TSSs located intergenic region without cognate gene were annotated as orphan (o).
The literature and database entries suggest additional TSSs located further upstream, for example at position −45.
In addition, this strategy overlooks genuine TSSs located within genes and in antisense orientation.
"Secondary TSSs" were assigned to TSSs located on intergenic regions and separated more than 500 bp from the adjacent annotated ORFs.
After a complete review of the TSSs set and comparison to the raw RNA-seq data set, 644 manually determined TSSs were added to the group of TSSs, while 731 false-positive TSSs and 119 TSSs located upstream of tRNA or rRNA genes were removed.
The two DPE-L elements preferentially enhance TAF1-dependent transcription from TSS located at the 5' end of the cluster of multiple start sites in the class I promoter, while GLE increases transcription from all TSSs.
The existence of alternative promoters for a single gene is one explanation that could account for multiple TSS, as exemplified by pstS, which encodes a phosphate-binding periplasmic protein, and was previously shown to have two TSS located downstream from two PhoB binding motifs (B1 and B2) corresponding to TSS S8 and S9 of Figure 1d [ 37].
TSS located within an annotated gene were designated internal TSS (iTSS) and when the TSS was located on the antisense strand in, or within 100 bp of an annotated gene, it was designated an antisense TSS (aTSS).
To find alternative TSSs located in one TSS region, an increase (2-fold) of +TAP signal at least 5 nucleotides in a TSS region was detected.
Alternatively, post-transcriptional processing of the 5′-UTR of mRNAs may account for multiple mTSS located downstream of TSS associated with conserved promoter motifs, as is plausible for six TSS of pstS (S1, S3, S4, S5, S8, and S9 in Figure 1d) and four TSS of ctrA (S1, S3, S4, and S5 in Figure 1d).
