Difference between revisions of "Part:BBa K1062004"

Revision as of 23:54, 11 October 2018

Csy4

Csy4 is an enzyme that is essential to the creation of gRNAs. Csy4 is a member of CRISPR family.

Sequence and Features

Usage and Biology

Conjugation Project

Synthetic Circuit

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.We tested five Csy4s individually by microplate reader. We have tested them every two hours. The green lines in all the images represent the control group, “miniToe only” group and the green lines keep stable which means the miniToe structure can close the expression of downstream genes. And the test groups show different characteristics. As we can see in Fig.3-A, the Csy4-WT shows the same result with the first system. The switch turn off when the system without IPTG (as the blue line shows). And the expression level is the highest among all the test groups which indicates the highest enzyme activity by Csy4-WT (Fig.3-F). In the Fig.3-B, the tendency of increase of fluorescence intensities by Csy4-Q104A is almost same with Csy4-WT. And the expression level is lower than Csy4-WT. So the Csy4-Y176F is. What is special is Csy4-H29A. We have mentioned Csy4-H29A before. The active site of Csy4 contains an essential histidine residue (H29) that functions as a general base during RNA strand scission. Mutation of H29 to alanine inactivates Csy4 without affecting substrate binding affinity or specificity. So Csy4-H29A is a dead-Csy4 which has high binding affinity but has lowest capabilities of cleavage as we can see in Fig.3-E. In summary, we put all the test groups together in Fig.3-F. See more information and result about Csy4 family in our part page!The Csy4-H29A, a member of Csy4 family!

Fig.2 Four key sites of wild type Csy4.

Fig.3 The fluorescence intensities of sfGFP by microplate reader. A. Csy4-WT&miniToe-WT. B. Csy4-Q104A&miniToe-WT. C. Csy4-Y176F&miniToe-WT. D. Csy4-F155A&miniToe-WT. E. Csy4-H29A&miniToe-WT. A-E. The blue line is test group with IPTG. The yellow line is test group without IPTG. The green line is a control group which only has miniToe structure without Csy4s. F. The summary of different test groups which indicates the capabilities of Csy4 mutants.