Dual genetic selection of the theophylline riboswitch with altered aptamer specificity for caffeine.

The aptamer domain of the theophylline riboswitch was randomized to generate a library containing millions of different variants. Dual genetic selection utilizing the cat-upp fusion gene was performed for the library, which successfully led to the identification of a caffeine-specific synthetic riboswitch. When a chloramphenicol-resistance gene was expressed under control of this riboswitch, E. coli cells showed chloramphenicol resistance only in the presence of caffeine. When inserted upstream of the gfpuv or lacZ gene, the caffeine riboswitch induced the expression of green fluorescent protein or ß-galactosidase in the presence of caffeine, respectively. When tested with various concentrations of caffeine, the ß-galactosidase activity was proportional to the amount of caffeine, clearly indicating the caffeine-dependent gene regulation by the caffeine riboswitch.

Rationally-designed fluorescent lysine riboswitch probes.

Two fluorescent lysine amide analogs, in which the carboxyl end of lysine was covalently attached to dansyl or NBD groups through an ethylene glycol-based linker, were rationally designed and synthesized. Both probes showed high binding affinity to the lysine riboswitch in vitro and their fluorescence intensities decreased by riboswitch binding.

Selective N-terminal fluorescent labeling of proteins using 4-chloro-7-nitrobenzofurazan: a method to distinguish protein N-terminal acetylation.

A fluorogenic derivatization method was developed to distinguish the protein N-terminal acetylation status. The unacetylated protein selectively reacted with 4-chloro-7-nitrobenzofurazan (NBD-Cl) at neutral pH to provide high fluorescence. In contrast, the protein with N-terminal acetylation was essentially nonfluorescent under the same conditions despite the presence of many internal lysine residues. Fluorescence of the NBD-labeled protein was very stable, and only micromolar concentrations of proteins were required for reliable detection. This method also provides a general and practical way to quantify proteins when their N-terminal amino group is available.

RimJ-mediated context-dependent N-terminal acetylation of the recombinant Z-domain protein in Escherichia coli.

N-terminal acetylation of the recombinant Z-domain protein depends on E. coli strains, expression vectors and amino acid residues near the N-terminus, and is enhanced by a high cellular level of RimJ.

In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy.

In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.

A genetically encoded infrared probe.

An orthogonal tRNA/aminoacyl-tRNA synthetase pair has been evolved that makes it possible to selectively and efficiently incorporate para-cyanophenylalanine (pCNPhe) into proteins in E. coli at sites specified by the amber nonsense codon, TAG. Substitution of pCNPhe for histidine-64 in myoglobin (Mb) affords a sensitive vibrational probe of ligand binding. This methodology provides a useful infrared reporter of protein structure, biomolecular interactions, and conformational changes.

The genetic incorporation of a distance probe into proteins in Escherichia coli.

The unnatural amino acid p-nitrophenylalanine (pNO2-Phe) was genetically introduced into proteins in Escherichia coli in response to the amber nonsense codon with high fidelity and efficiency by means of an evolved tRNA/aminoacyl-tRNA synthetase pair from Methanocuccus jannaschii. It was shown that pNO2-Phe efficiently quenches the intrinsic fluorescence of Trp in a distance-dependent manner in a model GCN4 basic region leucine zipper (bZIP) protein. Thus, the pNO2-Phe/Trp pair should be a useful biophysical probe of protein structure and function.

Decarboxylative Claisen condensation catalyzed by in vitro selected ribozymes.

The in vitro selection of RNAs catalyzing the decarboxylative Claisen condensation provides evidence for the synthesis of fatty acids, the building blocks of lipids and membranes, in the "RNA world".

Efficient incorporation of unnatural amino acids into proteins in Escherichia coli.

We have developed a single-plasmid system for the efficient bacterial expression of mutant proteins containing unnatural amino acids at specific sites designated by amber nonsense codons. In this system, multiple copies of a gene encoding an amber suppressor tRNA derived from a Methanocaldococcus jannaschii tyrosyl-tRNA (MjtRNATyrCUA) are expressed under control of the proK promoter and terminator, and a gene encoding the desired mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS) is expressed under control of a mutant glnS (glnS') promoter.

Efficient one-step syntheses of isoprenoid conjugates of nucleoside 5'-diphosphates.

[reaction: see text] Isoprenoid conjugates of nucleoside 5'-diphosphates were efficiently synthesized by one-step nucleophilic displacement reactions of either isoprenyl chlorides or isopentenyl tosylate with nucleoside 5'-diphosphates.