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Two-step purification of tryptophan-accepting tRNA from Bacillus stearothermophilus☆

Published on Dec 1, 1985in Analytical Biochemistry2.507
· DOI :10.1016/0003-2697(85)90213-1
Charles W. Carter1
Estimated H-index: 1
(UNC: University of North Carolina at Chapel Hill),
David C. Green2
Estimated H-index: 2
(UNC: University of North Carolina at Chapel Hill)
+ 1 AuthorsLaurie Betts18
Estimated H-index: 18
(UNC: University of North Carolina at Chapel Hill)
Abstract
Abstract Tryptophan-accepting tRNA has been purified essentially to homogeneity from Bacillus stearothermophilus . Crude tRNA was chromatographed first on benzoylated DEAE-cellulose and then on Sepharose 4B with reverse salt gradient elution. The product has tryptophan acceptor activity in excess of 2 nmol [ 14 C]tryptophan per A 260 unit. This procedure avoids costly aminoacylation, a step characteristic of other one- and two-step procedures. In two separate purifications 7 and 11 mg of tRNA trp were prepared from 750 and 1000 g of frozen cells, respectively. This yield compares favorably with that from other procedures. The pure tRNA trp has been crystallized under several different conditions.
  • References (8)
  • Citations (5)
References8
Newest
#1Charles W. Carter (UNC: University of North Carolina at Chapel Hill)H-Index: 36
#2David C. Green (UNC: University of North Carolina at Chapel Hill)H-Index: 2
We have used chromatofocusing as the final stage in purifying tryptophanyl-tRNA synthetase from Bacillus stearothermophilus. Partially purified protein containing approximately 12% (w/w) synthetase was applied to the resin as a precipitate suspended in application buffer since both the desired enzyme and other proteins in this fraction were insoluble at low ionic strength. Nevertheless, homogeneous enzyme of higher specific activity than that previously published was achieved using a linear pH g...
8 CitationsSource
We have studied the crystallization of Bacillus ste- arothermophilus tryptophan tRNA synthetase. Exper- imental conditions used in 35 different crystallization trials were chosen by systematic assignment of varia- bles in accordance with the two principles of randomi- zation and balance, which are characteristic of incom- plete factorial experiments. Polymorphism in the re- sulting precipitated, microcrystalline, or crystalline protein samples was identified and rated numerically on a rough but ...
332 Citations
Abstract A solid phase procedure has been developed for the rapid isolation of all 20 species of tRNA from Escherichia coli. The overall yields for a single preparation cycle ranged from 62 to 96%, the average being 80%. The values for the amino acid acceptor activities of the tRNA species equaled those reported in the literature for highly purified tRNAs. Starting from crude tRNA, a given tRNA species can easily be isolated in less than 2 h. One milliliter of the resin, which is reusable, is su...
19 Citations
#1Larry C. Waters (ORNL: Oak Ridge National Laboratory)H-Index: 20
#2Wen-Kuang YangH-Index: 3
Last. J. L. Nichols (ORNL: Oak Ridge National Laboratory)H-Index: 1
view all 4 authors...
Abstract Tryptophan transfer RNA from chick cells chromatographs differently in the reversed-phase column chromatographic system 5 depending upon whether it is aminoacylated or not. This property was utilized to prepare pure tryptophan tRNA. Oligonucleotide fingerprints of tryptophan tRNA purified in this manner are identical with those reported for "spot 1" RNA isolated from Rous sarcoma virus.
7 Citations
Abstract The molecular weight of tryptophanyl-tRNA synthetase of Escherichia coli is 74,000 by Sephadex gel filtration and 81,000 by sucrose gradient centrifugation. Both sodium dodecyl sulfate polyacrylamide gel electrophoresis and agarose gel filtration in 8 m urea reveal subunits of 37,000 in molecular weight. In addition only one protein band is observed after polyacrylamide disc gel electrophoresis in the presence if 8 m urea. By these criteria the enzyme is a dimer composed of subunits ide...
32 Citations
#1A.D. Kelmers (ORNL: Oak Ridge National Laboratory)H-Index: 3
#2D.E. Heatherly (ORNL: Oak Ridge National Laboratory)H-Index: 1
Abstract A small reversed-phase chromatographic system for the separation of [ 3 H] or [ 14 C] aminoacyl-tRNA's is described. This system has the advantages of speed (chromatographic runs can be completed in 30 to 60 min), sensitivity (samples containing as little as 3.5 × 10 −9 gm or 60 × 16 −6 A 260 unit of phenylalanyl-tRNA have been resolved as a chromatographic peak), and excellent resolution of isoaccepting tRNA's. The system has been applied to samples of E. coli tRNA's and calf liver tRN...
140 CitationsSource
#1Ian. GillamH-Index: 2
#2Blew DH-Index: 1
Last. G. M. TenerH-Index: 16
view all 5 authors...
190 CitationsSource
#1S P ColowickH-Index: 1
#2M D JoshiH-Index: 1
Last. N O KaplanH-Index: 1
view all 4 authors...
26k Citations
Cited By5
Newest
#1Sylvie Doublié (UNC: University of North Carolina at Chapel Hill)H-Index: 32
#2Gérard Bricogne (LMB: Laboratory of Molecular Biology)H-Index: 41
Last. Charles W. Carter (UNC: University of North Carolina at Chapel Hill)H-Index: 36
view all 4 authors...
Abstract Background: Tryptophanyl-tRNA synthetase (TrpRS) catalyzes activation of tryptophan by ATP and transfer to tRNA Trp , ensuring translation of the genetic code for tryptophan. Interest focuses on mechanisms for specific recognition of both amino acid and tRNA substrates. Results Maximum-entropy methods enabled us to solve the TrpRS structure. Its three parts, a canonical dinucleotide-binding fold, a dimer interface, and a helical domain, have enough structural homology to tyrosyl-tRNA sy...
144 CitationsSource
#1Hong Xue (U of T: University of Toronto)H-Index: 1
#2Wenyan Shen (U of T: University of Toronto)H-Index: 1
Last. J. Tze-Fei Wong (HKUST: Hong Kong University of Science and Technology)H-Index: 1
view all 3 authors...
Abstract To study the effects of evolutionary sequence changes on the molecular interactions of tRNA inside the cell, the Bacillus subtilis tRNA Trp gene has been cloned into Escherichia coli JM109 under the control of the lac promotor. Hyperexpression of the gene in minimal medium upon induction yielded 28% of total tRNA in the form of B. subtilis tRNA Trp . The tRNA Trp gene product was purified by the use of a single Vydac C 4 high-performance liquid chromatography (HPLC) matrix. This experim...
13 CitationsSource
#1Charles W. Carter (UNC: University of North Carolina at Chapel Hill)H-Index: 1
Statistically designed experiments for screening crystal growth conditions greatly reduce the amount of sample necessary to find conditions for crystal growth and lead naturally to a useful database for improving crystallization conditions in cases where the initial trials do not produce adequate results. Designed experiments simultaneously vary all the factors to be screened, and this results in a substantial increase in sampling efficiency. Essential features and examples of two-level factoria...
50 CitationsSource
#1W. William Wilson (MSU: Mississippi State University)H-Index: 20
Various analytical techniques may be valuable as precrystallization assays for monitoring and screening various protein crystal growth conditions. Dynamic light scattering (DLS) offers unique capabilities for such studies. DLS provides a measure of the translational diffusion coefficient, D , of the protein molecules. Assuming a spherical model for the protein, its hydrodynamic diameter, d , can then be estimated. DLS can be used to monitor crystallization events as a function of time, creating ...
37 CitationsSource
#1Charles W. Carter (UNC: University of North Carolina at Chapel Hill)H-Index: 1
#2Eric T. Baldwin (UNC: University of North Carolina at Chapel Hill)H-Index: 1
Last. Lloyd Frick (UNC: University of North Carolina at Chapel Hill)H-Index: 8
view all 3 authors...
Abstract Statistical design of crystallization experiments greatly reduces the amount of protein necessary to find conditions for crystal growth and leads naturally to a useful data base for improving crystallization conditions in cases where the initial trials do not produce adequate results. Although it is counterintuitive to vary simultaneously all the factors to be screened, this apparent loss of control over experimental parameters actually costs very little in terms of the statistical stre...
75 CitationsSource