The method used two unique Epicentre enzymes, RNA 5'-Polyphosphatase and Terminator Exonuclease, as part of a procedure to isolate and process mRNA from bacterial total RNA. The survey generated important information by locating transcription starts, 5'-untranslated regions, and other regulatory regions found in the transcriptome, enabling a comparison of two otherwise closely related organisms.
The technique outlined in the report is similar to that in the ExactSTART™ 5' & 3' RACE Kit. Total RNA was treated with Terminator Exonuclease to remove rRNA and degraded mRNA, enriching the sample in full-length mRNA containing 5'-triphosphorylated ends. The mRNA was treated with 5'-RNA Polyphosphatase to convert the 5'-triphosphates into 5'-monophosphates. The 5'-monophosphorylated mRNA was ligated to an adaptor molecule containing Illumina sequences using T4 RNA Ligase, and then reverse-transcribed using a modified Illumina RT primer. The resulting library was PCR-amplified and sequenced on an Illumina GAII sequencer.
The data generated were used to perform comparative genomic/gene expression studies between the two closely related organisms; the researchers identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Comparison of promoter regions and other regulatory sites revealed that 70% of the primary transcripts were expressed during exponential growth; this similarity changed dramatically when comparing TSS and regulatory elements.
The authors conclude that their comparative approach provides "a starting point for the determination of conserved and specific features of the transcriptional output of closely related bacteria at single nucleotide resolution."
Kim D et al. (2012). Comparative Analysis of Regulatory Elements between Escherichia coli and Klebsiella pneumoniae by Genome-Wide Transcription Start Site Profiling. PLoS Genetics, 8 (8) PMID: 22912590