Part:BBa_K4162005

Hammerhead ribozyme

Introduction

Hammerhead ribozyme was first found in the genome of viruses and viroids. It involved in the processing of RNA transcripts based on rolling-circle replication. The tandem copy of RNA sequence will be generated in the roll ring replication, and the self-cleaving activity of ribozyme can ensure the generation of RNA copy of unit length.^[1]

The secondary structure of hammerhead ribozyme resembles a hammer. According to different open helix tips, hammerhead ribozymes can be divided into three types: TypeⅠ, Type II and Type III. The catalytic center of ribozyme consists of 15 highly conserved bases surrounded by three helixs(HelixⅠ, Helix II and Helix III). The long-range interaction between HelixⅠ and Helix II can help stabilize the conformation of the catalytic center of the enzyme and improve the catalytic efficiency (Figure 1).^[2] While working, ribozymes utilize a network of defined hydrogen bonds, ionic and hydrophobic interactions to generate catalytic pockets, which capitalize on steric constraints to generate in-line cleavage alignments and general acid-base chemistry to catalyze site-specific cleavage of the phosphodiester backbone.^[3]

Figure 1. Overall tertiary structure of different types of hammerhead ribozymes.Blue marks highly conserved bases. The black arrow marks the digestion site. Red marks the long-range interaction between HelixⅠand Helix II.(Jimenez, Randi M et al. 2015)

We construct a ribozyme-assisted polycistronic co-expression system (pRAP) by inserting ribozyme sequences between CDSs in a polycistron. In the pRAP system, the RNA sequences of hammerhead ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons in vivo. Self-interaction of the polycistron can be nullified and each cistron can initiate translation with comparable efficiency. Besides, we can precisely manage this co-expression system by adjusting the RBS strength of individual mono-cistrons. In our project, we used ribozyme to build our crtEBIY biobrick.

Characterization

Successful protein expression

As many researches indicate, the major problem of polycistronic vectors, which contain two or more target genes under one promoter, is the much lower expression of the downstream genes compared with that of the first gene next to the promoter^[4]. The tail of the coding sequence (CDS) can interfere with the head of the ribosome binding site (RBS), which can hinder RBS from combining to ribosomes. Such shortage occurred when we assembled crtEBIY sequentially only to find incomplete expressions of our target proteins.

File:T--Fudan--定性1图片不要用中文做文件名.png

Figure 1. 图标题. 图注引用的不要忘了写某某人某某年的出处

定性1小结

定性2标题

定性2文字

File:T--Fudan--定性2图片不要用中文做文件名.png

Figure 2. 图标题. 图注引用的不要忘了写某某人某某年的出处

定性2小结

定性3标题

定性3文字

File:T--Fudan--定性3图片不要用中文做文件名.png

Figure 3. 图标题. 图注引用的不要忘了写某某人某某年的出处

定性3小结

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

References

↑ Ferré-D'Amaré, A. R., & Scott, W. G. (2010). Small self-cleaving ribozymes. Cold Spring Harbor perspectives in biology, 2(10), a003574. https://doi.org/10.1101/cshperspect.a003574
↑ Jimenez, R. M., Polanco, J. A., & Lupták, A. (2015). Chemistry and Biology of Self-Cleaving Ribozymes. Trends in biochemical sciences, 40(11), 648–661. https://doi.org/10.1016/j.tibs.2015.09.001
↑ Ren, A., Micura, R., & Patel, D. J. (2017). Structure-based mechanistic insights into catalysis by small self-cleaving ribozymes. Current opinion in chemical biology, 41, 71–83.
↑ Kim, K. J., Kim, H. E., Lee, K. H., Han, W., Yi, M. J., Jeong, J., & Oh, B. H. (2004). Two-promoter vector is highly efficient for overproduction of protein complexes. Protein science: a publication of the Protein Society, 13(6), 1698–1703.

[1] Ferré-D'Amaré, A. R., & Scott, W. G. (2010). Small self-cleaving ribozymes. Cold Spring Harbor perspectives in biology, 2(10), a003574. https://doi.org/10.1101/cshperspect.a003574

[2] Jimenez, R. M., Polanco, J. A., & Lupták, A. (2015). Chemistry and Biology of Self-Cleaving Ribozymes. Trends in biochemical sciences, 40(11), 648–661. https://doi.org/10.1016/j.tibs.2015.09.001

[3] Ren, A., Micura, R., & Patel, D. J. (2017). Structure-based mechanistic insights into catalysis by small self-cleaving ribozymes. Current opinion in chemical biology, 41, 71–83.

[4] Kim, K. J., Kim, H. E., Lee, K. H., Han, W., Yi, M. J., Jeong, J., & Oh, B. H. (2004). Two-promoter vector is highly efficient for overproduction of protein complexes. Protein science: a publication of the Protein Society, 13(6), 1698–1703.

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