Figure 1. The ONBY ncAA used for our photocaging project. When exposed to 365 nm light, the ONB group is released, resulting in a normal tyrosine amino acid.
We recreated a light-activatable T7 RNA polymerase (RNAP) for the spatio-temporal control of protein expression. The light-activatable T7 RNAP was created by mutating a tyrosine codon at position 639 of a domain crucial for the polymerization of RNA during transcription. Y639 was mutated to an amber codon, allowing us to incorporate a ncAA at this position. We used ortho-nitrobenzyl tyrosine (ONBY), which is a "photocaged" ncAA (Figure 1). Thus, if our synthetase/tRNA pair works, position 639 should contain ONBY in place of tyrosine. This work is essentially a recapitulation of earlier work done by [Chou et al. 2010].
When the photocaged ONBY is incorporated at position 639, RNAP activity is inhibited. The polymerase can become activated only upon de-caging of the ONB group from the ONBY. This is accomplished by exposing the cells to 365 nm light. When exposed to 365 nm light, the ONB group is released, resulting in a normal tyrosine amino acid at position 639. T7 RNAP was selected because of its orthogonal nature, which allows us to selectively induce the expression of specific genes that are preceded by the T7 RNAP promoter. Because T7 promoters are not natively found in E. coli, a gene downstream of a T7 promoter may be exclusively expressed through the introduction of 365 nm light.
Background
Tyrosine residue 639 (Y639) was specifically targeted because it lies on a crucial position on the O-helix domain of T7 RNAP and has been proven to be essential for polymerization. (REFERENCE) The Y639 residue in the O-helix is responsible for two major roles. First, this tyrosine residue discriminates between deoxyribose and ribose substrates using the Tyrosine-OH. Second, Y639 is responsible for moving newly synthesized RNA out of the catalytic site and preparing for the next NTP to be inserted. These functions of the O-helix were shown to be essential through mutational analysis. Introducing a bulky group such as ONBY in place of tyrosine renders the enzyme nonfunctional in several ways. First, the native tyrosine-OH is not there anymore to coordinate Mg2+, which plays an essential role in discriminating between deoxyribose and ribose substrates. Additionally, because of the sterics of the ONBY molecule itself, it blocks incoming nucleotides from entering the active site. Because the loss of this tyrosine residue in the active site leads to a non-functional polymerase, Y639 proved to be a good candidate for incorporating a photocaged amino acid (Reference; You are referring to a specific result).
Figure 2. The caged T7 RNAP is decaged via exposure to 365 nm light. SOURCE OF IMAGE? NEED REFERENCE
Incorporation of ONBY at position 639 of T7 RNAP halts activity because of the bulky nature of ONBY (REFERENCE). This ONB side group effectively renders T7 RNAP inactive. However, the bulky ONB group is able to be removed through irradiation with 365 nm light. The wavelength of light used to "decage" the amino acid proved to be another advantage of this system because 365 nm light is not toxic to the cell (accurate?). Once the ONB group is removed, a normal tyrosine residue is left in its place, restoring T7 RNA polymerase activity.
In order to incorporate the ncAA into amberless E.coli (which is described [here]), a Methanocaldoccus jannaschii tyrosyl-tRNA synthetase/tRNA pair was previously mutated to selectively charge and incorporate ONBY. Six residues (Tyr 32, Leu 65, Phe 108, Gln 109, Asp 158, and Leu 162) on the original synthetase were randomized and the library was selected for its ability to charge ONBY while discriminating against other canonical amino acids. The resulting mutant ONBY synthetase contained five mutations (reference). The Asp 158→Ser 158 and Tyr 32→Gly 32 mutations are believed to result in the loss of hydrogen bonds with the natural substrate, which would disfavor binding to tyrosine. Additionally, the Tyr 32→Gly 32 and Leu 65→Gly 65 mutations are believed to increase the size of the substrate-binding pocket to accommodate the bulky o-nitrobenzyl group (Deiters et al. 2006). The fifth mutation is ?? ARE THESE MUTATIONS CORRECT? Y32G is listed twice, which is fine, but then we should list the other mutations and what their roles are or that their roles are unknown.
Experimental Methods
Mention overall scheme, outline here... Then, ~"specific details are below" or something similar.
In order to create an in vivo light-activated GFP expression system, two plasmids were constructed and transformed into amberless E.coli cells. The first plasmid contains the tRNA and synthetase pair, which is necessary to incorporate ONBY into the amber stop codon of the T7 RNAP. The second plasmid contains the coding sequence for T7 RNAP which has a mutation on the Tyrosine 639 residue. This plasmid also contains a GFP coding sequence bound to an upstream T7 promoter.
Once these components were assembled by Gibson Assembly, the two plasmids were transformed via electroporation into aliquots of Amberless E.coli.
After transformation, the amberless E. coli cells containing both the synthetase/tRNA pair plasmid and the amber T7 RNAP/GFP plasmid were assayed for their ability to express GFP upon irradiation with 365 nm light in different intervals of time. The cells were irradiated for 0 minutes, 1 minute, 5 minutes, 15 minutes, and 30 minutes in order to observe the temporal control of GFP expression.
After irradiation with light, these cells were allowed to grow overnight before taking fluorescent measurements. This additional growth was necessary to allow the "decaged" T7 RNAP to polymerize mRNA transcripts of the GFP coding sequence.
Preparation & Growth of Cultures
Picked Colonies
PC 1: pQE/ONBY
PC 2: pQE/ONBY
PC 3: MJH117/ONBY
PC 4: MJH117/ONBY
PC 5: aT7/ONBY
PC 6: aT7/ONBY
Antibiotics for PC 1-4: Zeo, Kan, Gent
Antibiotics for PC 5-6: Zeo, Crb, Gent
Conditions for each picked colony
(-) IPTG, (-) ONBY
(+) IPTG, (-) ONBY
(+) IPTG, (+) ONBY
Inoculate each tube with designated PC; add 1 uL of cells into each tube
Grow cultures for 2 hours
Add IPTG to all (+) IPTG culture tubes.... Add ONBY???
Grow another 2-4 hours
Decaging ortho-nitrobenzyl tyrosine
Take cultures out and irradiate with 365 nm light
Place all (+) IPTG (+) ONBY cells into wells of a 12-well plate
Place (+) IPTG (-) ONBY of PC 1, 3, 5 into wells
Place (-) IPTG (-) ONBY of PC 3 & 4 to use as a control for growth without ONBY (*this will allow us to see how much the ONBY solution decreases growth rate of cells)
Place negative control into 12 well plate
Irradiate using handheld blacklight placed directly over the wells; irradiate cells and take out at every time point (0 min, 1 min, 5 min, 15 min, 30 min) and transfer to wells in a 96 well plate to test fluorescence
Measuring Fluorescence
Results
Figure 3. GFP fluorescence observed after exposure to 365 nm light for the time indicated.reexport this figure
To demonstrate the function of this system, cells were grown in culture... These cells were then exposed to varying amounts of 365 nm light, ranging from 0 to 30 minutes. During this exposure, the ONB functional group at position ONBY639 should be released, resulting in Y639.
After this exposure, the cells were grown for an additional [1 hour?], allowing the newly decaged T7 RNAP to transcribe the GFP reporter gene, which ultimately results in fluorescence.
References
Chou, C., Young, D. D. and Deiters, A. (2010), Photocaged T7 RNA Polymerase for the Light Activation of Transcription and Gene Function in Pro- and Eukaryotic Cells. ChemBioChem, 11: 972–977. doi: 10.1002/cbic.201000041
Deiters, A., Groff, D., Ryu, Y., Xie, J. and Schultz, P. G. (2006), A Genetically Encoded Photocaged Tyrosine. Angew. Chem., 118: 2794–2797. doi: 10.1002/ange.200600264
P. A. Osumidavis, N. Sreerama, D. B. Volkin, C. R. Middaugh, R. W. Woody, A. Y. M. Woody, J. Mol. Biol. 1994, J. Mol. Biol. 1994, 237, 5 – 19.
"
