The tyrosine residue which is replaced by ONBY lies on a crucial position on the O-helix and has been proved to be essential for polymerization. The Tyr639 residue in the O-helix is responsible for two major roles. First, this tyrosine residue discriminates between deoxyribose and ribose substrates. Second, Tyr639 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. Because the loss of this tyrosine residue in the active site leads to a non-functional polymerase, Tyr639 proved to be a good candidate for incorporating a photocaged amino acid.
Because of the bulky nature of ONBY, its incorporation at position 639 essentially halted the activity of the T7 polymerase. 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 because 365 nm light is not toxic to the cell. Once the ONB group is removed, a normal tyrosine residue is left which restores RNA polymerase activity.
In order to incorporate the non-canonical amino acid into the amberless E.coli (which is described [here]), a Methanococcus jannaschii synthetase/tRNA pair had to be 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 selected for its ability to charge ONBY while discriminating against other canonical amino acids. The Asp 158→Ser 158 and Tyr 32→Gly 32 mutations were likely involved in the loss of hydrogen bonds with the natural substrate, which would disfavor binding to natural tyrosine. On the other hand, the Tyr 32→Gly 32 and Leu 65→Gly 65 mutations likely increase the size of the substrate-binding pocket to accommodate the bulky o-nitrobenzyl group.
Experimental Methods
Note: I will elaborate in more words in the following sections
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
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
NEED TO EDIT: THIS WAS CUT AND PASTED FROM A PAPER WE WROTE.
BELOW: BETTER IMAGE resolution
"
