Incorporation of nonnatural amino acids into proteins by using five-base codon-anticodon pairs.

A novel strategy for the incorporation of nonnatural amino acids into proteins was developed by using five-base codon-anticodon pairs. The streptavidin mRNA containing five-base codon CGGUA and the chemically aminoacylated tRNA with five-base anticodon UACCG were prepared, and added into E. coli in vitro translation system. As a result, the nonnatural amino acid was successfully incorporated into desired position of the protein. Other five-base codons CGGN1N2, where N1 and N2 indicate one of four nucleotides, were also available for the incorporation of the nonnatural amino acid.     

Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids

In vitro selection and directed evolution of peptides from mRNA display are powerful strategies to find novel peptide ligands that bind to target biomolecules. In this study, we expanded the mRNA display method to include multiple nonnatural amino acids by introducing three different four-base codons at a randomly selected single position on the mRNA. Another nonnatural amino acid may be introduced by suppressing an amber codon that may appear from a (NNK)_n nucleotide sequence on the mRNA. The mRNA display was expressed in an Escherichia coli in vitro translation system in the presence of three types of tRNAs carrying different four-base anticodons and a tRNA carrying an amber anticodon, the tRNAs being chemically aminoacylated with different nonnatural amino acids. The complexity of the starting mRNA-displayed peptide library was estimated to be 1.1 × 10¹² molecules. The effectiveness of the four-base codon mediated mRNA display method was demonstrated in the selection of biocytin-containing peptides on streptavidin-coated beads. Moreover, a novel streptavidin-binding nonnatural peptide containing benzoylphenylalanine was obtained from the nonnatural peptide library. The nonnatural peptide library from the four-base codon mediated mRNA display provides much wider functional and structural diversity than conventional peptide libraries that are constituted from 20 naturally occurring amino acids.     

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.     

Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit

During translation, some +1 frameshift mRNA sites are decoded by frameshift suppressor tRNAs that contain an extra base in their anticodon loops. Similarly engineered tRNAs have been used to insert nonnatural amino acids into proteins. Here, we report crystal structures of two anticodon stem-loops (ASLs) from tRNAs known to facilitate +1 frameshifting bound to the 30S ribosomal subunit with their cognate mRNAs. ASL(CCCG) and ASL(ACCC) (5'-3' nomenclature) form unpredicted anticodon-codon interactions where the anticodon base 34 at the wobble position contacts either the fourth codon base or the third and fourth codon bases. In addition, we report the structure of ASL(ACGA) bound to the 30S ribosomal subunit with its cognate mRNA. The tRNA containing this ASL was previously shown to be unable to facilitate +1 frameshifting in competition with normal tRNAs (Hohsaka et al. 2001), and interestingly, it displays a normal anticodon-codon interaction. These structures show that the expanded anticodon loop of +1 frameshift promoting tRNAs are flexible enough to adopt conformations that allow three bases of the anticodon to span four bases of the mRNA. Therefore it appears that normal triplet pairing is not an absolute constraint of the decoding center.     

Incorporation of nonnatural amino acids into proteins by using various four-base codons in an Escherichia coli in vitro translation system

Incorporation of nonnatural amino acids into proteins is a powerful technique in protein research. Amber suppression has been used to this end, but this strategy does not allow multiple incorporation of nonnatural amino acids into single proteins. In this article, we developed an alternative strategy for nonnatural mutagenesis by using four-base codons. The four-base codons AGGU, CGGU, CCCU, CUCU, CUAU, and GGGU were successfully decoded by the nitrophenylalanyl-tRNA containing the complementary four-base anticodons in an Escherichia coli in vitro translation system. The most efficient four-base decoding was observed for the GGGU codon, which yielded 86% of the full-length protein containing nitrophenylalanine relative to the wild-type protein. Moreover, highly efficient incorporation of two different nonnatural amino acids was achieved by using a set of two four-base codons, CGGG and GGGU. This work shows that the four-base codon strategy is more advantageous than the amber suppression strategy in efficiency and versatility.     

Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon

An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a K_m = 47.1 μM and a k_cat = 0.151 s⁻¹, which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNA^Cys with the GCA anticodon (26.9 μM and 0.111 s⁻¹). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.     

Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment

Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNA^Opt. We suspected a modification of the tRNA^Opt_AUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNA^Opt_AUG is converted to inosine. We identified tRNA^Opt_AUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms.     

A multivariate study of the relationship between the genetic code and the physical-chemical properties of amino acids

Summary The 20 naturally occurring amino acids are characterized by 20 variables: pK_{NH 2}, pK_COOH, pI, molecular weight, substituent van der Waals volume, seven¹H and¹³C nuclear magnetic resonance shift variables, and eight hydrophobicity-hydrophilicity scales. The 20-dimensional data set is reduced to a few new dimensions by principal components analysis. The three first principal components reveal relationships between the properties of the amino acids and the genetic code. Thus the amino acids coded for by adenosine (A), uracil (U), or cytosine (C) in their second codon position (corresponding to U, A, or G in the second anticodon position) are grouped in these components. No grouping was detected for the amino acids coded for by guanine (G) in the second codon position (corresponding to C in the second anticodon position). The results show that a relationship exists between the physical-chemical properties of the amino acids and which of the A (U), U (A), or C (G) nucleotide is used in the second codon (anticodon) position. The amino acids coded for by G (C) in the second codon (anticodon) position do not participate in this relationship.     

Insights into Factorless Translational Initiation by the tRNA-Like Pseudoknot Domain of a Viral IRES

The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNA_i-independent IRES translation.     

Mutational alterations of tryptophan-specific transfer RNA that generate translation suppressors of the UAA, UAG and UGA nonsense codons

The recessive lethal amber suppressor su⁺7(UAG-1) in Escherichia coli inserts glutamine in response to the UAG codon. The genetic analysis presented in this paper shows that the su^?7 precursor allele can give rise to suppressors of the UGA codon as well as of the UAG codon. This observation suggests that the su^?7 gene normally codes for transfer RNA^Trp, a tRNA whose anticodon can be modified by single base changes to forms that can translate either UAG or UGA. The chemical findings presented in the accompanying paper (Yaniv et al., 1974) are wholly in accord with this interpretation. Thus, a single base substitution in the anticodon sequence of a tRNA can affect both the coding specificity of the molecule and also the amino acid acceptor specificity.     

Synthesis and in situ insertion of a site-specific fluorescently labeled membrane protein into cell-sized liposomes

The integral membrane protein bacteriorhodopsin, containing a fluorescent amino acid at a specific position, was synthesized in the presence of hydrated lipid films using an in vitro translation system expanded with a four-base codon/anticodon pair. Cell-sized liposomes with the labeled protein inserted into the liposome membranes were generated after the translation reaction. This study also demonstrated that this labeling method could be used to analyze the dynamic properties of membrane proteins in situ by fluorescence correlation spectroscopy.     

Quadruplet codons: implications for code expansion and the specification of translation step size

One of the requirements for engineering expansion of the genetic code is a unique codon which is available for specifying the new amino acid. The potential of the quadruplet UAGA in Escherichia coli to specify a single amino acid residue in the presence of a mutant tRNA(Leu) molecule containing the extra nucleotide, U, at position 33.5 of its anticodon loop has been examined. With this mRNA-tRNA combination and at least partial inactivation of release factor 1, the UAGA quadruplet specifies a leucine residue with an efficiency of 13 to 26 %. The decoding properties of tRNA(Leu) with U at position 33.5 of its eight-membered anticodon loop, and a counterpart with A at position 33.5, strongly suggest that in both cases their anticodon loop bases stack in alternative conformations. The identity of the codon immediately 5' of the UAGA quadruplet influences the efficiency of quadruplet translation via the properties of its cognate tRNA. When there is the potential for the anticodon of this tRNA to dissociate from pairing with its codon and to re-pair to mRNA at a nearby 3' closely matched codon, the efficiency of quadruplet translation at UAGA is reduced. Evidence is presented which suggests that when there is a purine base at position 32 of this 5' flanking tRNA, it influences decoding of the UAGA quadruplet.     

Maintenance of the correct open reading frame by the ribosome

During translation, a string of non-overlapping triplet codons in messenger RNA is decoded into protein. The ability of a ribosome to decode mRNA without shifting between reading frames is a strict requirement for accurate protein biosynthesis. Despite enormous progress in understanding the mechanism of transfer RNA selection, the mechanism by which the correct reading frame is maintained remains unclear. In this report, evidence is presented that supports the idea that the translational frame is controlled mainly by the stability of codon–anticodon interactions at the P site. The relative instability of such interactions may lead to dissociation of the P-site tRNA from its codon, and formation of a complex with an overlapping codon, the process known as P-site tRNA slippage. We propose that this process is central to all known cases of +1 ribosomal frameshifting, including that required for the decoding of the yeast transposable element Ty3. An earlier model for the decoding of this element proposed 'out-of-frame' binding of A-site tRNA without preceding P-site tRNA slippage.     

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.     

Codon-Anticodon Recognition in the Bacillus subtilis glyQS T Box Riboswitch: RNA-DEPENDENT CODON SELECTION OUTSIDE THE RIBOSOME*

Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the “Specifier Sequence,” in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNA^Gly anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3′ of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system.     

Codon:Anticodon and anticodon:Anticodon interaction. Evaluation of equilibrium and kinetic parameters of complexes involving a G:U wobble

In order to learn about the effect of the G:U wobble interaction we characterized the codon:anticodon binding between triplets: UUC, UUU and yeast tRNA^Phe (anticodon GmAA) as well as the anticodon:anticodon binding between Escherichia coli tRNA^Glu₂, E. coli tRNA^Lys (anticodons: mam⁵s²UUC, and mam⁵s²UUU, respectively) and tRNA^Phe from yeast and E. coli (anticodon GAA) using equilibrium fluorescence titrations and temperature jump measurements with fluorescence and absorption detection. The difference in stability constants between complexes involving a G:U pair rather than a usual G:C basepair is in the range of one order of magnitude and is mainly due to the shorter lifetime of the complex involving G:U in the wobble position. This difference is more pronounced when the codon triplet is structured, i.e., is built in the anticodon loop of a tRNA. The reaction enthalpies of the anticodon:anticodon complexes involving G:U mismatching were found to be about 4 kcal/mol smaller, and the melting temperatures more than 20°C lower, than those of the corresponding complexes with the G:C basepair. The results are discussed in terms of different strategies that might be used in the cell in order to minimize the effect of different lifetimes of codon-tRNA complexes. Differences in these lifetimes may be used for the modulation of the translation efficiency.     

Specificity of the ribosomal A site for aminoacyl-tRNAs

Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2′-deoxynucleotide-substituted mRNAs are used to demonstrate the presence of a lower threshold in k_off values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed. However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold. We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.     

Study of codon bias perspective of fungal xylanase gene by multivariate analysis

Fungal xylanases has important applications in food, baking, pulp and paper industries in addition to various other industries. Xylanases are produced extensively by both bacterial and fungal sources and has tremendous potential of being active at extremes of temperature and pH. In the present study an effort has been made to explore the codon bias perspective of this potential enzyme using bioinformatics tools. Multivariate analysis has been used as a tool to study codon bias perspectives of xylanases. It was further observed that the codon usage of xylanases genes from different fungal sources is not similar and to reveal this phenomenon the relative synonymous codon usage (RSCU) and base composition variation in fungal xylanase genes were also studied. The codon biasing data like GC content at third position (GC_3S), effective codon number (N_C), codon adaptive index (CAI) were further analyzed with statistical softwares like Sigma1plot 9.0 and Systat 11.0. Furthermore, study of translation selection was also performed to verify the influences of codon usage variation among the 94 xylanase genes. In the present study xylanase gene from 12 organisms were analyzed and codon usages of all xylanases from each organism were compared separately. Analysis indicates biased codon among all 12 fungi taken for study with Aspergillus nidulans, Chaetomium globosum, Aspergillus terreus and Aspergillus clavatus showing maximum biasing. N_C plot and correspondence analysis on relative synonymous codon usage indicate that mutation bias and translation selection influences codon usage variation in fungal xylanase gene. To reveal the relative synonymous codon usage and base composition variation in xylanase, 94 genes from 12 fungi were used as model system.