Impact of Nonnatural Amino Acid Mutagenesis on the in Vivo Function and Binding Modes of a Transcriptional Activator

Majmudar, Chinmay Y.; Lee, Lori W.; Lancia, Jody K.; Nwokoye, Adaora; Wang, Qian; Wands, Amberlyn M.; Wang, Lei; Mapp, Anna K.

doi:10.1021/ja904378z

Cited by 40 publications

(73 citation statements)

References 33 publications

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“…Therefore, Bpa incorporation at position Phe849 in Gal4 was selected for the combined covalent chemical capture-MuDPIT experiments. 16 Initial optimization studies with this approach examined the sugar-dependent changes of the complex between Gal4 and Gal80 at a genomically integrated Gal1-Lacz reporter gene. 19 Importantly, earlier work by our group demonstrated that Bpa-containing Gal4 mutants retain responsiveness to changes in nutrient availability (glucose, raffinose, and raffinose-galactose).…”

Section: Resultsmentioning

confidence: 99%

“…As there is considerable evidence that transcriptional activators use distinct but overlapping sequences within their activation domains to contact an array of binding partners, future experiments with a cross-linking amino acid placed at distinct positions will likely lead to the identification of additional coactivator targets. 15,16,36,47 …”

Section: Discussionmentioning

confidence: 99%

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Discovery of Enzymatic Targets of Transcriptional Activators via in Vivo Covalent Chemical Capture

Dugan¹,

Majmudar²,

Pricer³

et al. 2016

J. Am. Chem. Soc.

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The network of activator protein-protein interactions (PPIs) that underpin transcription initiation is poorly defined, particularly in the cellular context. The transient nature of these contacts and the often low abundance of the participants present significant experimental hurdles. Through the coupling of in vivo covalent chemical capture and shotgun LC-MS/MS (MuDPIT) analysis, we can trap the PPIs of transcriptional activators in a cellular setting and identify the binding partners in an unbiased fashion. Using this approach, we discover that the prototypical activators Gal4 and VP16 target the Snf1 (AMPK) kinase complex via direct interactions with both the core enzymatic subunit Snf1 and the exchangeable subunit Gal83. Further, we use a tandem reversible formaldehyde and irreversible covalent chemical capture approach (TRIC) to capture the Gal4-Snf1 interaction at the Gal1 promoter in live yeast. Together, these data support a critical role for activator PPIs in both the recruitment and positioning of important enzymatic complexes at a gene promoter and represent a technical advancement in the discovery of new cellular binding targets of transcriptional activators.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Discovery of Enzymatic Targets of Transcriptional Activators via in Vivo Covalent Chemical Capture

Dugan¹,

Majmudar²,

Pricer³

et al. 2016

J. Am. Chem. Soc.

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“…To test this idea, we carried out in vivo site-specific cross-linking using enhanced nonsense suppression (29,38,39) to incorporate the photo-reactive amino acid Bpa in place of the endogenous amino acid. Focusing first on Tim23, we incorporated Bpa at seven distinct sites (Ile-124, Asn-127, Leu-134, Val-137, Leu-138, Asn-139, and Ile-141) loop 1 (residues 122-145) (Fig.…”

Section: Loop 1 Of Tim23mentioning

confidence: 99%

“…Here, we report the results of genetic and coimmunoprecipitation approaches, coupled with site-specific in vivo cross-linking experiments. Cross-linking was performed by engineering the incorporation of a photo-activatable nonnatural amino acid p-benzoyl-L-phenyalanine (Bpa) in place of specific residues of components of the TIM23 and PAM complexes (28,29). This cross-linking approach has several important advantages.…”

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confidence: 99%

Architecture of the TIM23 Inner Mitochondrial Translocon and Interactions with the Matrix Import Motor

Ting

Schilke

Hayashi

et al. 2014

Journal of Biological Chemistry

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Background: Mitochondrial import motor associates with inner membrane translocase TIM23 by undefined interactions. Results: Site-specific photo-cross-linking was detected between matrix loops of TIM23 subunits Tim23 and Tim17 with Tim44 and Pam17, respectively. Conclusion: Motor components are tethered to translocon via the scaffold Tim44, with regulatory protein Pam17 having independent interaction site. Significance: Defined motor translocon architecture provides foundation for mechanistic understanding of efficient regulated translocation.

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“…28 This dramatic improvement has enabled the community to start using Uaas in yeast for research. 46 …”

Section: Introductionmentioning

confidence: 99%

Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts

Wang

2017

Acc. Chem. Res.

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Conspectus Expansion of the genetic code allows unnatural amino acids (Uaas) to be site-specifically incorporated into proteins in live biological systems, thus enabling novel properties selectively introduced into target proteins in vivo for basic biological studies and for engineering of novel biological functions. Orthogonal components including tRNA and aminoacyl-tRNA synthetase (aaRS) are expressed in live cells to decode a unique codon (often the amber stop codon UAG) as the desired Uaa. Initially developed in E. coli, this methodology has now been expanded in multiple eukaryotic cells and animals. In this account, we focus on addressing various biological challenges for rewriting the genetic code, describing impacts of code expansion on cell physiology, and discussing implications for fundamental studies of code evolution. Specifically, a general method using the type-3 polymerase III promoter was developed to efficiently express prokaryotic tRNAs as orthogonal tRNAs and a transfer strategy was devised to generate Uaa-specific aaRS for use in eukaryotic cells and animals. The aaRSs have been found to be highly amenable for engineering substrate specificity toward Uaas that are structurally far deviating from the native amino acid, dramatically increasing the stereochemical diversity of Uaas accessible. Preparation of the Uaa in ester or dipeptide format markedly increases the bioavailability of Uaas to cells and animals. Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism of eukaryotic cells, degrades mRNA containing a premature stop codon. Inhibition of NMD increases Uaa incorporation efficiency in yeast and C. elegans. In bacteria, release factor one (RF1) competes with the orthogonal tRNA for the amber stop codon to terminate protein translation, leading to low Uaa incorporation efficiency. Contradictory to the paradigm that RF1 is essential, it is discovered that RF1 is actually nonessential in E. coli. Knockout of RF1 dramatically increases Uaa incorporation efficiency and enables Uaa incorporation at multiple sites, making it feasible to use Uaa for directed evolution. Using these strategies, the genetic code has been effectively expanded in yeast, mammalian cells, stem cells, worms, fruit flies, zebrafish, and mice. It is also intriguing to find out that the legitimate UAG codons terminating endogenous genes are not efficiently suppressed by the orthogonal tRNA/aaRS in E. coli. Moreover, E. coli responds to amber suppression pressure promptly using transposon insertion to inactivate the introduced orthogonal aaRS. Persistent amber suppression evading transposon inactivation leads to global proteomic changes with a notable up-regulation of a previously uncharacterized protein YdiI, for which an unexpected function of expelling plasmids is discovered. Genome integration of the orthogonal tRNA/aaRS in mice results in minor changes in RNA transcripts but no significant physiological impairment. Lastly, the RF1 knockout E. coli strains afford a previously unavailable model organism for st...

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Impact of Nonnatural Amino Acid Mutagenesis on the in Vivo Function and Binding Modes of a Transcriptional Activator

Cited by 40 publications

References 33 publications

Discovery of Enzymatic Targets of Transcriptional Activators via in Vivo Covalent Chemical Capture

Discovery of Enzymatic Targets of Transcriptional Activators via in Vivo Covalent Chemical Capture

Architecture of the TIM23 Inner Mitochondrial Translocon and Interactions with the Matrix Import Motor

Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts

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