Part:BBa_K1150001

VP16

VP16
Function	transactivator
Use in	Mammalian cells and Herpes simplex virus-1
RFC standard	RFC 25
Backbone	pSB1C3
Submitted by	[http://2013.igem.org/Team:Freiburg Freiburg 2013]

Virus Protein 16 (VP16) is a transcription factor encoded by the UL48 gene of Herpes simplex virus-1 (HSV-1).

Through complex formation with cellular host factors VP16 can bind to a common regulatory element in the upstream promoter region of immediate-early genes [1]. Due to the transactivating function of VP-16 these genes can then be expressed.

VP16 consists of 490 amino acids with two very important functional domains: a core domain in its central region which is necessary for the indirect DNA binding and a carboxy-terminal transactivation domain [2], [3]; see Fig.1. The transactivation domain (TAD) of VP16 can be fused to a DNA-binding domain (DBD) of another protein in order to gain expression of a desired target gene [1].

Fig. 1: Domain structure of VP16. The different functional domains of VP16 are shown. Taken from Hirai et al., 2010.

Transactivation domain (TAD) of VP16

The TAD of VP16 is one of the most efficient TADs. It is widely fused to host transcription factors in order to increase their activity or, next to this, to other transcription factors to study the mechanism of gene regulation [1]. In our case the TAD of VP16 was cloned behind a double mutated Cas9 gene (which encodes for a non-cutting dCas9 protein with specific DNA binding properties) to activate our genes of interest. For more information see BBa_K1150019 and BBa_K1150020.

Biology

VP16 is originally a part of the virus particle from HSV and is released into an animal cell while infection. VP16 first binds via its core domain to the host nuclear protein which then recruits another host nuclear protein, Oct-1, to form a functional protein complex. This complex then binds to its target DNA sequence in the promoter region of immediate-early genes where VP16 activates these genes through interactions between its TAD and many different transcription factors and other proteins, such as histone acetyltransferases; see Fig.2. Through these multiple interactions the transactivation domain of VP16 facilitates the assembly of the pre-initiation complex [5], [1]. Also other TAD containing proteins share this magnitude of binding partners but VP16 is one of the most effective once [1].

Fig. 2: Interaction partners of VP16. Proteins are shown that interact with VP16 in the phase of gene activation. Taken from Hirai et al., 2010

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]

Protein data table for BioBrick BBa_K1150001 automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)  ATGGCCGGCGCGCGTACG ... TACGGTGGGACCGGT  ORF from nucleotide position -8 to 378 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below) 1  101  MAGARTKNNYGSTIEGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSA PYGALDMADFEFEQMFTDALGIDEYGGTG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features) None of the supported features appeared in the sequence
Amino acid composition: Ala (A) 16 (12.4%) Arg (R) 4 (3.1%) Asn (N) 2 (1.6%) Asp (D) 20 (15.5%) Cys (C) 0 (0.0%) Gln (Q) 1 (0.8%) Glu (E) 8 (6.2%) Gly (G) 16 (12.4%) His (H) 4 (3.1%) Ile (I) 2 (1.6%) Leu (L) 15 (11.6%) Lys (K) 1 (0.8%) Met (M) 5 (3.9%) Phe (F) 6 (4.7%) Pro (P) 10 (7.8%) Ser (S) 6 (4.7%) Thr (T) 8 (6.2%) Trp (W) 0 (0.0%) Tyr (Y) 3 (2.3%) Val (V) 2 (1.6%)
Amino acid counting Total number: 129 Positively charged (Arg+Lys): 5 (3.9%) Negatively charged (Asp+Glu): 28 (21.7%) Aromatic (Phe+His+Try+Tyr): 13 (10.1%)	Biochemical parameters Atomic composition: C585H881N153O206S5 Molecular mass [Da]: 13513.6 Theoretical pI: 3.85 Extinction coefficient at 280 nm [M-1 cm-1]: 4470 / 4470 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges
Codon usage Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals Codon quality (CAI): good (0.70) good (0.67) acceptable (0.50) acceptable (0.57) excellent (0.81) good (0.72)
Alignments (obtained from PredictProtein.org)    There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
Predictions (obtained from PredictProtein.org)
There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment.

References

[1] WEIR, J. (2001). Regulation of herpes simplex virus gene expression. Gene 271: 117-130.

[2] GREAVES, R.; O’HARE, P. (1989). Separation of requirements for protein-DNA complex assembly from those for functional activity in the herpes simplex virus regulatory protein Vmw65. J Virol 63: 1641-1650.

[3] TRIEZENBERG, S. J.; KINGSBURY, R. C.; MCKNIGHT, S. L. (1988). Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev 2: 718-729.

[4] HIRAI, H.; TANI, T; KIKYO, N. (2010). Structure and functions of powerful transactivators: VP16, MyoD and FoxA. Int. J. Dev. Biol. 54: 1589-1596.

[5] WALKER, R.; GREAVES, R.; O’HARE, P. (1993). Transcriptional activation by the acidic domain and involves additional determinants distinctof Vmw65 requires the integrity of the domain from those necessary for TFIIB binding. Molecular and Cellular Biology 13: 5233-5244.

[6] THOMPSON, R.; PRESTON, C.; SAWTELLl, N. (2009). De Novo Synthesis of VP16 Coordinates the Exit from HSV Latency In Vivo. PLoS Pathog 5: e1000352.

Functional Parameters: Austin_UTexas

BBa_K1150001 parameters

Burden Imposed by this Part:

Burden Value: 2.4 ± 6.4%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.