Part:BBa_K3179001

L7AeO(a optimized L7Ae which is a transcriptional level repressor)

L7Ae binds C/D boxes of sRNAs and guides RNA post-transcriptional modification; also binds KT-15 motif in 23S rRNA. L7Ae targets a specific sequence at the 5' end of the RNA, termed 2x-kturn. We use L7Ae to suppress another component 2xKturn-E1. Cooperating with another mRNA to play the role of miRNA sensor, identifying miRNA. We mutant one of its k-turn combine region, and reduce its cytotoxicity.

Usage and Biology

According to the three-position structure of the protein, we fixed a point mutation in the α-helical domain of protein-mRNA binding, mutated the C at position 109 to A, interrupted the α-helix, and obtained L7AeO.

Figure 3: L7Ae structure with K-turn from PDB

We characterizated L7Ae and constructed an L7Ae mutation to study the suppression efficiency of the L7Ae-Kturn System.During the measurement, we have proved that inhibition efficiency of L7AeO is significantly lower than that of L7Ae, but still higher than the control group. It is probably due to the fact that the structural change of the mutant only resulted in a decrease in the binding efficiency between L7Ae and Kturn structure, but the active site was not completely inactivated. Therefore, L7AeO is still possible to combine with Kturn structure in high concentration, and suppress the translation of mRNA.

Cytotoxicity test

SYSU-CHINA tested the cytotoxicity of L7Ae, which was previously considered to be slightly cytotoxic, but the opposite result was found in our experiments.Neither L7Ae nor its mutants showed significant cytotoxicity.

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]