RRM
2013 Rustbelt RNA Meeting
RRM
|
|
|
|
|
|
Poster abstracts
Abstract:
To expand our ability to utilize our previously developed comparative analysis of RNA digests (CARD) approach for tRNA analysis,1,2 we must establish reference (baseline) information related to tRNA modifications from beyond the proteobacteria phylum. One phylum of interest is the actinobacteria, which are gram-positive bacteria typically having a high G+C content. To establish a reference set of modifications for us to work with in this phylum, we are presently investigating Streptomyces griseus, which is best known for producing streptomycin. An LC-MS/MS analysis of the modified nucleosides from S. griseus tRNAs yielded 19 unique post-transcriptional modifications. In comparison to Bacillus subtilis, another gram-positive bacterium that is a member of the firmicutes phylum, S. griseus was found to lack mo5U, m5U, s4U, cmnm5U, cmnm5s2U, k2C and Q modified nucleosides. One of the more interesting absences in S. griseus is m5U (rT). This finding was particularly surprising as a BLAST analysis of the TrmA gene from Escherichia coli reveals a gene in S. griseus with high homology. To verify the nucleotide identity at position 54, we used LC-MS/MS to analyze RNase T1 digested S. griseus tRNAs. These experiments reveal that N-54 (in the conserved TPC loop) is either an unmodified uridine or possibly pseudouridine. These findings along with the other differences in S. griseus tRNA modifications will be presented.
References:
1. Siwei Li and Patrick A. Limbach (2012). Method for comparative analysis of ribonucleic acids using isotope labeling and mass spectrometry. Analytical chemistry, 84 8607–8613.
2. Siwei Li and Patrick A. Limbach (2013). Mass spectrometry sequencing of transfer ribonucleic acids by the comparative analysis of RNA digests (CARD) approach. The Analyst, 138 1386–1394.
Keywords: tRNA modification, Streptomyces griseus, mass spectrometry
