Team:OUC-China/Model

Team OUC-China: Main

Overview

The aim of our project is to develop a better post-transcriptional regulation strategy and use it in monocistron and polycistron. We build models to design and predict our work.

miniToe —— a better transcriptional regulate strategy

To achieve a better post-transcriptional regulation strategy, we design a system which is composed of an RNA endoribonuclease (Csy4) and an RNA module named miniToe. We model to describe the dynamics of the miniToe system and point out the way to achieve different regulation level. The ODE and molecular dynamics are two main tools to explore it. We use the ODE to describe the reaction curve and the molecular dynamics give some explanations to experimental data.

Below you can follow the several questions we point out to have a better understanding of model work and the miniToe system. We will discuss some structures of Csy4 in different stage (Q1), some structures of miniToe system in different stage (Q2), the reaction order and some keys of miniToe system (Q3), the simulation of ODE model (Q4), some significant symbol in molecular dynamics (Q5) and the way to different regulation level (Q6).

Q1 : What does the structure of Csy4?

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The endoribonuclease Csy4 from CRISPR family is the main role of miniToe system. Csy4 (Cas6f) is a 21.4 kDa protein which recognizes and cleaves a specific 22nt RNA hairpin which consists of an N-terminal ferredoxin-like domain and a C-terminal domain. This later domain constitutes most of the recognition interactions with the RNA. The RNA adopts a stem-loop structure (the specific 22nt RNA hairpin) with five base pairs in A-form helical stem capped by GUAUA loop containing a sheared G11-A15 base pair and a bulged nucleotide U14. In the binding structure of Csy4-RNA complex, the RNA stem-loop straddles the β-hairpin formed by strands β6-7 of Csy4. The Fig.1 and Fig.2 shows two structure of Csy4: with and without hairpin bound.

Fig.1 The structure of Csy4 without hairpin bound (PDB ID: 4AL5, resolution 2.0 A)

Fig.2 The structure of Csy4 with hairpin bound (PDB ID: 4AL5, resolution 2.0 A)

Q2 : What does the structure of miniToe structure?

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Based on function of Csy4, we design a new cis-regulatory RNA element named miniToe which can be recognized by Csy4. The whole system works as a translational activator including three modular parts:

1. A cis-repressive RNA (crRNA) to serve as translation suppressor by pairing with RBS as the critical part of miniToe structure.
2. A Csy4 site as a linker between cis-repressive RNA and RBS, which can be specifically cleaved upon Csy4 function.
3. A CRISPR endoribonuclease Csy4.

Fig.2-1 is the secondary structure of miniToe.

Fig.2 The structure of miniToe.

The Fig.4 and Fig.5 show that the two complex of miniToe structure: with and without specific site of hairpin cleaved, which is called the precursor complex and precursor complex respectively.

Fig.4 The precursor complex of wild-type Csy4

Fig.5 The product complex of wild-type Csy4

Q3 : What is the reaction order of miniToe system?

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Fig.6 The working process of miniToe system

All the reaction happened in our first system, miniToe, can be described chronologically by following five main steps in Fig.6:

(1)The miniToe structure is produced and accumulated.
(2)The Csy4 is produced with IPTG induced.
(3)The Csy4 binds to the miniToe structure and form the Csy4-miniToe complex
(4)The Csy4 cleave the special site and divide the miniToe structure into two parts: the Csy4-crRNA complex and the mRNA of sfGFP.
(5)The sfGFP is produced.

From the description above, we can get four key problems in our system to make sure that our system can work successfully:

(1)Does the Csy4 dock correctly with the miniToe structure (hairpin)?
(2)How about the ability of binding between the Csy4 and miniToe structure (hairpin)?
(3)How about the ability of cleavage between the Csy4 and miniToe structure (hairpin)?
(4)Does cis-repressive RNA release from the RBS?

Q4 : How about the simulation result of the ODE model?

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According to the work process we build an ODEs model and simulate our miniToe system for 30h, the result can be seen in the Fig.7.

Fig.7 The dynamics of sfGFP by model prediction

We compare the experimental data to the simulation, find it fit perfectly in Fig.7

Fig.8 The comparison between experimental data and simulation data

To see more details

Q5 : What does the structure of Csy4?

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The endoribonuclease Csy4 from CRISPR family is the main role of miniToe system. Csy4 (Cas6f) is a 21.4 kDa protein which recognizes and cleaves a specific 22nt RNA hairpin which consists of an N-terminal ferredoxin-like domain and a C-terminal domain. This later domain constitutes most of the recognition interactions with the RNA. The RNA adopts a stem-loop structure (the specific 22nt RNA hairpin) with five base pairs in A-form helical stem capped by GUAUA loop containing a sheared G11-A15 base pair and a bulged nucleotide U14. In the binding structure of Csy4-RNA complex, the RNA stem-loop straddles the β-hairpin formed by strands β6-7 of Csy4. The Fig.1 and Fig.2 shows two structure of Csy4 in the different stage: with and without hairpin bound.

Fig.1 The structure of Csy4 without hairpin bound (PDB ID: 4AL5, resolution 2.0 A)

Q6 : What does the structure of Csy4?

click to see more

click to see less

The endoribonuclease Csy4 from CRISPR family is the main role of miniToe system. Csy4 (Cas6f) is a 21.4 kDa protein which recognizes and cleaves a specific 22nt RNA hairpin which consists of an N-terminal ferredoxin-like domain and a C-terminal domain. This later domain constitutes most of the recognition interactions with the RNA. The RNA adopts a stem-loop structure (the specific 22nt RNA hairpin) with five base pairs in A-form helical stem capped by GUAUA loop containing a sheared G11-A15 base pair and a bulged nucleotide U14. In the binding structure of Csy4-RNA complex, the RNA stem-loop straddles the β-hairpin formed by strands β6-7 of Csy4. The Fig.1 and Fig.2 shows two structure of Csy4 in the different stage: with and without hairpin bound.

Fig.1 The structure of Csy4 without hairpin bound (PDB ID: 4AL5, resolution 2.0 A)