Part:BBa_K4140005

L7AE

Part Description

A protein called L7Ae that binds to RNA inhibits the targeted transcript's ability to be translated. Transcriptional repressors take longer to suppress the intended construct than L7Ae that regulate gene expression at the translational level. As it specifically targets a sequence on the 5' end of the RNA termed the 2x-kturn. L7Ae can be used to build more complicated genetic circuits that are regulated at both the translational and transcriptional levels

Usage

L7Ae that binds to RNA inhibits the targeted transcript's ability to be translated. As it specifically targets a sequence on the 5' end of the RNA termed the 2x-kturn. as a post transcriptional modifier we use it to repress the translation of cas12g protein in case of high levels of phenylalanine and TyrR that leads to inhibiting CRISPR system activity and preserving the expression of PAH in the therapeutic circuit or lacZ alpha in the diagnostic circuit as shown in figure 1.

Figure 1. (shows the usage of TL7Ae in our circuit design)

Characterization of Mutational Landscape

After creating a multiple sequence alignment of the protein sequence and predicting mutational landscapes, the effect of these mutations on the evolutionary fitness of the protein is tested. The prediction of the mutational landscape by saturation mutagenesis of the L7Ae protein. The (N99D) mutation, as depicted in the chart, had the greatest score when compared to other mutations. On the other hand, it's clear that the (A60M) had the least evolutionary fitness for LacZ protein. As displayed in Figure(1)

Figure 1. (shows the mutational landscape of the L7Ae protein)

Literature Characterization

In order for our regulatory CRISPR-based system, which L7Ae attaches to, to operate as a regulator, domain the RNA of k-turn should be unstructured in the absence of L7Ae. In this figure, we examine how L7Ae affects both the native sequence and the mutant version of the k-turn. and in-line probing for both original and mutant RNA at concentrations of 0, 2.5, 5, and 10 M AfL7Ae, respectively. Positions of reactivity in the native 5′-UTR RNA in the presence of L7Ae are shown by the black circles drawn on the fluorogram.

Figure.1 Using in-line probing, analyze the conformational change in the A. fulgidus 5′-UTR RNA caused by the interaction of AfL7Ae protein.

References

1.uang L, Lilley DMJ. 2018. The kink-turn in the structural biology of RNA. Q Rev Biophys 51: 1–32.doi:10.1017/S0033583518000033 2.Huang L, Lilley DMJ. 2013. The molecular recognition of kink turn structure by the L7Ae class of proteins. RNA 19: 1703–1710.doi:10.1261/rna.041517.113





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]