N-terminal labeling of proteins using initiator tRNA

Methodology based on tRNA mediated protein engineering is described for the introduction of fluorophores and other labels at the N-terminus of proteins produced in cell-free translation systems. One method for low-level (trace) N-terminal labeling is based on the use of an Escherichia coli initiator tRNA(fMet) misaminoacylated with methionine modified at the alpha-amino group. In addition to the normal formyl group, the protein translational machinery incorporates the fluorophore BODIPY-FL and the affinity tag biotin at an N-terminal end of the nascent protein. A second method for higher N-terminal labeling uses a chemically aminoacylated amber initiator suppressor tRNA and a DNA template which contains a complementary amber (UAG) codon instead of the normal initiation (AUG) codon. This more versatile approach is demonstrated using a variety of N-terminal markers including fluorescein, biotin, PC-biotin, and a novel dual marker conjugate (Biotin/BODIPY-FL).     

Retroviral gag gene amber codon suppression is caused by an intrinsic cis-acting component of the viral mRNA.

In some type C retroviruses, translation of the pol gene appears to require translational suppression of the proximal gag amber codon. To identify the region of the viral nucleic acid responsible for synthesis of the pol gene products, a 300-base-pair DNA fragment containing the stop codon from a type C murine virus (AK virus) was inserted into the Escherichia coli lacZ gene such that the translational reading frame was maintained. Introduction of the resulting fusion gene into cells resulted in the suppression of the viral stop codon. As measured by beta-galactosidase production, suppression occurred at a frequency of approximately 10%. Suppression could occur in at least several vertebrate cell types and was not augmented by virus replication or the expression of viral gene products. This indicates that gag amber codon suppression does not require augmented levels of suppressor tRNA species.     

Expanding the genetic code of Caenorhabditis elegans using bacterial aminoacyl-tRNA synthetase/tRNA pairs

The genetic code specifies 20 common amino acids and is largely preserved in both single and multicellular organisms. Unnatural amino acids (Uaas) have been genetically incorporated into proteins by using engineered orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pairs, enabling new research capabilities and precision inaccessible with common amino acids. We show here that Escherichia coli tyrosyl and leucyl amber suppressor tRNA/RS pairs can be evolved to incorporate different Uaas in response to the amber stop codon UAG into various proteins in Caenorhabditis elegans. To accurately report Uaa incorporation in worms, we found that it is crucial to integrate the UAG-containing reporter gene into the genome rather than to express it on an extrachromosomal array from which variable expression can lead to reporter activation independent of the amber-suppressing tRNA/RS. Synthesizing a Uaa in a dipeptide drives Uaa uptake and bioavailability. Uaa incorporation has dosage, temporal, tRNA copy, and temperature dependencies similar to those of endogenous amber suppression. Uaa incorporation efficiency was improved by impairing the nonsense-mediated mRNA decay pathway through knockdown of smg-1. We have generated stable transgenic worms capable of genetically encoding Uaas, enabling Uaa exploitation to address complex biological problems within a metazoan. We anticipate our strategies will be generally extendable to other multicellular organisms.     

RNA cleavage linked with ribosomal action

Ribonuclease LS in Escherichia coli is a potential antagonist of bacteriophage T4. When T4 dmd is mutated, this RNase efficiently cleaves T4 mRNAs and leads to the silencing of late genes, thus blocking T4 growth. We previously found that, when two consecutive ochre codons were placed in the open reading frame of T4 soc, RNase LS cleaved soc mRNA at a specific site downstream of the ochre codons. Here, we demonstrate that RNase LS cleaves soc RNA at the same site even when only a single ochre codon is present or is replaced with either an amber or an opal codon. On the other hand, disruption of the Shine-Dalgarno sequence, a ribosome-binding site required for the initiation of translation, eliminates the cleavage. These results strongly suggest that RNase LS cleaves in a manner dependent on translation termination. Consistent with this suggestion, the cleavage dependency on an amber codon was considerably reduced in the presence of amber-codon-suppressing tRNA. Instead, two other cleavages that depend on translation of the region containing the target sites occurred farther downstream. Additional analysis suggests that an interaction of the ribosome with a stop codon might affect the site of cleavage by RNase LS in an mRNA molecule. This effect of the ribosome could reflect remodeling of the high-order structure of the mRNA molecule.     

Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coli.

Site-directed incorporation of the amino acid analogue p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNA(Phe)amber/phenylalanyl-tRNA synthetase pair was expressed in an analogue-resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64-75% as p-F-Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p-F-Phe incorporation was 11-21-fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8-12 mg/L of culture, corresponding to about two thirds of the expression level of the wild-type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F-NMR spectroscopy and other sample-intensive methods. The use of a nonessential "21st" tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.     

Molecular modeling of positioning of human release factor eRF1 relative to mRNA stop-codon explains a proximity of the eRF1 C-domain to stop-codon in ribosomal complex

A properties of atomic models of structure of eukaryotic triple complex eRF1 . mRNA . tRNAPhe containing human class-1 polypeptide release factor eRF1 at the A-site of human 80S ribosome, mRNA and P-site tRNAPhe, obtained before, are considered. The stricture of the complex is described using high resolution NMR structure of eRF1 M-domain. The structural properties of distribution of chemical cross-links are investigated, which allows us to choose correct model of positioning of the eRF1 molecule in ribosome A-site relative to stop codon of mRNA. A distributions of crosslinks between photoactivatable perfluoroaryl azide group of modified nucleotides of mRNA analogues and eRF1 molecule are modeled via molecular dynamics method. Twelve different mRNA analogues with modified nucleotides of stop signal in positions +4 to +9 with respect to the first nucleotide of the P-site codon are modeled. It was shown that only one of the two models of complex eRFI . mRNA . tRNA gives cross-link distribution in a good agreement with experimental data. A new features of the final structure of triple complex eRF1 . mRNA . tRNA is spatial proximity of stop-codon nucleotides to the C-domain of the eRF1, which explains previously obtained cross-link experimental data.     

Escherichia coli JA221 can suppress the UAG stop signal

The Escherichia coli strain JA221 can suppress the UAG stop codon, although the existence of an amber suppressor tRNA has not previously been described for this strain. When using a plasmid to express α-sarcin, which has TAG as its stop signal, two proteins were obtained: a smaller protein corresponding in size to that of the expected protein, and a larger protein, which could be accounted for by the presence of a second stop codon (TGA) 18 base pairs downstream of the original. This feature of strain JA221 must therefore be considered when using this strain as a host for the production of recombinant proteins.     

Site-specific incorporation of non-natural amino acids into proteins in mammalian cells with an expanded genetic code

We describe a detailed protocol for incorporating non-natural amino acids, 3-iodo-L-tyrosine (IY) and p-benzoyl-L-phenylalanine (pBpa), into proteins in response to the amber codon (the UAG stop codon) in mammalian cells. These amino acids, IY and pBpa, are applicable for structure determination and the analysis of a network of protein-protein interactions, respectively. This method involves (i) the mutagenesis of the gene encoding the protein of interest to create an amber codon at the desired site, (ii) the expression in mammalian cells of the bacterial pair of an amber suppressor tRNA and an aminoacyl-tRNA synthetase specific to IY or pBpa and (iii) the supplementation of the growth medium with these amino acids. The amber mutant gene, together with these bacterial tRNA and synthetase genes, is introduced into mammalian cells. Culturing these cells for 16-40 h allows the expression of the full-length product from the mutant gene, which contains the non-natural amino acid at the introduced amber position. This method is implemented using the conventional tools for molecular biology and treating cultured mammalian cells. This protocol takes 5-6 d for plasmid construction and 3-4 d for incorporating the non-natural amino acids into proteins.     

Site-specific insertion of spin-labeled L-amino acids in Xenopus oocytes

Site-specific insertion of modified amino acids in proteins expressed in living cells is an emerging field holding great promise for elucidating protein structure-function relationships, expression levels, localization, and activation states in a complex milieu. To evaluate the efficiency of amino acids modified to carry either a nitroxide spin probe or a fluorescence probe, we have developed a screen using the levels of functional luciferase protein expressed in Xenopus oocytes. Natural and modified amino acids were targeted to position 14 in firefly luciferase using an amber mutation or introducing the four-codon nucleotide GGGU. Using the amber stop codon, the incorporation efficiencies of injected tRNA charged with the native phenylalanine residue, a fluorescent NBD-alanine, or nitroxide-labeled cysteine and tyrosine amino acids ranged from 1% to 18%. While the NBD-amino acid derivative gave higher incorporation levels, the EPR signals from the spin-labeled amino acids allow for the direct assessment of aminoacylation extent and stability. Applying the four-base codon for the first time in Xenopus oocytes, we found the incorporation efficiencies were significantly lowered compared to results using the three-base amber codon. The studies presented here provide quantitative assessment of protein expression levels when using nonsense suppression to site-specifically label proteins with spectroscopic probes in oocytes. Finally, the effect of a 77-base RNA aptamer known to inhibit the eucaryotic release factor of protein synthesis was tested for its influence on nonsense incorporation in Xenopus oocytes. The combination of A34 and charged suppressor tRNA produced a 3-fold increase in the expressed TAG(14)-luciferase level, compared to the use of charged suppressor tRNA alone.     

Nonsense suppression in eukaryotes: the use of the Xenopus oocyte as an in vivo assay system.

Amber, ochre, and opal nonsense suppressor tRNAs isolated from yeast were injected into Xenopus laevis oocytes together with purified mRNAs (globin mRNA from rabbit, tobacco mosaic virus-RNA). Yeast opal suppressor tRNA is able to read the UGA stop codon of the rabbit beta-globin mRNA, thus producing a readthrough protein. A large readthrough product is also obtained upon coinjection of yeast amber or ochre suppressor tRNA with TMV-RNA. The amount of readthrough product is dependent on the amount of injected suppressor tRNA. The suppression of the terminator codon of TMV-RNA is not susceptible to Mg++ concentration or polyamine addition. Therefore, the Xenopus laevis oocyte provides a simple, sensitive, and well buffered in vivo screening system for all three types of eukaryotic nonsense suppressor tRNAs.     

Nonsense suppression in Dictyostelium discoideum

We describe the generation of Dictyostelium discoideum cell lines that carry different suppressor tRNA genes. These genes were constructed by primer-directed mutagenesis changing a tRNA(Trp)(CCA) gene from D. discoideum to a tRNA(Trp)(amber) gene and changing a tRNA(Glu)(UUC) gene from D. discoideum to a tRNA(Glu)(ochre) as well as a tRNA(Glu)(amber) gene. These genes were stably integrated into the D. discoideum genome together with a reporter gene. An actin 6::lacZ gene fusion carrying corresponding translational stop signals served as a reported. Active beta-galactosidase is expressed only in D. discoideum strains that contain, in addition to the reporter, a functional suppressor tRNA. Both amber suppressors are active in D. discoideum without interfering significantly with cell growth and development. We failed, however, to establish cell lines containing a functional tRNA(Glu)(ochre) suppressor. This may be due to the fact that nearly every message from D. discoideum known so far terminates with UAA. Therefore a tRNA capable of reading this termination codon may not be compatible with cell growth.     

Analysis of the roles of tRNA structure, ribosomal protein L9, and the bacteriophage T4 gene 60 bypassing signals during ribosome slippage on mRNA

A 50-nucleotide coding gap divides bacteriophage T4 gene 60 into two open reading frames. In response to cis-acting stimulatory signals encrypted in the mRNA, the anticodon of the ribosome-bound peptidyl tRNA dissociates from a GGA codon at the end of the first open reading frame and pairs with a GGA codon 47 nucleotides downstream just before the second open reading frame. Mutations affecting ribosomal protein L9 or tRNA(Gly)(2), the tRNA that decodes GGA, alter the efficiency of bypassing. To understand the mechanism of ribosome slippage, this work analyzes the influence of these bypassing signals and mutant translational components on -1 frameshifting at G GGA and hopping over a stop codon immediately flanked by two GGA glycine codons (stop-hopping). Mutant variants of tRNA(Gly)(2) that impair bypassing mediate stop-hopping with unexpected landing specificities, suggesting that these variants are defective in ribosomal P-site codon-anticodon pairing. In a direct competition between -1 frameshifting and stop-hopping, the absence of L9 promotes stop-hopping at the expense of -1 frameshifting without substantially impairing the ability of mutant tRNA(Gly)(2) variants to re-pair with the mRNA by sub-optimal pairing. These observations suggest that L9 defects may stimulate ribosome slippage by enhancing mRNA movement through the ribosome rather than by inducing an extended pause in translation or by destabilizing P-site pairing.Two of the bypassing signals, a cis-acting nascent peptide encoded by the first open reading frame and a stemloop signal located in the 5' portion of the coding gap, stimulate peptidyl-tRNA slippage independently of the rest of the gene 60 context. Evidence is presented suggesting that the nascent peptide signal may stimulate bypassing by destabilizing P-site pairing.     

An in vitro DNA virus for in vitro protein evolution.

In vitro virus is a molecular construct for in vitro protein evolution, which requires some mechanism to link phenotype to genotype. The first in vitro virus was realized by bonding a nascent protein with its coding mRNA via puromycin in in vitro translation. We report a new construct of in vitro DNA virus. The virion was a covalent cDNA-protein fusion, and virion formation did not require any modification of mRNA. Due to intactness of mRNA, this type of in vitro DNA virus will take the next step toward in vitro autonomous evolution, just like in vivo viral evolution in a cellstat.     

Efficient suppression of the amber codon in E. coli in vitro translation system

An mRNA encoding the esterase from Alicyclobacillus acidocaldarius with catalytically essential serine codon (ACG) replaced by an amber (UAG) codon was used to study the suppression in in vitro translation system. Suppression of UAG by tRNA(Ser(CUA)) was monitored by determination of the full-length and active esterase. It was shown that commonly used increase of suppressor tRNA concentration inhibits protein production and therefore limits suppression. In situ deactivation of release factor by specific antibodies leads to efficient suppression already at low suppressor tRNA concentration and allows an in vitro synthesis of fully active enzyme in high yield undistinguishable from wild-type protein.     

Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3'-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA(Phe), targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3'-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.     

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal,ochre, and amber suppressor tRNAs

Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNA^Cys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies.     

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.