Phenylalanyl‐tRNA synthetase from <i>Thermus thermophilus</i> can attach two molecules of phenylalanine to tRNA<sup>Phe</sup>

Степанов, В. П.; Moor, Nina; Ankilova, V.N.; Lavrik, Olga I.

doi:10.1016/0014-5793(92)81099-8

Cited by 22 publications

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“…While ribosomal protein synthesis ordinarily involves (mono)aminoacylated tRNAs, Stepanov et al have described the formation of bis-(2 ,3 -O-phenylalanyl)-tRNAs by phenylalanyl-tRNA synthetase from Thermus thermophilus. 1,2 This enzyme was shown to form a tandemly activated tRNA using T. thermophilus tRNA Phe as substrate, as well as Escherichia coli tRNA Phe . Recently, we reported that it is possible to use such tandemly activated tRNAs as participants in protein synthesis in spite of the fact that they exhibit surprising chemical stability.…”

Section: Introductionmentioning

confidence: 99%

Modeling the reactive properties of tandemly activated tRNAs

2008

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Section: Introductionmentioning

confidence: 99%

Modeling the reactive properties of tandemly activated tRNAs

2008

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“…Stepanov et al (6,7) have described the formation of bis(2Ј,3Ј-O-phenylalanyl)-tRNAs by phenylalanyl-tRNA synthetase from Thermus thermophilus; the enzyme was shown to form a tandemly activated tRNA using T. thermophilus tRNA Phe as substrate, as well as Escherichia coli tRNA Phe and an RNA transcript identical in sequence with E. coli tRNA Phe . While the formation of bisphenylalanyl-tRNAs by T. thermophilus phenylalanyl-tRNA synthetase has been known for some time, possible functions of bisacylated tRNAs have apparently not been studied.…”

Section: From the Departments Of Chemistry And Biology University Ofmentioning

confidence: 99%

Tandemly Activated tRNAs as Participants in Protein Synthesis

Wang

Zhou

Lodder

et al. 2006

Journal of Biological Chemistry

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While all studies of protein synthesis to date have employed monoaminoacylated transfer RNAs, there have been reports that bisphenylalanyltRNA is formed by Thermus thermophilus phenylalanyl-tRNA synthetase. Such tandemly activated tRNAs have now been prepared by chemicoenzymatic techniques and are shown to function in both prokaryotic and mammalian protein synthesizing systems. They exhibit characteristics consistent with their possible utility under extreme conditions in natural systems and have important potential advantages for protein elaboration in cell free systems. Mechanistically, the bisaminoacylated tRNAs bind to the ribosomal A-site and utilize the aminoacyl moiety attached to the 3 -position of the terminal adenosine for addition to the growing polypeptide chain. Following translocation to the P-site and transfer of the formed peptidyl moiety, the donor tRNA dissociates from the ribosome as a monoaminoacylated tRNA capable of functioning in a subsequent polypeptide elongation step.Aminoacyl-tRNAs are key intermediates in protein biosynthesis. These adaptor molecules (1) not only faithfully recognize the appropriate mRNA codon but also transfer aminoacyl residues onto the growing peptide chain, thus bridging the information gap between nucleic acids and proteins. The amino acid residue is attached to the 2Ј(3Ј)-hydroxyl group of the 3Ј-terminal adenosine residue of tRNA via an ester linkage. The esterification of a tRNA with an amino acid is mediated by an aminoacyl-tRNA synthetase. There are 20 different aminoacyl-tRNA synthetases, each having a cognate amino acid and substrate tRNA. While the attachment of the amino acid occurs specifically either on the 2Ј-or 3Ј-OH group of the 3Ј-terminal adenosine moiety in tRNA (2), the resulting aminoacyl ester is quite activated and equilibriates rapidly between the 2Ј-and 3Ј-positions (3-5).Stepanov et al. (6, 7) have described the formation of bis(2Ј,3Ј-O-phenylalanyl)-tRNAs by phenylalanyl-tRNA synthetase from Thermus thermophilus; the enzyme was shown to form a tandemly activated tRNA using T. thermophilus tRNA Phe as substrate, as well as Escherichia coli tRNA Phe and an RNA transcript identical in sequence with E. coli tRNA Phe . While the formation of bisphenylalanyl-tRNAs by T. thermophilus phenylalanyl-tRNA synthetase has been known for some time, possible functions of bisacylated tRNAs have apparently not been studied. This may reflect the difficulty in obtaining such species by conventional methods on a preparative scale.Presently, we have prepared several tRNAs having amino acids attached to both the 2Ј-and 3Ј-positions of the 3Ј-terminal adenosine moiety. The ability of these tandemly activated tRNAs to participate in protein synthesis is described, as is an analysis of the mechanistic details of that participation. EXPERIMENTAL PROCEDURESConstruction of Expression Plasmids-Plasmids pThis15 and pTHD8 were used to express wild-type DHFR 2 in different in vitro translation systems. Plasmid pThis15 was used in a rabbit reticulocyte lysate system (8),...

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“…The most important recognition cletnents are G34, A35 and A 3 6 The rccognilion set also includes nucleotides in the variable pocket (U20 and U59), at the acceptor cnd (A73) and in the tKNA central corc (G10, C25, A26, C44 and U45). Many of the recognition nucleotides arc also among the residues comprising the identity set determined in viva using an amber suppressor tRNhPh' [I I].…”

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Recognition of tRNA^Phe by Phenylalanyl‐tRNA Synthetase of Thermus Thermophilus

Moor

Ankilova

Lavrik

1995

European Journal of Biochemistry

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The tRNAYh' nucleotides required for recognition by phenylalanyl-tRNA synlhetase of lhermus thermophilus have been determined using Escherichia roli tRNAph' transcripts with various mutations. The anticodon nucleotides are shown to be the most important recognition elements. The discriininator nucleotide, A73, involved in thc recognition sel of yeast, E. r d i and human phenylalanyl-tRNA synthetases contributes only slightly to tRNAPh' recognition by Th. tht,rrrmphilus phenylaliinyl-tRNA synthetase. Nucleotide 20 and some tertiary nucleotides, including the conserved GI 9 . CS6 base pair, are proposed to participate in stabilization of the precise tRNA conformation required for efficient aminoxcylation. The role of the 3'-CCA terminus, corninon to all tRNAs, in the spccific intcraction of tRNA with phenylalanyltRNA synthetase is discussed.Keyworh: tRNA; aminoacyl-tRNA synthetase; 1'7 transcript; IKNA recognition.Although all aminoacyl-tRNA synthctases have a coinrnon function in protein biosynthesis, namely to catalyze the aminoacylation of tRNA with one specific cognate amino acid, they are widely diverse in structure. All phenylalanyl-tRNA synihetases (tRNA'"" synthetases) known have a rare (for aminoacyltRNA synthetases) subunit structure of the (I$, type [I], excepting mitochondria1 tRNAPhc synthetase from Succ:harnmyces cerevisiae 121. Recent X-ray crystallographic analysis of a number of aminoacyl-tRNA synthetases and computer comparison of amino acid sequences have revealed two groups of aininoacyl-tRNA synthetases [31. tKNA"hr synthetases belong to the second class of aminoacyl-tRNA synthetases. For all enzymes of this class, tRNA aminoacylation occurs on the 3'-hydroxyl group of the 3'-terminal adenosine residue. However, tRNAphe synthetase iicylates tRNAYh' on the 2'-hydroxyl group [4, 51. tRNAPh' synthetase of Thermus thermophilus can attach the phenylalanyl moiety to both 2'-OH and 3'-OH groups simultaneously 161, tRNAPhe synthetase of Th. thermoyhilus is the biggest aminoacyl-tRNA synthetase crystallized to data. A high-resolution Xray study o f this enzyme and its cocrystals with tRNAPhcis now in progress [7]. tRNA'"* synthetase i s one enzyme for which the tRNA recognition set is known for evolutionary diverged species. The recognition patterns for yeast [S] and human [9] tRNAph' synthetases seem to be very similar and include three anticodon nucleotides (G34, A35 and A36), G20 and A73. The only difference between them is the importance of one or two anticodon stem base pairs for the recognition of tRNAPhr by the human enzyme.

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Phenylalanyl‐tRNA synthetase from Thermus thermophilus can attach two molecules of phenylalanine to tRNA^Phe

Cited by 22 publications

References 9 publications

Modeling the reactive properties of tandemly activated tRNAs

Modeling the reactive properties of tandemly activated tRNAs

Tandemly Activated tRNAs as Participants in Protein Synthesis

Recognition of tRNA^Phe by Phenylalanyl‐tRNA Synthetase of Thermus Thermophilus

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Phenylalanyl‐tRNA synthetase from Thermus thermophilus can attach two molecules of phenylalanine to tRNAPhe

Cited by 22 publications

References 9 publications

Modeling the reactive properties of tandemly activated tRNAs

Modeling the reactive properties of tandemly activated tRNAs

Tandemly Activated tRNAs as Participants in Protein Synthesis

Recognition of tRNAPhe by Phenylalanyl‐tRNA Synthetase of Thermus Thermophilus

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Phenylalanyl‐tRNA synthetase from Thermus thermophilus can attach two molecules of phenylalanine to tRNA^Phe

Recognition of tRNA^Phe by Phenylalanyl‐tRNA Synthetase of Thermus Thermophilus