Part:BBa_K2282001

AmilCP (from BBa_K592009) containing the DS box sequence at the N-terminus

Usage and Biology

AmilCP is a coral chromoprotein which displays a strong blue color. The DSBox (Downstream Box) sequence increases the translation of mRNA at low temperatures (Mitta et al, 1997). At low temperatures translation of most transcripts is strongly reduced. The DSbox is a cis-acting mRNA element enabling assembly of the translation pre-initiation complex, likely through interaction with ribosomal protein S1 (Studer et al 2006, Duval et al, 2013, Qu et al 2012).

Source of this part

The DS Box sequence (Mitta et al, 1997) from the E.coli genome was inserted in the amilCP coding sequence after the first two residues. The DSBox size as defined in Mitta et al, 1997 is 14bp. In order to avoid introducing a frame shift error we added an adenine nucleotide at the 5’ end to increase the size from 14bp to 15bp by completing the 5’ codon.

Design consideration

Adding 15 nucleotides to a protein coding sequence raised some concern about its effect on protein stability and activity. We addressed this problem with molecular modelling to optimize the placement of the DS Box (see our wiki) and confirmed the results in the lab.

References

Masanori Mitta et al, Deletion analysis of cspA of Escherichia coli requirement of the AT-rich UP element for cspA transcription and the downstream box in the coding region for its cold shock induction, Molecular Microbiology (1997) 26(2), 321–335

Studer, Sean M., and Simpson Joseph. “Unfolding of mRNA Secondary Structure by the Bacterial Translation Initiation Complex.” Molecular Cell 22, no. 1 (April 7, 2006): 105–15. doi:10.1016/j.molcel.2006.02.014.

Duval, Mélodie, Alexey Korepanov, Olivier Fuchsbauer, Pierre Fechter, Andrea Haller, Attilio Fabbretti, Laurence Choulier, et al. “Escherichia Coli Ribosomal Protein S1 Unfolds Structured mRNAs Onto the Ribosome for Active Translation Initiation.” PLOS Biology 11, no. 12 (December 10, 2013): e1001731. doi:10.1371/journal.pbio.1001731.

Qu, Xiaohui, Laura Lancaster, Harry F. Noller, Carlos Bustamante, and Ignacio Tinoco. “Ribosomal Protein S1 Unwinds Double-Stranded RNA in Multiple Steps.” Proceedings of the National Academy of Sciences 109, no. 36 (September 4, 2012): 14458–63. doi:10.1073/pnas.1208950109.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]