Shape-selective RNA recognition by cysteinyl-tRNA synthetase

Hauenstein, Scott; Zhang, Chunmei; Hou, Ya‐Ming; Perona, John J.

doi:10.1038/nsmb849

Cited by 84 publications

(109 citation statements)

References 60 publications

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“…The lack of PyC fluorescence change indicates little binding interaction between the CCA end and the enzyme active site and/or no major alterations of the acceptor end region of the enzyme-tRNA binary complex. This result, together with the change of K d from the binary complex (K d = 0.3 6 0.1 mM) to the ternary complex (K d = 2.2 6 0.2 mM), is consistent with the notion that formation of the ternary complex leads to structural rearrangements that bring the 39 end of the tRNA into close contact with CysRS, as had been inferred from structural analysis (Hauenstein et al 2004). Thus, comparison of the PyC fluorescence, which monitors the CCA sequence, with the synthetase intrinsic fluorescence, which monitors the entire tRNA molecule, sheds new light on the conformational rearrangement of the synthetase-tRNA complex during aminoacylation.…”

Section: Equilibrium Binding Of the Cysrs-trna Cys Complexsupporting

confidence: 87%

“…Previously, the intrinsic tryptophan fluorescence of E. coli CysRS had been used to determine the enzyme affinity for tRNA Cys in the presence of CysAMS (59-O-[N-(L-cysteinyl) sulfamoyl] adenosine), which is an analog of the Cys-AMP intermediate of the aminoacylation reaction. The K d value determined from this study was 1.9 6 0.1 mM (Hauenstein et al 2004), closely similar to the K d value determined by using PyC as a probe. The similarity in K d values validates the use of the PyC probe in measuring the CysRS-tRNA Cys interaction.…”

Section: Equilibrium Binding Of the Cysrs-trna Cys Complexsupporting

confidence: 83%

“…As shown in the cocrystal structure of the complex, the CCA end enters into the ATP-binding site in the absence of ATP or an analog (Hauenstein et al 2004). Thus, the binding interaction was monitored in the presence of saturating concentrations of cysteine and the nonhydrolyzable AMPcPP (where the a-and b-phosphates are bridged by a CH 2 group).…”

Section: Equilibrium Binding Of the Cysrs-trna Cys Complexmentioning

confidence: 99%

“…In the editing site, however, class I aaRS enzymes flip the hairpin conformation of CCA into a straight conformation (e.g., Silvian et al 1999), whereas class II aaRS enzymes fold the CCA sequence into a hairpin (e.g., Dock-Bregeon et al 2004). Additional examples of CCA mobility are provided by the changes in conformation within tRNA-aaRS complexes that are induced by addition of cognate amino acid (Sherlin and Perona 2003) or by correct aaRS recognition of the anticodon loop (Hauenstein et al 2004). Similarly, chemical tRNA footprinting (Moazed and Noller 1989a, b), kinetic (Dorner et al 2006;Pan et al 2007), and FRET (Ermolenko et al 2007) studies have provided evidence that the CCA ends of A and P site tRNAs move into hybrid states during the elongation cycle of protein synthesis, in which the CCA ends of tRNAs move toward the P and E sites on the large subunit, while the anticodons of tRNAs remain in A and P sites on the small subunit.…”

Section: Introductionmentioning

confidence: 99%

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Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

Zhang¹,

Liu²,

Christian³

et al. 2008

RNA

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All mature tRNA molecules have the conserved CCA sequence at the 39 end with a range of dynamic conformations that are important for tRNA functions. We present here the details of a general approach to fluorescent labeling of the CCA sequence with the fluorescent base analog pyrrolo-C (PyC) at position 75 as a molecular probe for monitoring the dynamics of the tRNA 39 end. Using Escherichia coli tRNACys as an example, we achieve such labeling by first synthesizing the tRNA as a transcript up to C74 and then employing the tRNA CCA-adding enzyme to incorporate PyC75 and A76, using pyrrolo-CTP (PyCTP) and ATP as the respective substrates. PyC-labeled full-length tRNA Cys , separated from the unlabeled precursor tRNA by reverse phase highpressure liquid chromatography, is an efficient substrate for aminoacylation by E. coli cysteinyl-tRNA synthetase (CysRS). Fluorescence binding measurement of the PyC-labeled tRNACys with E. coli CysRS reveals an equilibrium K d closely similar to the value determined from the fluorescence of intrinsic enzyme tryptophans. Kinetic measurements of translocation of the PyClabeled tRNA from the ribosomal A to P sites identify a kinetic intermediate with a rate of formation and decay similar to the values reported for tRNAs labeled with the fluorescent proflavin at the tertiary core. These results highlight the potential of PyC to probe the dynamics of the tRNA CCA end in reactions ranging from aminoacylation to those on the ribosome.

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Section: Equilibrium Binding Of the Cysrs-trna Cys Complexsupporting

confidence: 87%

Section: Equilibrium Binding Of the Cysrs-trna Cys Complexsupporting

confidence: 83%

Section: Equilibrium Binding Of the Cysrs-trna Cys Complexmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

Zhang¹,

Liu²,

Christian³

et al. 2008

RNA

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“…This could be the mark of a recent assignment, or even a codon swapping phenomenon (Szathmary 1991). On the other hand, tRNA Cys , whose primary site of aminoacylation was also measured to be both 29OH and 39OH (Sprinzl and Cramer 1975;Hecht and Chinault 1976), has been shown to be recognized by CysRS (a Class I aaRS) in a purely Class I mode by crystallographic studies of the CysRS-tRNA Cys complex (Hauenstein et al 2004). Also, more recent detailed kinetic studies showed that k cat (29OH) > >k cat (39OH) by a factor of about 20 for CysRS (Shitivelband and Hou 2005).…”

Section: Asymmetric Pattern In a Colored Codon Tablementioning

confidence: 99%

An asymmetric underlying rule in the assignment of codons: Possible clue to a quick early evolution of the genetic code via successive binary choices

Delarue¹

2006

RNA

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Aminoacyl-tRNA synthetases (aaRSs) are responsible for creating the pool of correctly charged aminoacyl-tRNAs that are necessary for the translation of genetic information (mRNA) by the ribosome. Each aaRS belongs to either one of only two classes with two different mechanisms of aminoacylation, making use of either the 29OH (Class I) or the 39OH (Class II) of the terminal A76 of the tRNA and approaching the tRNA either from the minor groove (29OH) or the major groove (39OH). Here, an asymmetric pattern typical of differentiation is uncovered in the partition of the codon repertoire, as defined by the mechanism of aminoacylation of each corresponding tRNA. This pattern can be reproduced in a unique cascade of successive binary decisions that progressively reduces codon ambiguity. The deduced order of differentiation is manifestly driven by the reduction of translation errors. A simple rule can be defined, decoding each codon sequence in its binary class, thereby providing both the code and the key to decode it. Assuming that the partition into two mechanisms of tRNA aminoacylation is a relic that dates back to the invention of the genetic code in the RNA World, a model for the assignment of amino acids in the codon table can be derived. The model implies that the stop codon was always there, as the codon whose tRNA cannot be charged with any amino acid, and makes the prediction of an ultimate differentiation step, which is found to correspond to the codon assignment of the 22nd amino acid pyrrolysine in archaebacteria.

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The Chemistry of Selenocysteine

Metanis¹,

Beld²,

Hilvert³

2011

Patai's Chemistry of Functional Groups

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Selenocysteine is a redox active amino acid. It is inserted co‐translationally into a number of natural proteins, providing them with a range of interesting enzymatic activities; it can also be incorporated into artificial peptides and proteins as a structural and mechanistic probe. In this chapter, the chemistry and biochemistry of this novel amino acid is reviewed. In addition to the (bio)synthesis of selenocysteine, selenopeptides and selenoproteins, the roles selenium plays in enzyme catalysis and its utility for diverse biotechnological applications are discussed.

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Shape-selective RNA recognition by cysteinyl-tRNA synthetase

Cited by 84 publications

References 60 publications

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

An asymmetric underlying rule in the assignment of codons: Possible clue to a quick early evolution of the genetic code via successive binary choices

The Chemistry of Selenocysteine

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