Part:BBa_K5466010

LexA-VP16

LexA-VP16 is a synthetic transcription factor formed by the fusion of LexA DNA binding domain (BD) and Herpesviurs VP16 activation domain (AD). Due to its structure, it can exert potent transcriptional activation on DNA sequences harboring LexA operators followed by native core promoter elements.

SV40 NLS was attached to the C-terminal.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 300
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Orthogonality

To avoid unpredictable interference with the functionality of synthetic systems, the concept of orthogonality is crucial. An orthogonal system should deliver its intended functions with minimal, or ideally no, cross-talk with the host organism. This means that the system's functionality should not be affected by the host organism, nor should it impact the host in any unintended ways.

Synthetic transcription factor

The LexA DNA-binding domain (DBD), from a transcription factor of Escherichia coli, has been repeatedly used in various molecular studies. This domain is characterized by its ability to bind to the consensus sequence CTGTATATATATACAG. The activation domain comes from VP16, a transcription factor of herpes simplex virus, as its activation domain is one of the most potent and is widely fused to transcription factors to enhance their activity. Additionally, VP16 interacts with numerous proteins involved in gene activation. These multiple interactions indicate that the VP16 transactivation domain facilitates the assembly of the pre-initiation complex at several distinct steps. It also recruits histone acetyltransferases, contributing to chromatin decondensation. This effect of VP16 on chromatin decondensation during gene activation appears to have a widespread impact, as it can be detected using fluorescence microscopy.

The fusion of these two domains results in a powerful and orthogonal synthetic transcription factor.

Nuclear localization signals (NLS) are typically short peptides that function as signal sequences, facilitating the transport of proteins from the cytoplasm to the nucleus. VP40 is classified as a classical NLS, usually containing four or more positively charged residues, specifically arginine (R) or lysine (K).

References

Hirai, H., Tani, T., & Kikyo, N. (2010). Structure and functions of powerful transactivators: VP16, MyoD and FoxA. The International Journal Of Developmental Biology, 54(11-12), 1589-1596. https://doi.org/10.1387/ijdb.103194hh

Rantasalo, A., Czeizler, E., Virtanen, R., Rousu, J., Lähdesmäki, H., Penttilä, M., Jäntti, J., & Mojzita, D. (2016). Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae. PLoS ONE, 11(2), e0148320. https://doi.org/10.1371/journal.pone.0148320