Difference between revisions of "Part:BBa K4162005"
(→‎Usage and Biology)
Line 17: Line 17:
 
===Usage and Biology===
 
===Usage and Biology===
  
−
Hammerhead ribozyme can perform self-cleaving efficiently. While constructing polycistrons in ''E. coli'', ribozyme sequence should be inserted between two coding sequences. After the transcription of CDS is completed, the ribozyme will conduct self-cleaving and separate the long piece of mRNA into segments corresponding to each CDS. Therefore, the translation process of target proteins will depend on their respective RBS strength.
+
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.
  
 
===Characterization===
 
===Characterization===

Revision as of 11:21, 11 October 2022


Hammerhead ribozyme


Introduction

2021 Fudan

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)

Usage and Biology

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.

Characterization

定性1标题

定性1文字

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

  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.