Transcription Start Site (TSS): is the nucleotide/base in the gene from which the transcription begins. In other words, it is the first nucleotide that you see in a newly  transcribed RNA or the nucleotide directly attached to the 7-methyl Guanosine cap.

A gene whose transcription is initiated by a TATA box usually have a fixed TSS.  Approximately 70-80% of pol II promoter sequences contain a TATA or TATA-like element, with a consensus of TATAA, about 25-30 bases upstream of the TSS.

Broadly there are two ways of finding a TSS

1. In Silico

2. Experimentally 

TSS can be predicted using various software's, but let me warn you that these programs have very high false positive results. There are no software's or programs available which can predict a TSS with hundred percent accuracy.

The following programs can be used to find TSS In Silico:

PROMOTER 2.0 : It has been developed as an evolution of simulated transcription factors that interact with sequences in promoter regions. It builds on principles that are common to neural networks and genetic algorithms.

CorePromoter : This page contains software tools designed to predict putative Transcriptional Start Site (TSS) and hence the core-promoter defined in the interval (-60,+40).

Eponine: Eponine is a probabilistic method for detecting transcription start sites (TSS) in mammalian genomic sequence, with good specificity and excellent positional accuracy. Eponine models consist of a set of DNA weight matrices recognizing specific sequence motifs. Each of these is associated with a position distribution relative to the transcription start site.

TSS for a gene can also be found by searching different databases which has full 5'end of the gene. These databases can be searched by using different queries.

EST database: The EST database at NCBI has EST clones, some of which has been obtained by 5'RACE experiments. The RNA for such experiments are isolated by methods, such as cap-trapper or oligo-caping, which makes sure that you get the complete 5'end (TSS) of the gene.

DBTSS : This is a database of transcription start sites. It includes cDNA clones obtained by Oligo -capping method and represent a true TSS. Search this database by gene name, accession number etc.

Experimental:

TSS of a gene can be found experimentally. These are the most reliable methods compared to the one discussed above.

PRIMER EXTENSION: This method uses AMV Reverse Transcriptase and an end-labeled primer to reverse transcribe the target RNA into cDNA. The amount of cDNA synthesized is proportional to the amount of target RNA, and the length of the cDNA product reflects the distance from the primer to the 5´end of the RNA. Primer extension products are typically analyzed on denaturing polyacrylamide gels, and the use of labeled DNA markers allow the size of the primer extension product to be determined. But, the biggest disadvantage of this technique is, it will not work for the genes which has high GC content in their 5' region. (Kits available)

5' RACE: 5' Rapid amplification of cDNA ends is also a Reverse Transcriptase based method. There are various modified methods are available. RACE techniques couple cDNA synthesis with a method that attaches a "known" sequence to the end of the gene to enable PCR amplification. The PCR amplified product is cloned and several clones  will be analyzed by sequencing. (Several kits available)

S1 NUCLEASE MAPPING: In S1 Nuclease mapping, a DNA probe labeled at its 5'-end and which overlaps the Gene 5'-end is hybridized to the RNA. S1 nuclease, which is specific for either ssDNA or RNA, is used to digest the single-stranded DNA. The resulting 5'-labeled DNA probe is then "sized" via PAGE. Its size pinpoints the 5'-end of the Gene.

RIBONUCLEASE PROTECTION ASSAY (RPA): This method is same in principle as S1 nuclease mapping but here a labeled RNA probe which overlaps the gene 5'end is hybridized to the RNA. RNase, which is specific for ssRNA, is used to digest the RNA which is not hybridzed and remain single stranded. The resulting RNA:RNA hybrid is sized via denaturing PAGE.

Once we know the TSS, the next step is to find a promoter in the region upstream of the TSS.

Back to Promoter page

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