Difference between revisions of "Part:BBa K1062004"

2018 OUC-China

Csy4 (Csy6f), a member of CRISPR 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.

Fig.1 The Csy4/Hairpin complex.

Background of 2018 OUC-China' project

This year, we design a toolkit focused on translational regulation, which is composed of a RNA endoribonuclease (Csy4) and a RNA module (hairpin). In our project, the cleavage function 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.

We want to achieve precise expression of proteins by using different Csy4 mutants. The aim is using one system to realize diverse expression. We focus on the sites which play an important role in binding and cleavage. Gln104 is located in the linker segment connecting the body of Csy4 to the arginine-rich area, which makes sequence-specific hydrogen bonding contacts in the major groove of the RNA stem to nucleotides G20 and A19. His29 is in its deprotonated form and functions as a general base during cleavage by activating the 2′-hydroxyl nucleophile through proton abstraction. The side chain of Tyr176 points into the active site and stacks on top of the His29 imidazole group, which plays a role in orienting His 29. Phe155 is to recognize the ssRNA-dsRNA junctions in RNA hairpin. Based on the molecular simulation and the theory of fluctuations, four mutants are chosen rationally: Q104A, H29A, Y176F, F155A.

This year, we have created a brand new family called Csy4 family on the basis of an existing part BBa_K1062004. We redesign four Csy4 mutants by point mutation to form this family, whose capabilities of cleavage and recognition are different from each other. As an important role in our project miniToe family, we have tested them by several ways. We have proved that our system can work well by using Csy4 family. Now Csy4 family is an improvement and has been shown to work well in our system. See more information in our part page!Csy4-H29A

Fig.2 Four key sites of wild type Csy4.