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

Moor, Nina; Репкова, М. Н.; Yamkovoy, V.I.; Lavrik, Olga I.

doi:10.1016/0014-5793(94)00841-8

Cited by 11 publications

(10 citation statements)

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“…We have studied the role of the universally conserved 3'terminal C C A sequence in the spccific interaction of Th. thernzophitus tRNA"'C with homologous tRNA"" synthetase [24]. T h e data clearly demonstrate the iinportance of the C C A end nucleotides for efficient intcraclinn of tRNh"'" with thc synthetase.…”

Section: Discussionmentioning

confidence: 66%

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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Section: Discussionmentioning

confidence: 66%

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

Moor

Ankilova

Lavrik

1995

European Journal of Biochemistry

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“…The data on affinity crosslinking of T. thermophilus PheRS with tRNA Phe derivatives bearing a reactive nucleotide at the 3' end give clear indication of the acceptor end rearrangement in the presence of other substrates (Vasil'eva et al, 2000). The kinetic data on the substrate activity of tRNA Phe s substituted at position 76 suggest functional importance of base-specific contacts of the terminal adenosine for the productive interaction of tRNA Phe with PheRS: their disruption is reflected predominantly in a reduction in the rate of catalytic transformation (Moor et al, 1994;Vasil'eva et al, 2000).…”

Section: A Vasil'eva Et Almentioning

confidence: 97%

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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“…The 3'-CCA sequence is a universal feature of all tRNAs and is important in many aspects of tRNA function [22], including aminoacylation [23,24] and interaction with large subunit rRNA [25]. This tRNA also contains several modified nucleosides that are highly conserved among tRNAs and are thought to be important for tRNA structure and function [26][27][28][29][30][31][32][33].…”

Section: Discussionmentioning

confidence: 99%

“…So far, how-h/LN. Schnare et al/FEBS Letters 362 (1995) [24][25][26][27][28] A 8 ever, such editing has not been detected in analyses designed to investigate this possibility (J. Edqvist, unpublished results). In summary, we have isolated and fully characterized a tRNA Met from T. pyriformis mitochondria.…”

Section: Discussionmentioning

confidence: 99%

Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNA^Met from Tetrahymena pyriformis

1995

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We have now isolated and characterized the smallest T. pyriformis mitochondrial tRNA and we show here that it has the primary sequence that we had predicted from the trnM sequence. The transcript also contains a Y-terminal CCA sequence, not encoded in the mtDNA, as well as several modified nucleosides characteristic of known functional tRNAs. Materials and methodsT. pyriformis, amicronucleate strain ST (obtained from Y. Suyama, Department of Biology, University of Pennsylvania), was grown at 28°C with constant shaking in 500 ml NeWs medium [9]. The culture was chilled on ice for 10 15 min, following which cells were collected by centrifugation at 2000 rpm for 5 min, resuspended, and centrifuged at 2000 rpm for 5 min through a layer of ice-cold homogenizing medium (0.35 M sucrose, 10 mM Tris-HC1, pH 7.2, 2 mM EDTA) [10]. Resuspended cells in 50 ml homogenizing medium were disrupted by three passages through a hand emulsifier (nozzle unscrewed 2.5 turns) [11]. Unbroken cells and debris were removed by centrifugation at 3000 rpm (IEC) for 5 min. A mitochondrial pellet was recovered from the resulting supernatant by centrifugation at 8000 rpm for 5 min and washed twice by resuspension in 50 ml homogenizing medium followed by centrifugation (5 min, 8000 rpm). The final mitochondrial pellet was resuspended in 10 ml 0.15 M NaC1, 0.1 M EDTA (pH 9.0) [12] at room temperature. Ten ml of 4% SDS in the same buffer were added and the mitochondrial lysate was then extracted three times with phenol/cresol [13] at room temperature. Nucleic acids were precipitated by addition of 2 vols. of 95% ethanol. For preparation of a post-ribosomal supernatant [14], purified mitochondria were resuspended in buffer (100 mM KC1, 10 mM MgCI 2, 10 mM Tris-HCl, pH 7.4) and lysed at a final concentration of 2% Triton X-100. After removal of membrane fragments by centrifugation at 10,000 rpm for 10 rain (IEC), the clarified mitochondrial lysate was centrifuged at 42,000 rpm for 90 min in a Ti50 rotor to sediment ribosomes. Transfer RNA was isolated from the supernatant by detergent phenol/cresol extraction.Nucleic acid samples were either used directly for 3'-end-labeling or, alternatively, the RNA samples (5 ~tg post-ribosomal supernatant RNA or 20/lg total nucleic acid) were dissolved in 7.5/11 NMF/urea loading buffer [15] containing 0.1% (w/v) xylene cyanol or Bromophenol blue and heated to 65°C for 5 min. After electrophoresis in a thin (0.5 ram) 6% polyacrylamide gel containing 7 M urea, the smallest tRNA was visualized by ethidium bromide staining and eluted [16] in the presence of 20 gtg linear polyacrylamide carrier (prepared as in [17], extracted with phenol, precipitated with ethanol and re-dissolved at 2.5 flg//.tl). The unlabeled tRNA was either re-purified by gel-electrophoresis and then subjected to modified nucleotide analysis [16] or used directly for end-labeling. 3'-End-labeled tRNA [18] was purified by gel-electrophoresis and used for chemical sequence analysis [18]; both 3'-and 5'-endlabeled tRNAs were subjected to enzymatic s...

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

Cited by 11 publications

References 11 publications

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

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

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

Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNA^Met from Tetrahymena pyriformis

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

Cited by 11 publications

References 11 publications

Recognition of tRNAPhe by Phenylalanyl‐tRNA Synthetase of Thermus Thermophilus

Recognition of tRNAPhe by Phenylalanyl‐tRNA Synthetase of Thermus Thermophilus

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

Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNAMet from Tetrahymena pyriformis

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

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

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

Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNA^Met from Tetrahymena pyriformis