Translating a nucleotide sequence
Often, when analysing new sequence we wish to translate a nucleotide sequence to amino acids to look for potential coding regions.
In this part of the exercise we will use a web based translation tool at the Swiss ExPASy (Expert Protein Analysis System) Molecular Biology Server.
You may have noticed that there is also a translate option in the BCM tools we used earlier and also translation tools exist at EBI and MBS.
However, our use of the tool at ExPASy serves as an introduction to the ExPASy site.
The ExPASy site is maintained by the Swiss Institute of Bioinformatics and contains a multitude of useful tools for protein analysis.
Click here to bring up the main page for the server.
You will be presented with a large table of options – have a look round to see if any of these will be useful to your research and then click here to take us to the Translate tool.
Again, we will be using our trusty glutamate racemase sequence. Copy it to the clipboard from its Word document or click here to get it. JUST COPY THE SEQUENCE, NOT THE ID HEADER (the id header is the line with > on it!)
Step 1: Paste your FASTA formatted sequence into the box labelled ‘Please enter a DNA or RNA sequence in the box below (numbers and blanks are ignored)’.
Step 2: Underneath the sequence entry box is a drop down list that allows you to vary the output format – have a look at the possible options.
Most commonly you’ll want to analyse any resulting amino acid sequence using the BLAST tool (see section 2) so we’ll set the output format to ‘Compact (“M”,”-“,no spaces)’
Click on the grey ‘Translate sequence’ button to perform the translation. (show me)
Step 3: You’ll be presented with the results of translation for all six possible reading frames for you sequence (three on the top strand (5’3’) and three for the complementary strand (3’5’)).
Look at the sequences. Which frame possesses the longest open reading frame?
Step 4: We can now look at our chosen frame in more detail – click on the blue label of the frame to examine (show me).
Step 5: You will see a close up view of the translation for that frame, showing the possible start codons highlighted in blue. Clicking on any of these start codons will produce a ‘virtual’ Swiss-prot type entry for your chosen protein sequence.
Click on the first Methionine in the sequence to try this (show me)
The sequence from this virtual Swiss-Prot entry can be copied and used to paste into other protein sequence analysis tools.
Copy your virtual entry and save it into your Word document.
Step 6: If you interested in 2d gels for functional characterisation of proteins then click on the coloured link labelled ‘DR’ in the entry.
There are a number of options at the bottom of the screen that now let you submit your sequence straight to BLAST (see section 2) and directly to other sequence analysis tools.
Try some if you are feeling brave. The ScanProsite tool is a good example. However, we’ll cover motif searching in section 3 so you’re on your own if you want to explore further from here!