Team:Austin Texas/photocage
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- | 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 which | + | 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 the O-helix ('''figure??? of helix?'''), which is 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 [reference authors/paper]. |
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=Background= | =Background= | ||
- | Tyrosine residue 639 (Y639) was specifically targeted because it lies on a crucial position on the O-helix and has been | + | 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). |
[[Image:Uncaging_of_ONBY.jpg | 300px|left|thumb| Figure 2. The caged T7 RNAP is decaged via exposure to 365 nm light. '''SOURCE OF IMAGE? NEED REFERENCE''']] | [[Image:Uncaging_of_ONBY.jpg | 300px|left|thumb| Figure 2. The caged T7 RNAP is decaged via exposure to 365 nm light. '''SOURCE OF IMAGE? NEED REFERENCE''']] | ||
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- | 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, | + | 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= | =Experimental Methods= | ||
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+ | * 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 | ||
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Revision as of 23:47, 14 October 2014
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