, Post transcriptional 5-end labeling, Template-dependent nucleotide incorporation