Part:BBa_K2615005

Csy4-Y176F, the No.3 member of Csy4 family.

Csy4 is a 21.4 kDa protein that binds and cleaves at the 3' side of a stable RNA hairpin structure via sequence- and structure-specific contacts. Csy4 binds its substrate RNA with extremely high affinity and functions as a single-turnover enzyme. Tight binding is mediated exclusively by interactions upstream of the scissile phosphate that allow Csy4 to remain bound to its product. Substrate specificity is achieved by RNA major groove contacts that are highly sensitive to helical geometry, as well as a strict preference for guanosine adjacent to the scissile phosphate in the active site. A highly basic a-helix docks into the major groove of the hairpin and contains multiple arginine residues that form a network of hydrogen.

The cleavage of Cys4 releases a cis-repressive RNA module (crRNA,paired with RBS) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. A Ribosome Binding Site (RBS) is an RNA sequence to which ribosomes can bind and initiate translation. Combined RBS and Cys4, it can be more convenient for other iGEMers to use this composite part without putting a RBS aequence on the upstream of Cys4 coding sequence.

To realize different expression level of downstream gene, according to molecular dynamics and the theory of fluctuations, we design several variants of Cys4. Ensuring that varient Cys4 can bind substrate RNA, guranteeing molecular docking, we consider the cleavage and affinity activity of Cys4. Critical active site residues(Tyr176) have been implicated in properly positioning the G20 ribose in an orientation that is compatible with nucleophilic attack on the downstream phosphodiester bond, so we replace Tyr176 with Phe.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 377
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 93