Predicting the transcription start site (TSS) in bacteria can be challenging. One approach is to use position weight matrices (PWMs) to define conserved motifs at the sigma-factor binding site¹. However, this method is known to result in a large number of false positive predictions³.

For Lactococcus lactis NZ9000, you could try using the PePPER webserver for prediction of prokaryote promoter elements and regulons⁷. This webserver offers an all-in-one method of data mining for transcription factors (TFs), transcription factor binding sites (TFBSs), promoters, and regulons for any bacterial genome⁷.

(1) Improved prediction of bacterial transcription start sites. https://pubmed.ncbi.nlm.nih.gov/16287942/.

(2) Improved prediction of bacterial transcription start sites. https://academic.oup.com/bioinformatics/article/22/2/142/424439.

(3) PePPER: a webserver for prediction of prokaryote promoter elements and .... https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-13-299.

(4) ORFfinder Home - NCBI. https://www.ncbi.nlm.nih.gov/orffinder/.

(5) The prediction of bacterial transcription start sites using SVMs. https://pubmed.ncbi.nlm.nih.gov/17117497/.

(6) THE PREDICTION OF BACTERIAL TRANSCRIPTION START SITES USING SVMS .... https://www.worldscientific.com/doi/abs/10.1142/S0129065706000767.

(7) Genome sequences of Lactococcus lactis MG1363 (revised) and NZ9000 and .... https://pubmed.ncbi.nlm.nih.gov/20639323/.

(8) Genome-scale exploration of transcriptional regulation in the nisin Z .... https://pubmed.ncbi.nlm.nih.gov/32123183/.

@Dr. Antonov. Thank you.

The concensus -10 region of a bacterial promoter is TATAAT.

Are TSS always 6-7bp away from the -10 sequences?

And do they always start from nucleotide G?

This is what i wanted to know too..

Predicting the transcription start site (TSS) in bacteria is a challenging task because there is no universal prokaryote transcription initiation DNA motif ³. However, some common features can be used to identify potential TSSs, such as:

- The presence of a Pribnow box (TATAAT) 10 base pairs upstream of the TSS and a conserved sequence (TTGACA) 35 base pairs upstream of the TSS. These are the binding sites for the sigma-70 factor that recruits RNA polymerase to initiate transcription ².

- The presence of a ribosome binding site (RBS) or Shine-Dalgarno sequence (AGGAGG) a few base pairs upstream of the start codon. This is the binding site for the 16S rRNA of the ribosome that initiates translation ².

- The presence of a start codon (ATG, GTG, or TTG) that encodes for methionine or valine as the first amino acid of the protein ².

There are some tools available online that can help you to predict TSSs in bacteria based on these features and other criteria, such as:

- PePPER: a webserver for prediction of prokaryote promoter elements and regulons ². This tool allows you to input a DNA sequence and search for potential promoters and TSSs using a combination of PWMs, SVMs, and distance models. You can also compare your results with existing regulon data from different databases.

- BPROM: a bacterial promoter prediction program . This tool allows you to input a DNA sequence and search for potential promoters and TSSs using PWMs and statistical models. You can also select different sigma factors and species-specific parameters to improve your predictions.

- TSSAR: an automated tool to annotate TSSs in prokaryotic genomes . This tool allows you to input a genome sequence and search for potential TSSs using RNA-seq data and machine learning algorithms. You can also visualize your results on a genome browser and download them in different formats.

(1) Identification of Translation Start Sites in Bacterial Genomes. https://pubmed.ncbi.nlm.nih.gov/33765270/.

(2) PePPER: a webserver for prediction of prokaryote promoter elements and .... https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-13-299.

(3) Improved prediction of bacterial transcription start sites .... https://academic.oup.com/bioinformatics/article/22/2/142/424439.

The consensus -10 region of a bacterial promoter is TATAAT ¹. However, the distance between the -10 region and the TSS is not always 6-7 bp. It can vary from 3 to 12 bp depending on the promoter class and the sigma factor involved ². For example, for sigma-70 promoters, the distance is usually 7 bp, but for sigma-28 promoters, it can be as short as 3 bp ².

The nucleotide at the TSS is not always G either. It can be any of the four bases, but G and A are more common than C and T ³. The frequency of each base at the TSS depends on the promoter class and the sigma factor involved as well ³. For example, for sigma-70 promoters, G is the most frequent base (about 60%), followed by A (about 30%), C (about 7%), and T (about 3%) ³.

(1) Addgene: Promoters. https://www.addgene.org/mol-bio-reference/promoters/.

(2) bTSSfinder: a novel tool for the prediction of promoters in .... https://academic.oup.com/bioinformatics/article/33/3/334/2584478.

(3) Genome-wide determination of transcription start sites reveals new .... https://pubmed.ncbi.nlm.nih.gov/28412515/.

Don't forget that the transcription start site might be very far away from the ORF, especially if the ORF is part of an operon. So don't just look immediately upstream from the ORF.

You just have to sequence the vicinity of the beginning of your ORF