Difference between revisions of "Part:BBa K1062004:Experience"

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===Applications of BBa_K1062004===
 
===Applications of BBa_K1062004===
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In 2018 OUC-China's project, we used csy4 as a main role in our toolkit. We design a toolkit focused on translational regulation, which is composed of a RNA endoribonuclease (Csy4) and a RNA module (hairpin). Csy4 (Csy6f), a member of CRISPR family, recognizes a specific 16-nt nucleotide repetitive sequence(hairpin). The RNA module was constructed by inserting the 16nt Csy4 recognition site between a RBS and cis-repressive RNA element, which can be specifically cleaved upon Csy4 expression, so that the RBS is masked. Cleaved at the specific recognition site, it can release the masked RBS, thus endowing the programming of gene expression in the translation level with higher feasibility.
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The RBS and Csy4 recognize and cleave a 22nt hairpin.
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We found that csy4 is a perfect part for our toolkit. Because once the RNA/Csy4 complex formed, the structure become really stable and hard to separate which make our toolkit stable when it function.
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===User Reviews===
 
===User Reviews===
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<I>2018 OUC-China</I>
 
<I>2018 OUC-China</I>
 
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In 2018 OUC-China's project, we used csy4 as a main role in our toolkit. We design a toolkit focused on translational regulation, which is composed of a RNA endoribonuclease (Csy4) and a RNA module (hairpin). Csy4 (Csy6f), a member of CRISPR family, recognizes a specific 16-nt nucleotide repetitive sequence(hairpin). The RNA module was constructed by inserting the 16nt Csy4 recognition site between a RBS and cis-repressive RNA element, which can be specifically cleaved upon Csy4 expression, so that the RBS is masked. Cleaved at the specific recognition site, it can release the masked RBS, thus endowing the programming of gene expression in the translation level with higher feasibility.
 
  
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We found that csy4 is a perfect part for our toolkit. Because once the RNA/Csy4 complex formed, the structure become really stable and hard to separate which make our toolkit stable when it function.
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===[https://parts.igem.org/Part:BBa_K2615003 Csy4 (Csy6f)], a member of CRISPR family.===
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<p>
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  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.
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<br>
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[[Image:T--OUC-China--complex.jpg|center|thumb|250px|'''Fig.1  The Csy4/Hairpin complex.''']]
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</p>
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===Background of 2018 OUC-China' project===
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<p>
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  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 ([https://parts.igem.org/Part:BBa_K2615020 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.
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<br>
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<br>
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  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: [https://parts.igem.org/Part:BBa_K2615004 Q104A], [https://parts.igem.org/Part:BBa_K2615007 H29A], [https://parts.igem.org/Part:BBa_K2615005 Y176F], [https://parts.igem.org/Part:BBa_K2615006 F155A].
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<br>
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[[Image:T--OUC-China--Csy4complex.jpg|center|thumb|400px|'''Fig.2  Four key sites of wild type Csy4.''']]
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</p>

Latest revision as of 12:12, 11 September 2018


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1062004

The RBS and Csy4 recognize and cleave a 22nt hairpin.

User Reviews

UNIQ1abfdbc7f1e997e2-partinfo-00000000-QINU

•••

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.

Fig.2 Four key sites of wild type Csy4.