Motif discovery exercise

Choose a set of potentially coregulated genes in E. coli from the E. coli ORF table. If you don't have a favorite set of genes, you can copy one of the following lists of genes:
- E. coli ArgR regulon
- E. coli PurR regulon
- E. coli LexA regulon
Extract upstream sequences of these genes
- Copy and paste the gene names of your E. coli regulon into the web-based program Operon_upstream. Run this program to extract the upstream regions of the genes you selected from the E. coli genomic sequence. The default parameters are fine. Copy or save the text of the results to a file with suffix *.txt.
  (Click here to view the E. coli genomic sequence.)
Run AlignACE
- Download AlignACE (Please note: this is an old version of AlignACE. Click here to download the latest version) and other files used in this exercise. Save the file NT_exercise.zip in a new directory on your local drive. This zip file contains several Windows files. Double-click on NT_exercise.zip and extract all of the files contained within it.
  NOTE: READ ALIGNACE LICENSING AGREEMENT HERE.
- Run AlignACE. The input to AlignACE is a list of unaligned sequences in FASTA format. Save the FASTA format upstream regions you obtained with the Operon_upstream program in Step 2 to a file with the suffix *.txt (i.e. argR.txt) on your local drive. Open a command prompt window and type the following command in your directory:
  - AlignACE -iyour_file.txt -g0.5
  (0.5 is the GC content of the E. coli genome). Just type "AlignACE" to view a list of all of the available command-line options.
  NOTE: For 2 kb of input sequence, AlignACE should take about 30 minutes on a 400 MHz processor. However, you should be able to see the first motifs showing up in the output file after ~5 minutes. The run time is approximately linear with the amount of input sequence.
Analyze the motifs found by AlignACE
- Generate sequence logos for each motif in your AlignACE output file in PostScript format using a web-based interface. In the web interface, select your AlignACE output file on your local drive (or use the file argR1.ace in the directory your downloaded if your job has not finished running yet). Copy the output in text format to a file with suffix *.ps in your directory. (sample file in the directory your downloaded: argR1.ps).
  To view the resulting PostScript file, download Ghostview if you do not already have a PostScript viewer on your system. (Save the file gsv27550.exe to your local drive and simply double-click to install).
- Run ScanACE from the command prompt to search for other instances of this motif in the genome. The ScanACE executable is located in the folder you downloaded with AlignACE. Type ScanACE at the command prompt in your directory to view the command-line options. Example:
  - Scanace -iyour_file.ace -zEC.fna -aecoli_ORF.txt -s200 -n1 -b
  Note: ScanACE should take about 30 seconds on a 400 MHz processor to search across the E. coli genome (CPU time linear with size of the genome).
- Calculate the site specificity score using a web-based interface. In the web interace, select your ScanACE output file from your local drive, as well as the original .txt file that you used as input to AlignACE (or simply choose the sample files argR1.scn and argR1.txt from the file that you downloaded).
- Compare to known E. coli motifs with CompareACE. In the web interface, select your AlignACE output file from your local drive. (Alignments for known E. coli DNA-binding proteins are found here).

Abigail Manson McGuire
Harvard Medical School Genetics Dept., Church Lab Alpert Bldg. 512 200 Longwood Ave. Boston, MA. 02115	Phone: (617) 432-0503 Fax: (617) 432-7266