Enzymatic Incorporation of ATP and CTP Analogues into the 3′ End of tRNA

Sprinzl, Mathias; Sternbach, Hans; Haar, Friedrich von der; Cramer, Friedrich

doi:10.1111/j.1432-1033.1977.tb11985.x

Cited by 79 publications

(48 citation statements)

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“…2B, lanes 1,3) or 2 mM concentration (data not shown). Inhibition by both 2Ј-deoxy and 2Ј-fluoro modification, but not by 2Ј-amino modification (Sprinzl et al 1977), suggests that the 2Ј hydroxyl group serves as a hydrogen bond donor but does not exclude the possibility that it also serves as an acceptor (Ortoleva-Donnelly et al 1998;Ryder and Strobel 1999). The highly electronegative 2Ј-fluoro substitution favors the C3Ј-endo conformation of ribose (Ryder and Strobel 1999), but this seems unlikely to explain inhibition because unsubstituted ribose also favors the C3Ј-endo conformation.…”

Section: Incorporation Of Ctp Analogs Into Trna-d and Trna-dcmentioning

confidence: 93%

“…Indeed, introduction of nucleotide analogs into 3Ј-terminal tRNA substrates by the CCA-adding enzyme itself has helped to understand the mechanism of CCA addition (Sprinzl et al 1972(Sprinzl et al , 1973a(Sprinzl et al , 1973b(Sprinzl et al , 1977Cramer et al 1975;Sprinzl and Cramer 1979). However, these pioneering studies of the CCA-adding enzyme often used very high concentrations of nucleotide analogs (5 mM instead of 30 to 200 µM) and nearly stoichiometric levels of enzyme (1:1 instead of 1:100; Sprinzl et al 1973bSprinzl et al , 1977Zachau and Hertz 1974) thus potentially generating aberrant products caused by tRNA binding, rebinding, or slipping out of register on the enzyme.…”

Section: Introductionmentioning

confidence: 99%

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Use of nucleotide analogs by class I and class II CCA-adding enzymes (tRNA nucleotidyltransferase): Deciphering the basis for nucleotide selection

Cho¹,

Oyelere²,

Strobel³

et al. 2003

RNA

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We explored the specificity and nature of the nucleotide-binding pocket of the CCA-adding enzyme (tRNA nucleotidyltransferase) by using CTP and ATP analogs as substrates for a panel of class I and class II enzymes. Overall, class I and class II enzymes displayed remarkably similar substrate requirements, implying that the mechanism of CCA addition is conserved between enzyme classes despite the absence of obvious sequence homology outside the active site signature sequence. CTP substrates are more tolerant of base modifications than ATP substrates, but sugar modifications prevent incorporation of both CTP and ATP analogs by class I and class II enzymes. Use of CTP analogs (zebularine, pseudoisocytidine, 6-azacytidine, but not 6-azauridine) suggests that base modifications generally do not interfere with recognition or incorporation of CTP analogs by either class I or class II enzymes, and that UTP is excluded because N-3 is a positive determinant and/or O-4 is an antideterminant. Use of ATP analogs (N 6 -methyladenosine, diaminopurine, purine, 2-aminopurine, and 7-deaza-adenosine, but not guanosine, deoxyadenosine, 2-O-methyladenosine, 2-deoxy-2-fluoroadenosine, or inosine) suggests that base modifications generally do not interfere with recognition or incorporation of ATP analogs by either class I or class II enzymes, and that GTP is excluded because N-1 is a positive determinant and/or the 2-amino and 6-keto groups are antideterminants. We also found that the 3-terminal sequence of the growing tRNA substrate can affect the efficiency or specificity of subsequent nucleotide addition. Our data set should allow rigorous evaluation of structural hypotheses for nucleotide selection based on existing and future crystal structures.

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Section: Incorporation Of Ctp Analogs Into Trna-d and Trna-dcmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Use of nucleotide analogs by class I and class II CCA-adding enzymes (tRNA nucleotidyltransferase): Deciphering the basis for nucleotide selection

Cho¹,

Oyelere²,

Strobel³

et al. 2003

RNA

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“…Further, the preparations were tested with a crude mixture of aminoacyltRNA synthetases and 14C-labelled amino acids except isoleucine, yielding only background radioactivity. From these tRNA"' preparations the modified tRNA species tRNA1Ie-C-C-A(3'NH2) were prepared with the aid of nucleotidyltransferase from yeast by reaction of 3'-deoxy-3'-amino-ATP with tRNA"'-C-C and analyzed as described by Sprinzl et al [15], Cramer et al [16], Maelicke et al [17], Fraser and Rich [18] and Sternbach et al [19]. The preparations were also controlled by HPLC [14] and found to be free of tRNA"'-C-C-A as must be expected after the several steps of preparation and purification.…”

Section: Methodsmentioning

confidence: 99%

Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNAIle-C-C-A(3'NH2)

1987

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For discrimination between isoleucine and the other 19 naturally occuring amino acids by isoleucyl-tRNA synthetases from baker's yeast and from Escherichia coli MRE 600 discrimination factors have been determined from k,,, and K , values in aminoacylation of the modified tRNA1Ie-C-C-A(3'NH& Discrimination factors D 1 are products of an initial discrimination factor and a proof-reading factor: D 1 = Il . n1. From Initial discrimination factors ZI are directly related to hydrophobic interaction forces between the substrates and the enzymes. Plots of Gibbs free energy differences calculated from these factors are linearly related to the accessible surface areas of the amino acids. A hypothetical model of the binding site can be given in which selection of amino acids is achieved by hydrophobic forces and removal of steric hindrance.Identification of the cognate amino acids by aminoacyltRNA synthetases is one of the most complex processes in protein biosynthesis. According to the present stage of knowledge for discrimination between isoleucine and valine by isoleucyl-tRNA synthetases, a four-step recognition mechanism has been postulated, in which after an initial two-step physical discrimination misrecognized valine is rejected in a pre-and in a post-transfer hydrolytic proof-reading step If in the normal reaction catalyzed by the aminoacyl-[l -31.tRNA synthetases E + ATP + aa =S E . aa-AMP + PPi E . aa-AMP + tRNA + E + aa-tRNA + AMP the naturally occurring tRNA is substituted by tRNA-C-C-A(3'NH2), this modified substrate is easily aminoacylated with noncognate amino acids [4-61. The final products of these reactions are aminoacyl amides which are more stable against hydrolysis than the 0-aminoacyl esters obtained with the normal substrate [7,8]. This has been regarded as a reason for the lower specificities observed in aminoacylation of this modified tRNA because amides should not be hydrolyzed in a hydrolytic post-transfer proof-reading step [9,. The lack of one proof-reading step and the resulting less complex aminoacylation process caused us to do a systematic study on Correspondence to F. Cramer, Abteilung Chemie,

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“…3c, lanes 6 and 12). This is most probably the results of CMP incorporation into bulk tRNA by ATP(CTP)tRNA nucleotidyl transferase [21].…”

Section: Resultsmentioning

confidence: 99%

Affinity labeling of GTP‐binding proteins in cellular extracts

1992

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GTP-binding pro&s in cellular extracts from &schrrickio co/i, Thrrmtrs rhennophl/us, yeast. wheat germ or calf thymus were identified using in situ pcriodate-oxidized [@P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiffs base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamidc gel clectrophoresis and visualized by autoradiography. In addition cross-linking of [t@P]GTP with G'TP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-rus ~21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. Awr~ophihts and initiation factor 2 (lF2) from T. rhernwphi/us. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initibcion factors, and heterolrimeric signal transducing G-proteins.

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Enzymatic Incorporation of ATP and CTP Analogues into the 3′ End of tRNA

Cited by 79 publications

References 50 publications

Use of nucleotide analogs by class I and class II CCA-adding enzymes (tRNA nucleotidyltransferase): Deciphering the basis for nucleotide selection

Use of nucleotide analogs by class I and class II CCA-adding enzymes (tRNA nucleotidyltransferase): Deciphering the basis for nucleotide selection

Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNAIle-C-C-A(3'NH2)

Affinity labeling of GTP‐binding proteins in cellular extracts

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