Phenylalanyl‐tRNA synthetase from <i>Thermus thermophilus</i> HB8 Purification and properties of the crystallizing enzyme

Ankilova, V.N.; Reshetnikova, L.; Chernaya, M.M.; Lavrik, Olga I.

doi:10.1016/0014-5793(88)81403-0

Cited by 36 publications

(16 citation statements)

References 4 publications

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“…FRS from E. coli and Th. thermophilus were purified [5,6]. Oligoribonucleotides pCpCpA and pUpUpA were synthesized using the H-phosphonate procedure [7] followed by phosphorylation [8].…”

Section: Methodsmentioning

confidence: 99%

Alterations at the 3′‐CCA end of Escherichia coli and Thermus thermophilus tRNA^Phe do not abolish their acceptor activity

et al. 1994

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The 3'-CCA end of tRNA Phe from Escherichia coli and Thermus thermophilus was changed to AAA, CCC, UUU and UUA by the stepwise degradation procedure of the 3'-CCA end of tRNA Ph° followed by the ligation with oligoribotrinucleotides. Substrate activity of tRNA~h~, and tRNA~h~ in tRNA aminoacylation was shown, tRNA,~h~, is a bad substrate for E. coli and Th. thermophilus phenylalanyl-tRNA synthetases.Phe tRNAutm has no detectable activity in tRNA aminoacylation. Therefore the nature of the Y-end of tRNA Phe plays an important role in tRNA binding and its substrate efficiency. Nevertheless the CCA sequence at the 3'-end of tRNA Phe does not seem to be an absolute requirement for tRNA aminoacylation.

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“…FRS from E. coli and Th. thermophilus were purified [5,6]. Oligoribonucleotides pCpCpA and pUpUpA were synthesized using the H-phosphonate procedure [7] followed by phosphorylation [8].…”

Section: Methodsmentioning

confidence: 99%

Alterations at the 3′‐CCA end of Escherichia coli and Thermus thermophilus tRNA^Phe do not abolish their acceptor activity

et al. 1994

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“…Comparison of the purification outputs of different thermophilic synthetases indicate that the cellular concentration of Gly-tRNA synthetase is high and similar to those of Phe-, Met-and Thr-tRNA synthetases. Like GlytRNA synthetase, Phe-tRNA synthetase was isolated with a high yield (Ankilova et al, 1988) in contrast to Met-tRNA synthetase and Thr-tRNA synthetase (Kohda et al, 1987;Zheltonosova et al, 1994). Asp-, Asn-, and Ala-tRNA synthetases are present in lower amounts in 7: thermophilus (Kern, D., unpublished results).…”

Section: Discussionmentioning

confidence: 99%

An Example of Non‐Conservation of Oligomeric Structure in Prokaryotic Aminoacyl‐tRNA Synthetases

Mazauric

Reinbolt

Lorber

et al. 1996

European Journal of Biochemistry

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Glycyl-tRNA synthetase (Gly-tRNA synthetase) from Thermus thermoplzilus was purified to homogeneity and with high yield using a five-step purification procedure in amounts sufficient to solve its crystallographic structure [Logan, D. Heat-stability measurements show that this synthetase keeps its ATP-PP, exchange and aminoacylation activities up to 70°C. Glycyladenylate strongly protects the enzyme against thermal inactivation at higher temperatures. Unexpectedly, tRNACly does not induce protection.Cross-aminoacylations reveal that the thermophilic Gly-tRNA synthetase charges heterologous E. coli tRNA"l~'G'C'~ and tRNA"lscCCC) and yeast tRNAG1y("C"' as efficiently as 7: thermophilus tRNA"IY. All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly-tRNA synthetases, the prokaryotic thermophilic enzyme does not possess a strict species specificity.The results are discussed in the context of the three-dimensional structure of the synthetase and in the view of the particular evolution of the glycinylation systems.

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“…The reaction mixtures were incubated at 37°C for 5 min prior to initiation by addition of PheRS to final concentrations ranging from 3 to 30 mg/ml. [ 3 H]phenylalanine incorporation into the trichloroacetic-acid-precipitable fraction was measured as described (Ankilova et al, 1988). K m and k cat values were calculated using the Microcal Origin 4.10 program.…”

Section: Trna Aminoacylationmentioning

confidence: 99%

“…The data allow us to propose how the specific complex formation may occur. PheRS with a specific activity of 350 units/mg was isolated from T. thermophilus HB8 as described (Ankilova et al, 1988). T7 RNA polymerase was isolated from E. coli BL21 harbouring the plasmid pAR1219.…”

Section: Introductionmentioning

confidence: 99%

tRNA discrimination by T. thermophilus phenylalanyl–tRNA synthetase at the binding step

Васильева

Ankilova

Lavrik

et al. 2002

J of Molecular Recognition

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The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs.

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Phenylalanyl‐tRNA synthetase from Thermus thermophilus HB8 Purification and properties of the crystallizing enzyme

Abstract: Phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 was isolated, characterized and crystallized. The enzyme is a tetramer of a&-type structure, its molecular mass being 264 kDa. (Thermus thermophilus)

Cited by 36 publications

References 4 publications

Alterations at the 3′‐CCA end of Escherichia coli and Thermus thermophilus tRNA^Phe do not abolish their acceptor activity

Alterations at the 3′‐CCA end of Escherichia coli and Thermus thermophilus tRNA^Phe do not abolish their acceptor activity

An Example of Non‐Conservation of Oligomeric Structure in Prokaryotic Aminoacyl‐tRNA Synthetases

tRNA discrimination by T. thermophilus phenylalanyl–tRNA synthetase at the binding step

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Phenylalanyl‐tRNA synthetase from Thermus thermophilus HB8 Purification and properties of the crystallizing enzyme

Abstract: Phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 was isolated, characterized and crystallized. The enzyme is a tetramer of a&-type structure, its molecular mass being 264 kDa. (Thermus thermophilus)

Cited by 36 publications

References 4 publications

Alterations at the 3′‐CCA end of Escherichia coli and Thermus thermophilus tRNAPhe do not abolish their acceptor activity

Alterations at the 3′‐CCA end of Escherichia coli and Thermus thermophilus tRNAPhe do not abolish their acceptor activity

An Example of Non‐Conservation of Oligomeric Structure in Prokaryotic Aminoacyl‐tRNA Synthetases

tRNA discrimination by T. thermophilus phenylalanyl–tRNA synthetase at the binding step

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Alterations at the 3′‐CCA end of Escherichia coli and Thermus thermophilus tRNA^Phe do not abolish their acceptor activity

Alterations at the 3′‐CCA end of Escherichia coli and Thermus thermophilus tRNA^Phe do not abolish their acceptor activity