Crystal structure of a eukaryotic initiator tRNA

Schevitz, Richard W.; Podjarny, A.; Krishnamachari, N.; Hughes, John Jay; Sigler, Paul B.; Sussman, Joel L.

doi:10.1038/278188a0

Cited by 160 publications

(71 citation statements)

References 28 publications

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“…Fig. 5 shows a comparison between the potentials calculated for all the phosphates of the backbone of the tRNAPhe molecule and the ApK values measured in this work for positions 8,34,47 and 76 (3' end) in the chains of representative tRNAs, each in four buffers (1,5,6,10) chosen so as to indicate the limits of the observed ApK. The scales of the calculated potentials and the measured ApK values are arbitrary.…”

Section: Discussionmentioning

confidence: 99%

Electrostatic potential of macromolecules measured by pK_a shift of a fluorophore

Friedrich

Woolley

Steinhäuser

1988

European Journal of Biochemistry

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The procedures developed earlier (Friedrich and Woolley, preceding paper in this journal) for probing electrostatic potential with the fluorescein label were applied to transfer RNA. By using tRNA species that contain chemically reactive bases we were able to label these bases with fluorescein derivatives and thus to 'map' the electrostatic potential around the molecule. Both the electrostatic potential and the fluorescence emission anisotropy data that were obtained at the same time could be understood in terms of the well-known, paradigmatic crystal structure of tRNAPhe. However, within the distribution of the various tRNA species, tRNA,M"' appeared to occupy an extreme position, which suggests a relation between the conformation in solution and the initiation function of this molecule. Comparison with theoretical predictions by others of the electrostatic potential map of tRNA showed agreement in respect of trends, but the values of the potentials measured were orders of magnitude lower than predicted. This we attribute primarily to solvation.We have demonstrated in the preceding paper [l] that the pK, of the fluorescein label can be used as a probe of electrostatic potential; the theoretical basis of this leads to the simple relationship y = -58.2 ApK,, where y is the potential at the position occupied by the fluorophore and ApK, is the shift in pK, of the bound fluorescein compared with a chemically identical fluorescein derivative not bound to a macromolecule. The method was applied to the 16s RNA molecule, with the fluorescein label bound at its 3' end.Here we investigate the electrostatics of the tRNA molecule. This molecule allows a much more detailed investigation, as the various tRNA species contain many chemically modified bases, at known positions, that can react specifically with various derivatives of fluorescein. Since these bases are found at different locations on the molecule, comparisons can be made and an overall picture can be built up. An advantage of tRNA is that it is less easily denatured than the larger RNA molecules and solution structures may readily be compared. Further, the electrostatics of this molecule have been investigated theoretically [2 -71 so that experimental results can be interpreted in more depth.Of the more than 200 tRNA sequences determined to date, no exception to the clover-leaf structure [8,9] has been found. The X-ray structure of tRNAPhe from yeast and that of tRNA,M"' from Escherichia coli are not greatly different [lo, 111 and it is highly probable that the yeast tRNAPhe crystal Correspondence to' P. Woolley, Kemisk Institut, Aarhus Universitet, DK-8000 k h u s , DenmarkAbbreviations. FITC, fluorescein isothiocyanate; FTSC, fluorescein thiosemicarbazide; IAFl, iodacetamidofluorescein; X, 3-(3-amino-3-carboxypropyl)-uridine; Q, (4,5-cis-dihydroxy-l-cyclopenten-3-yl aminomethyl)-7-deazoguanidine; s4U, 4-thiouridine; mams2U, 5-methylaminomethyl-2-thiouridine.

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Section: Discussionmentioning

confidence: 99%

Electrostatic potential of macromolecules measured by pK_a shift of a fluorophore

Friedrich

Woolley

Steinhäuser

1988

European Journal of Biochemistry

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“…Instead, it reflects changesininteractionsoccurring within thec omplex upon startc odon recognitiont hata re likely involved in mediatingt he response to AUGi dentification.The resultspresentedhereindicatethatthiscoupling is dependentu pont he presence of theinitiator-specificG:C base pairsi nt he anticodons temo ft RNA i . S1 nuclease digestions tudies (Wrede et al 1979;S eong and RajBhandary 1987b) as well as crystallographic studies (Schevitz et al 1979;Woo et al1980;Basavappa and Sigler 1991)h ave suggested that the three consecutiveG :C base pairsi nt he anticodon stem of initiator tRNA impart a unique conformation to the anticodon loop, one that has been described as ''skewed'' and may be required for proper codon:anticodon pairingb etween mRNA and the initiator tRNA. The progressive mutationofthe individual G:C base pairsi nt he anticodon stem of the Escherichia coli initiator tRNA to their corresponding elongatoridentities converted its S1 nuclease digestionp atternt ot hat of an elongator tRNA (Seong and RajBhandary 1987a), and such mutations have produced defects in the functioningo fm utanti nitiatort RNAs in vivo (Mandal et al 1996).…”

Section: Discussionmentioning

confidence: 99%

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation

Kapp

Kolitz

Lorsch³

2006

RNA

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All three kingdoms of life employ two methionine tRNAs, one for translation initiation and the other for insertion of methionines at internal positions within growing polypeptide chains. We have used ar econstituted yeast translation initiation system to explore the interactions of the initiator tRNA with the translation initiation machinery. Our data indicate that in addition to its previously characterized role in binding of the initiator tRNA to eukaryotic initiation factor 2( eIF2), the initiator-specific A1:U72 base pair at the top of the acceptor stem is important for the binding of the eIF2 d GTP d Met-tRNA i ternary complex to the 40S ribosomal subunit. We have also shown that the initiator-specific G:C base pairs in the anticodon stem of the initiator tRNA are required for the strong thermodynamic coupling between binding of the ternary complex and mRNA to the ribosome. This coupling reflects interactions that occur within the complex upon recognition of the start codon, suggesting that these initiatorspecific G:C pairs influence this step. The effect of these anticodon stem identity elements is influenced by bases in the Tloop of the tRNA, suggesting that conformational coupling between the D-loop-T-loop substructure and the anticodon stem of the initiator tRNA may occur during AUG codon selection in the ribosomal P-site, similar to the conformational coupling that occurs in A-site tRNAs engaged in mRNA decoding during the elongation phase of protein synthesis.

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“…Mutations of TRM6 and TRM61 cause derepression of GCN4 translation and its target genes because of the reduced level of initiator-tRNA Met Calvo et al 1999). The role of m 1 A58 in stabilizing the initiator-tRNA Met structure has been proposed to derive from its unique A54-A58 interaction (all other tRNAs in yeast have T54-A58 interaction) (Sigler 1975;Schevitz et al 1979;Basavappa and Sigler 1991). Alternatively, the absence of m 1 A58 leads to the formation of a nontRNA-like structure for the initiator-tRNA Met (Anderson and Droogmans 2005).…”

Section: Introductionmentioning

confidence: 99%

Genome-wide analysis of N¹-methyl-adenosine modification in human tRNAs

Saikia

Fu²,

Pavon-Eternod³

et al. 2010

RNA

112

103

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The N 1 -methyl-Adenosine (m 1 A58) modification at the conserved nucleotide 58 in the TCC loop is present in most eukaryotic tRNAs. In yeast, m 1 A58 modification is essential for viability because it is required for the stability of the initiator-tRNA Met . However, m 1 A58 modification is not required for the stability of several other tRNAs in yeast. This differential m 1 A58 response for different tRNA species raises the question of whether some tRNAs are hypomodified at A58 in normal cells, and how hypomodification at A58 may affect the stability and function of tRNA. Here, we apply a genomic approach to determine the presence of m 1 A58 hypomodified tRNAs in human cell lines and show how A58 hypomodification affects stability and involvement of tRNAs in translation. Our microarray-based method detects the presence of m

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Crystal structure of a eukaryotic initiator tRNA

Cited by 160 publications

References 28 publications

Electrostatic potential of macromolecules measured by pK_a shift of a fluorophore

Electrostatic potential of macromolecules measured by pK_a shift of a fluorophore

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation

Genome-wide analysis of N¹-methyl-adenosine modification in human tRNAs

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Crystal structure of a eukaryotic initiator tRNA

Cited by 160 publications

References 28 publications

Electrostatic potential of macromolecules measured by pKa shift of a fluorophore

Electrostatic potential of macromolecules measured by pKa shift of a fluorophore

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation

Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs

Contact Info

Product

Resources

About

Electrostatic potential of macromolecules measured by pK_a shift of a fluorophore

Electrostatic potential of macromolecules measured by pK_a shift of a fluorophore

Genome-wide analysis of N¹-methyl-adenosine modification in human tRNAs