How do signal sequences/peptides work in bacteria, and how can we use them?

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What are signal sequences?

Signal sequences are "leader" amino acids on the amino end of a polypeptide. They are recognized by soluble signal recognition proteins and docking proteins that facilitate the attachment of the ribosome-peptide complex to the cell membrane and the translocation of the polypeptide. The signal sequence is cleaved off of the preprotein by a signal peptidase that recognizes the signal sequence's 3-D conformation rather than sequence. The remaining "mature" protein then passes through the cell membrane. As of January 2008 no inhibitors of the signal pepsidase cleavage process have been described. Signal sequences are restricted to general rules about amino acid placement. The signal sequence itself, from amino to carboxy terminus, is described as follows: the positively-charged "n-region" of roughly 1-5 amino acids, the "core" or "h-region" of about 7-15 hydrophobic amino acids, and lastly the cleavage or carboxy-terminal of principally hydrophilic but rarely charged amino acids. Following the carboxy end of the signal sequence are the amino acids of the mature protein that will ultimately be transported across the designated membrane. Of important note is the (-3,-1)-rule, which states that the amino acids in the -1 and -3 positions must be uncharged and small in size (usually alanine). -3 and -1 refer to three amino acids and one amino acid, respectively, from the mature protein's first amino acid to the amino terminus of the signal sequence.

How can we use signal sequences in our project?

Signal sequences would allow us to remove the FSL from the protein. We would have to insert a signal sequence that E. coli can recognize between the FSL and the wild type peptide sequence. This would potentially allow us to remove the entire FSL and signal sequence from the protein before it is fully translated.

Questions That Need Addressed

  1. Will using this technique require that the reporter protein be excreted from the cell (which would pose a problem for antibiotic resistance proteins)?
  2. What are some examples of the use of signal sequences in E. coli, both in nature and the lab?
  3. What are the length constraints for signal sequences?
  4. How often is the signal sequence found in the E. coli genome, and will this pose a problem?
  5. Are signal sequences adaptable to different proteins (ie could we insert one before GFP)?
  6. How are signal peptidases regulated by E. coli?
  7. Is this the mechanism by which E. coli excretes proteins into its extracellular space?
  8. Will adding the FSL disable the signal sequence? The literature suggests that the amino end must carry an amino acid-generated positive charge. This may not be possible because the FSL must be on the amino end of the signal sequence in order to be removed.


  1. Leader chemical constraints, MOST RELEVANT, full text
  2. Highly relevant but partial paper--is there any chance of getting the full text for free?
  3. General information and predicting cleavage sites