Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.1.1.20 (phenylalanyl-tRNA synthetase)
 358 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A procedure for replacing residues 33-35 in the anticodon loop of yeast tRNATyr with any desired oligonucleotide has been developed. The three residues were removed by partial ribonuclease A digestion. An oligonucleotide was inserted into the gap in four steps by using RNA ligase, polynucleotide kinase, and pseT 1 polynucleotide kinase. The rate of aminoacylation of anticodon loop substituted tRNATyr by yeast tyrosyl-tRNA synthetase was found to depend upon the sequence of the oligonucleotide inserted. This suggests that the nucleotides in the anticodon loop of yeast tRNATyr are required for optimal aminoacylation. In addition, tRNATyr modified to have a phenylalanine anticodon was shown to be misacylated by yeast phenylalanyl-tRNA synthetase at a rate at least 10 times faster than unmodified tRNATyr. Thus, the anticodon is used by phenylalanyl-tRNA synthetase to distinguish between tRNAs.
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PMID:Aminoacylation of anticodon loop substituted yeast tyrosine transfer RNA. 384 56

Escherichia coli threonyl-tRNA synthetase (EC 6.1.1.3) expression has been examined in an acellular protein-synthesizing system programmed with a plasmid DNA carrying thrS, infC, pheS, and pheT, the gene for threonyl-tRNA synthetase, initiation factor 3, and the two protomers of phenylalanyl-tRNA synthetase (EC 6.1.1.20), respectively. The initial rate of synthesis of L-[35S]methionine-labeled threonyl-tRNA synthetase is markedly reduced by the addition of homogeneous RNase-free threonyl-tRNA synthetase to the assay, not by that of phenylanyl- or tyrosyl-tRNA synthetase (EC 6.1.1.1). The inhibition is 50% in the presence of 0.25 microM threonyl-tRNA synthetase and reaches 90% with 2 microM enzyme. Synthesis of mRNA in the acellular DNA-dependent protein-synthesizing system has been measured by molecular hybridization to gene-specific lambda DNA probes corresponding to thrS, pheS, and pheT. The addition to the assay of 2 microM threonyl-tRNA synthetase does not affect the extent of mRNA hybridizing to the thrS-specific DNA probe. This result is interpreted as reflecting an effect of the synthetase on its expression at the translational level. Analysis of the DNA sequence of the thrS gene predicts several potential secondary structures capable of forming in the thrS mRNA. One of these potential structures is a cloverleaf. The possible role of such structures in controlling expression of thrS is discussed.
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PMID:Autogenous repression of Escherichia coli threonyl-tRNA synthetase expression in vitro. 632 25

Tyrosyl-, arginyl-, leucyl-, and phenylalanyl-tRNA synthetase activities were measured in extracts from three root sections of 3-day-old pea seedlings. The sections 0 to 2, 3 to 7, and 8 to 22 millimeters from the root tip were chosen to represent the regions of cell division, elongation, and maturation, respectively. The specific activity for each aminoacyl-tRNA synthetase was highest in the 0- to 2-millimeter section and lowest in the 8 to 22 millimeter section. The changes in specific activity between the sections, however, varied with the particular synthetase. Tyrosyl-tRNA synthetase from each section was fractionated into two activity regions on a diethylaminoethyl cellulose column. Approximately 10, 22, and 44% of the total tyrosyl-tRNA synthetase activity in the 0 to 2, 3 to 7, and 8- to 22-millimeter sections, respectively, were associated with the first tyrosyl-tRNA synthetase region; the remaining activity was located in the second tyrosyl-tRNA synthetase region. Only one activity region for arginyl-tRNA synthetase was detected by diethylaminoethyl cellulose column fractionation.
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PMID:Changes in certain aminoacyl transfer ribonucleic Acid synthetase activities in developing pea roots. 1665 89

To guarantee specific tRNA and amino acid pairing, several aminoacyl-tRNA synthetases correct aminoacylation errors by deacylating or "editing" misaminoacylated tRNA. A previously developed variant of Escherichia coli tyrosyl-tRNA synthetase (iodoTyrRS) esterifies or "charges" tRNA(Tyr) with a nonnatural amino acid, 3-iodo-l-tyrosine, and with l-tyrosine less efficiently. In the present study, the editing domain of phenylalanyl-tRNA synthetase (PheRS) was transplanted into iodoTyrRS to edit tyrosyl-tRNA(Tyr) and thereby improve the overall specificity for 3-iodo-l-tyrosine. The beta-subunit fragments of the PheRSs from Pyrococcus horikoshii and two bacteria were tested for editing activity. The isolated B3/4 editing domain of the archaeal PheRS, which was exogenously added to the tyrosylation reaction with iodoTyrRS, efficiently reduced the production of tyrosyl-tRNA(Tyr). In addition, the transplantation of this domain into iodoTyrRS at the N terminus prevented tyrosyl-tRNA(Tyr) production most strongly among the tested fragments. We next transplanted this archaeal B3/4 editing domain into iodoTyrRS at several internal positions. Transplantation into the connective polypeptide in the Rossmann-fold domain generated a variant that efficiently charges tRNA(Tyr) with 3-iodo-l-tyrosine, but hardly produces tyrosyl-tRNA(Tyr). This variant, iodoTyrRS-ed, was used, together with an amber suppressor derived from tRNA(Tyr), in a wheat germ cell-free translation system and incorporated 3-iodo-l-tyrosine, but not l-tyrosine, in response to the amber codon. Thus, the editing-domain transplantation achieved unambiguous pairing between the tRNA and the nonnatural amino acid in an expanded genetic code.
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PMID:Transplantation of a tyrosine editing domain into a tyrosyl-tRNA synthetase variant enhances its specificity for a tyrosine analog. 1878 68

Translation of mRNAs by the ribosome is stereospecific, with only l-amino acids being incorporated into the nascent polypeptide chain. This stereospecificity results from the exclusion of d-amino acids at three steps during protein synthesis: (1) the aminoacylation of tRNA by aminoacyl-tRNA synthetases, (2) binding of aminoacyl-tRNAs to EF-Tu, and (3) recognition of aminoacyl-tRNAs by the ribosome. As a first step toward incorporating d-amino acids during protein synthesis, we have altered the enantioselectivity of tyrosyl-tRNA synthetase. This enzyme is unusual among aminoacyl-tRNA synthetases, as it can aminoacylate tRNA with d-tyrosine (albeit at a reduced rate compared to l-tyrosine). To change the enantioselectivity of tyrosyl-tRNA synthetase, we introduced the post-transfer editing domain from Pyrococcus horikoshii phenylalanyl-tRNA synthetase into the connective polypeptide 1 (CP1) domain of Geobacillus stearothermophilus tyrosyl-tRNA synthetase (henceforth designated TyrRS-FRSed). We show that the phenylalanyl-tRNA synthetase editing domain is stereospecific, hydrolyzing l-Tyr-tRNA(Tyr), but not d-Tyr-tRNA(Tyr). We further show that inserting the phenylalanyl-tRNA synthetase editing domain into the CP1 domain of tyrosyl-tRNA synthetase decreases the activity of the synthetic site in tyrosyl-tRNA synthetase. This decrease in activity is critical, as it prevents the rate of synthesis from overwhelming the ability of the editing domain to hydrolyze the l-Tyr-tRNA(Tyr) product. Overall, inserting the phenylalanyl-tRNA synthetase editing domain results in a 2-fold shift in the enantioselectivity of tyrosyl-tRNA synthetase toward the d-Tyr-tRNA(Tyr) product. When a 4-fold excess of d-tyrosine is used, approximately 40% of the tRNA(Tyr) is aminoacylated with d-tyrosine.
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PMID:Altering the Enantioselectivity of Tyrosyl-tRNA Synthetase by Insertion of a Stereospecific Editing Domain. 2689 Sep 80

d-Amino acids are excluded at three different steps during protein synthesis: the aminoacylation of tRNA, binding of aminoacyl-tRNAs to EF-Tu, and selection of the aminoacyl-tRNA by the ribosome. We previously altered the enantioselectivity of tyrosyl-tRNA synthetase (TyrRS) by inserting the editing domain from phenylalanyl-tRNA synthetase (FRSed) between Gly 161 and Ile 162 in tyrosyl-tRNA synthetase (the editing domain hydrolyzes l-Tyr-tRNA but not d-Tyr-tRNA). In this paper, we test the hypothesis that the enantioselectivity of this TyrRS-FRSed chimera can be shifted further toward the formation of d-Tyr-tRNA by introducing activating mutations into the editing site. Yokoyama and colleagues previously identified six alanine substitutions in phenylalanyl-tRNA synthetase that increase its editing activity.1 We have introduced these alanine substitutions into TyrRS-FRSed in various combinations, generating 14 different variants. To analyze their editing activity, we developed a continuous, spectrophotometric, steady-state post-transfer editing assay in which l-Tyr-tRNA is generated in situ, resulting in the release of one molecule of AMP during each editing cycle. Post-transfer editing is monitored by coupling the release of AMP to the reduction of NAD(+) (via the actions of AMP deaminase and IMP dehydrogenase), resulting in an increase in absorbance at 340 nm. In general, TyrRS-FRSed variants containing two activating mutations are the most active, with additional alanine substitutions decreasing the activity of the editing domain. Linear free energy relationships indicate that high kcat values are correlated with high binding affinities for l-Tyr-tRNA. Lastly, competition assays indicate that at least one TyrRS-FRSed variant (F145A/S211A) preferentially aminoacylates tRNA with d-tyrosine, demonstrating that the enantioselectivity of tyrosyl-tRNA synthetase can be inverted using hyperactive editing domains.
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PMID:Hyperactive Editing Domain Variants Switch the Stereospecificity of Tyrosyl-tRNA Synthetase. 2706 38