2009 Rustbelt RNA Meeting
RRM

 

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Poster number 53 submitted by Emily Maris

In vivo activity of B. thuringiensis and M. smithii Thg1 expressed in yeast

Emily Maris (Biochemistry Ohio State University), Maria Abad (Biochemistry Ohio State University), Jane Jackman (Biochemistry Ohio State University)

Abstract:
tRNAHis guanylyltransferase (Thg1) catalyzes the post-transcriptional addition of an essential G-1 residue to the 5' end of tRNAHis in eukaryotes. Thg1 homologs are found in some bacteria and archaea although the presence of Thg1 is not expected in these organisms due to a genomically encoded G-1. Intriguingly, in vitro activity assays show that bacterial and archaeal Thg1 add residues to the 5' end of tRNA through templated addition, as opposed to the non-templated addition observed in eukaryotes. Consistent with the differences between in vitro biochemical activities exhibited by yeast versus archaeal and bacterial Thg1, the majority of bacterial and archaeal Thg1 homologs will not support the growth of yeast thg1Ä strains with the notable exception of B. thuringiensis Thg1 (BtThg1) and M. smithii Thg1 (MsThg1), suggesting that both may have unique properties distinct from other archaeal and bacterial homologs.
The goal of this work is to further investigate the apparent discrepancy between the in vitro and in vivo activities exhibited by BtThg1 and MsThg1. First the growth rates of the thg1Ä strains containing yeast Thg1, BtThg1, or MsThg1 were compared and shown to be essentially the same, suggesting that BtThg1 and MsThg1 have the ability to act as efficiently as yeast Thg1 in vivo, despite their different in vitro activities. Second, tRNAs isolated from the same strains using a hot phenol method were probed for the presence of a G-1 using primer extension assays specific for tRNAHis; these assays showed that a single nucleotide was being added at the –1 position in all three strains. Unexpectedly, U-1 was also being incorporated into tRNAHis in vivo. Further assays will be done to determine if there are differences in the relative quantities of U-1 and G-1 addition and whether nucleotides are added to other tRNA substrates in vivo in any of these strains.

Keywords: yeast, tRNAHis, Thg1