Determination of the transcription start sites of heterologous promoters in Actinoplanes sp. SE50/110 by 5'-end specific transcriptome sequencing

The promoter structure influences binding and clearance of RNA polymerase and therefore substantially influences expression of a gene. A promoter usually consists of a -10 and a -35-region, an extended -10-motif and A+T-rich upstream promoter elements. Most of these elements are optional, whereas the -10-region is essential (Albersmeier et al. 2017). Knowledge about the transcription start sites (TSS) of genes allows genome-wide localization and determination of the promoter regions. In our group, a special protocol for the amplification of primary transcripts was developed, including the capture of primary transcripts, rewriting them into cDNA (complementary DNA) and amplification in the further course of the protocol (Pfeifer-Sancar et al. 2013). Here, TSS were manually determined with special regard to the heterologous promoters. For each construct, at least one and up to three different TSS were found, leading to the identification of one or several -10-core-hexamers. These were located mostly 6 to 7 nucleotides upstream of each TSS, which corresponds to the average distance of 6.4 nt described for Actinoplanes sp. SE50/110 by Schwientek et al. (2014).