In this practice we will use consensus sequences of several well-known binding sites to find potential binding sites in a set of putative promoter regions corresponding to coexpressed genes in a DNA-microarray experiment. Consensus sequences are extracted from: P. Bucher. Journal of molecular biology 212: 563-578 (1990)

ADVICE: It is very useful to open 2 or more browser windows, preserving this text in one of them and running the exercise using another one.

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Input sequences:
6 genes of Drosophila melanogaster.

WWW tools:
Regulatory Sequence Analysis Tools (RSA) by Jacques van Helden from SCMBB - Service de Conformation des Macromolécules Biologiques et de Bioinformatique (Université Libre de Bruxelles).

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Step 1: Exact matches of TATA-box consensus: STATAAAWR

• Read the promoter regions in fasta format.

• Connect to RSA tools server.

• In the menu (left frame), click over Pattern matching: dna-pattern (strings).

• Type the consensus pattern into the Query pattern box.

• Copy and paste the promoter regions into the Sequence box.

• Choose "direct only" in the Search strands selector.

• Click the button Go to submit the query.

• -- NO EXACT MATCHES WILL BE FOUND (go next step) --

Step 2: Partial matches of TATA-box consensus: STATAAAWR

• Repeat the process but increasing the number of allowed mismatches in the pattern (try 1, 2 and 3 in Substitutions box).

• Results will be displayed below the headline
  "PatID Strand Pattern SeqID Start End Matching_word Score"

• Click the button Feature map to enter a new menu about plotting the results.

• Click the button Go to obtain a graphical output of the reported matches. Browse across the interactive map.

Questions:

1. Real TATA-boxes are supposed to appear 20 bp before Transcription Start Site (TSS). How many of the TATA boxes are in this range? NOTE: TSS annotations might easily contain errors and therefore ranges and distances will be useless.
2. How many occurences will you get if you try 9 substitutions (everything)? Do you get one occurence in every position of the sequence? Why not? Think about the option prevent overlapping matches . Try switching it off.

Results:

Mismatches	Output	Map
1	X	X
2	X	X
3	X	X

Mismatches	no overlap	overlap
9	X	X

Extra work:

Try to find new putative binding sites with these other consensus:

• GC-box consensus: GGGCGG
• CAAT-box consensus: RRCCAATS