Recent Developments of Engineered Translational Machineries for the Incorporation of Non-Canonical Amino Acids into Polypeptides

Genetic code expansion and reprogramming methodologies allow us to incorporate non-canonical amino acids (ncAAs) bearing various functional groups, such as fluorescent groups, bioorthogonal functional groups, and post-translational modifications, into a desired position or multiple positions in polypeptides both in vitro and in vivo. In order to efficiently incorporate a wide range of ncAAs, several methodologies have been developed, such as orthogonal aminoacyl-tRNA-synthetase (AARS)–tRNA pairs, aminoacylation ribozymes, frame-shift suppression of quadruplet codons, and engineered ribosomes. More recently, it has been reported that an engineered translation system specifically utilizes an artificially built genetic code and functions orthogonally to naturally occurring counterpart. In this review we summarize recent advances in the field of ribosomal polypeptide synthesis containing ncAAs.     

PCR and magnetic bead-mediated target capture for the isolation of short interspersed nucleotide elements in fishes

Short interspersed nucleotide elements (SINEs), a type of retrotransposon, are widely distributed in various genomes with multiple copies arranged in different orientations, and cause changes to genes and genomes during evolutionary history. This can provide the basis for determining genome diversity, genetic variation and molecular phylogeny, etc. SINE DNA is transcribed into RNA by polymerase III from an internal promoter, which is composed of two conserved boxes, box A and box B. Here we present an approach to isolate novel SINEs based on these promoter elements. Box A of a SINE is obtained via PCR with only one primer identical to box B (B-PCR). Box B and its downstream sequence are acquired by PCR with one primer corresponding to box A (A-PCR). The SINE clone produced by A-PCR is selected as a template to label a probe with biotin. The full-length SINEs are isolated from the genomic pool through complex capture using the biotinylated probe bound to magnetic particles. Using this approach, a novel SINE family, Cn-SINE, from the genomes of Coilia nasus, was isolated. The members are 180-360 bp long. Sequence homology suggests that Cn-SINEs evolved from a leucine tRNA gene. This is the first report of a tRNA(Leu)-related SINE obtained without the use of a genomic library or inverse PCR. These results provide new insights into the origin of SINEs.     

Genetic Code Expansion by Degeneracy Reprogramming of Arginyl Codons

The genetic code in most organisms codes for 20 proteinogenic amino acids or translation stop. In order to encode more than 20 amino acids in the coding system, one of stop codons is usually reprogrammed to encode a non‐proteinogenic amino acid. Although this approach works, usually only one amino acid is added to the amino acid repertoire. In this study, we incorporated non‐proteinogenic amino acids into a protein by using a sense codon. As all the codons are allocated in the universal genetic code, we destroyed all the tRNA^Arg in a cell‐free protein synthesis system by using a tRNA^Arg‐specific tRNase, colicin D. Then by supplementing the system with tRNA_CCU, the translation system was partially restored. Through this creative destruction, reprogrammable codons were successfully created in the system to encode modified lysines along with the 20 proteinogenic amino acids.     

New Efficient Synthesis of tRNA Related Adenosines Bearing the Hydantoin Ring (ct6A,ms2ct6A) by Intramolecular Cyclization of N6-(N-Boc-α-aminoacyl)-adenosine Derivatives

A novel and efficient way for the synthesis of N⁶-hydantoin-modified adenosines, which utilizes readily available N⁶-(N-Boc-α-aminoacyl)-adenosine derivatives, was developed. The procedure is based on the epimerization-free, Tf₂O-mediated conversion of the Boc group into an isocyanate moiety, followed by intramolecular cyclization. Using this method two recently discovered hydantoin modified tRNA adenosines, that is, cyclic N⁶-threonylcarbamoyl-adenosine ( ct⁶A ) and 2-methylthio-N⁶-threonylcarbamoyladenosine ( ms²ct⁶A ) were prepared in good yields.     

Analysis of a 62 kb DNA sequence of chromosome X reveals 36 open reading frames and a gene cluster with a counterpart on chromosome XI

We have sequenced a 61,989 bp stretch located between genes RAD7 and FIP1 of Saccharomyces cerevisiae chromosome X. This stretch contains 36 open reading frames (ORFs) of at least 100 codons. Fourteen of these correspond to sequences previously published as HIT1, CDC8, YAP17, CBF1, NAT1, RPA12, CCT5, TOR1, RFC2, PEM2, CDC11, MIR1, STE18 and GRR1. The proteins deduced from four ORFs (YJR059w, YJR065c, YJR075w, YJR078w) have significant similarity to proteins of known function from yeast or other organisms, including S. cerevisiae serine/threonine-specific protein kinase, Schizosaccharomyces pombe Act2 protein, S. cerevisiae mannosyltransferase OCH1 protein and mouse indoleamine 2,3-dioxygenase, respectively. Four of the remaining 18 ORFs have similarity to proteins with unknown function, six are weakly similar to other known sequences, while another eight exhibit no similarity to any known sequence. In addition, three tRNA genes have been recognized. Three genes clustered within 22 kb (YJR059w, YJR061w and TOR1) have counterparts arranged within 15 kb on the left arm of chromosome XI. The sequence has been deposited in the Genome Sequence Data Base under Accession Number L47993.     

Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism

Transfer RNA-guanine transglycosylases (TGTs) are evolutionarily ancient enzymes, present in all kingdoms of life, catalyzing guanine exchange within their cognate tRNAs by modified 7-deazaguanine bases. Although distinct bases are incorporated into tRNA at different positions in a kingdom-specific manner, the catalytic subunits of TGTs are structurally well conserved. This review provides insight into the sequential steps along the reaction pathway, substrate specificity, and conformational adaptions of the binding pockets by comparison of TGT crystal structures in complex with RNA substrates of a eubacterial and an archaebacterial species. Substrate-binding modes indicate an evolutionarily conserved base-exchange mechanism with a conserved aspartate serving as a nucleophile through covalent binding to C1' of the guanosine ribose moiety in an intermediate state. A second conserved aspartate seems to control the spatial rearrangement of the ribose ring along the reaction pathway and supposedly operates as a general acid/base. Water molecules inside the binding pocket accommodating interaction sites subsequently occupied by polar atoms of substrates help to elucidate substrate-recognition and substrate-specificity features. This emphasizes the role of water molecules as general probes to map binding-site properties for structure-based drug design. Additionally, substrate-bound crystal structures allow the extraction of valuable information about the classification of the TGT superfamily into a subdivision of presumably homologous superfamilies adopting the triose-phosphate isomerase type barrel fold with a standard phosphate-binding motif.     

Analysis of a 42·5 kb DNA sequence of chromosome X reveals three tRNA genes and 14 new open reading frames including a gene most probably belonging to the family of ubiquitin–protein ligases

We have sequenced a 42,500 bp stretch located on chromosome X of Saccharomyces cerevisiae between the genes MET3 and CDC8. This stretch contains 24 open reading frames (ORFs) of at least 100 amino acids. Ten of these correspond to previously published sequences, whereas of the 14 remaining ORFs, only one, GTD892, has significant similarity to proteins from yeast or other organisms. It may belong to the family of ubiquitin–protein ligases and be involved in the ubiquitin-dependent proteolytic pathway. In addition, three tRNA genes were recognized, two of which had not been hitherto localized. The sequence has been deposited in the Genome Sequence Data Base under Accession Number L36344.