🧬 1. What is a promoter?
A promoter is a DNA region — usually located upstream of a gene — where
transcription is initiated. It contains:
- Core promoter (~40 bp around the TSS) — binds RNA polymerase II and general TFs.
- Proximal promoter (~250 bp upstream) — contains primary regulatory elements.
- Distal enhancers / silencers (up to several kb away) — modulate expression in a
tissue- and time-specific manner.
Key idea: Promoter activity is determined by the combinatorial binding of
transcription factors (TFs) to short DNA motifs.
✂ 2. Promoter Extraction — How and How Much?
You need to define which region to analyse. Common choices:
| Region | Typical size | When to use |
|---|
| Core promoter | −50 to +50 bp | Studying TSS, TATA box, Inr |
| Proximal promoter | −500 to +100 bp | Standard for most studies |
| Extended promoter | −1,000 to +100 bp | Default in PlantCARE / PLACE |
| Long-range | −2,000 to −3,000 bp | Enhancer / silencer discovery |
How to find the TSS
- From annotation — use NCBI / Ensembl / Phytozome gene models (5' UTR start).
- From experimental data — CAGE-seq, TSS-seq, or 5' RACE.
- Predicted — look for a TATA box approximately 25–35 bp upstream.
Warning: If you extract the wrong strand or the wrong TSS, all downstream motif
predictions will be wrong. Always verify the orientation of your gene.
🔬 3. Core Promoter Elements — Interpretation
| Element | Consensus | Position | Function |
|---|
| TATA box | TATAWAW | −25 to −35 | Binds TBP; positions RNA Pol II. Presence → focused TSS. |
| CAAT box | GGVCAATCT | −70 to −80 | Enhances transcription; binds NF-Y / CBF. |
| GC box | GGGCGG | Variable | Binds Sp1; often in housekeeping genes. |
| Inr (Initiator) | YYANWYY | Overlaps TSS | Positions the TSS precisely. |
| BRE | GSGCG | −35 to −40 | Binds TFIIB; stabilises preinitiation complex. |
| DPE | RGWCGTG | +25 to +30 | Binds TFIID; common in TATA-less promoters. |
What to look for in results
- Presence of a TATA box — indicates a regulated, tissue-specific promoter.
- Absence of TATA box + presence of GC box / CpG island — housekeeping or
broadly expressed gene.
- Multiple CAAT boxes — strong, constitutive expression.
- BRE / Inr clusters — precise TSS usage.
🎯 4. TF-Binding Site Prediction — Reading the Output
Each row in the TF-binding site table represents a predicted binding event
for a transcription factor whose consensus motif matches the sequence.
Motif families included in this tool
| Motif | Consensus | TF | Role |
|---|
| ABRE | ACGTG | bZIP (ABF/AREB) | ABA signalling, drought |
| DRE/CRT | RCCGAC | DREB/CBF | Cold & dehydration response |
| MYB | YAACKG | MYB | Secondary metabolism, stress |
| MYC | CANNTG | MYC (bHLH) | JA signalling, development |
| G-box | CACGTG | bZIP / bHLH | Light, ABA, stress |
| W-box | TTGAC | WRKY | Pathogen defence, senescence |
| GT element | GRWAAW | GT-1 | Light-responsive |
| I-box | GATAAG | GATA / bZIP | Light, nitrate |
| LTR | CCGAAA | ICE1 / CBF | Cold response |
| HSE | AGAAY | HSF | Heat-shock response |
| CArG box | CCWGG | MADS | Floral / developmental |
| AuxRR | TGTCTC | ARF | Auxin response |
| GARE | TAACRRA | GAMYB | GA response (seed) |
| E-box | CANNTG | bHLH | Development, JA |
| SORLIP | GCCAC | HY5 / bZIP | Light signalling |
| Skn-1 | GTCAT | WRKY / DOF | Endosperm expression |
| DOF core | AAAG | DOF | Seed, tissue-specific |
| NAC BS | CATGTG | NAC | Stress, development |
| TGACG | TGACG | TGA (bZIP) | SA / MeJA signalling |
| CGTCA | CGTCA | bHLH / MYC | MeJA signalling |
Reading the results table
- Motif — the element name (e.g. "W-box").
- TF / Family — the predicted binding factor(s).
- Position — 1-based coordinate in your input sequence.
- Strand — "+" means the motif matches the forward strand; "−" means the
reverse complement.
- Match — the actual sequence bound.
What the summary tables mean
| Observation | Interpretation |
|---|
| Many W-boxes | Likely pathogen / defence responsive. |
| Multiple ABREs + DREs | ABA- and drought-responsive promoter. |
| Many G-boxes + GT elements | Light-regulated promoter. |
| Cluster of DOF / Skn-1 | Seed / endosperm-specific expression. |
| Mixed hormone motifs (ABRE, TGACG, CGTCA) | Broad stress-responsive promoter. |
Remember: These predictions are computational. A predicted TF-binding site
does not prove that the TF binds in vivo. Validate with EMSA, ChIP-qPCR, or
yeast one-hybrid assays.
🔎 5. TF Identification — Confidence & Validation
The "TF identification" tab lists the actual transcription factors whose binding
motifs appear in your sequence. Each hit is given a confidence level:
| Confidence | Criteria | Recommended action |
|---|
| High | Motif ≥ 6 nt with an exact consensus | Prioritise for EMSA / ChIP. |
| Medium | Motif ≥ 6 nt with degeneracy | Validate with complementary tools. |
| Low | Short or highly degenerate motif | Treat as a weak hypothesis. |
Recommended validation experiments
- EMSA / gel shift — confirms TF–DNA binding in vitro.
- ChIP-qPCR — confirms TF–DNA binding in vivo.
- Yeast one-hybrid (Y1H) — screens cDNA library for binders.
- Dual-LUC reporter assay — tests whether the promoter drives expression and
whether the TF activates or represses it.
- Mutagenesis — mutate the predicted motif; if activity changes, the site is
functional.
Best practice: Combine in silico prediction (this tool) with
in vitro (EMSA) and in vivo (ChIP, dual-LUC) validation before drawing
conclusions.
🏝 6. CpG Islands & Methylation
A CpG island is a genomic region ≥ 200 bp with:
- GC content ≥ 50%, and
- observed/expected CpG ratio ≥ 0.6.
Why they matter
- ~70% of human promoters are associated with CpG islands.
- Unmethylated CpG islands → active, housekeeping-type promoters.
- Methylated CpG islands → silenced genes.
- In plants, CpG methylation also regulates transposons and imprinting.
Interpretation: A CpG island overlapping the TSS region is a strong indicator
of a broadly expressed gene with multiple TSSs.
🚀 7. Workflow & Pitfalls
Recommended workflow
- Retrieve the gene + upstream sequence (NCBI, Ensembl, Phytozome).
- Identify the TSS (annotation or TATA-box prediction).
- Extract −1,000 to +100 bp (Tab 1).
- Analyse core elements and CpG islands (Tab 2).
- Predict TF-binding sites (Tab 3).
- List candidate TFs (Tab 4) — sort by confidence.
- Cross-validate with PlantCARE / PLACE / JASPAR / MEME.
- Validate experimentally (EMSA, ChIP, dual-LUC).
Common pitfalls
Wrong strand: Extracting the reverse-complement of a gene gives meaningless motifs. Always verify orientation.
Short motifs: A 4–5 nt motif occurs by chance every ~256–1,024 bp. Don't over-interpret single hits.
Species specificity: Motifs trained on human/yeast may not hold in plants. Use plant-specific databases.
Motif ≠ function: A predicted binding site can be silent in vivo due to
chromatin state, methylation, or competing factors.
Best practice: Combine multiple tools (this one + PlantCARE + JASPAR + MEME),
and always confirm at least one prediction experimentally.
Recommended external resources
- PlantCARE — plant cis-acting regulatory elements
- PLACE — plant DNA motifs database
- JASPAR — open-access TF-binding profiles
- MEME Suite — motif discovery & scanning
- PlantTFDB — plant TF classification
- NCBI / Ensembl / Phytozome — sequence retrieval