Conformation in solution of yeast tRNAAsp transcripts deprived of modified nucleotides

Perret, Véronique; García, Ángela Domínguez; Puglisi, Joseph D.; Grosjean, Henri; Ebel, Jean‐Pierre; Florentz, Catherine; Giegé, Richard

doi:10.1016/0300-9084(90)90158-d

Cited by 127 publications

(113 citation statements)

References 39 publications

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“…Many studies reported how the lack of modification in tRNA in vitro transcripts can be compensated by an increased concentration of Mg 2+ ions to obtain functional structures (Sampson and Uhlenbeck 1988;Perret et al 1990;Maglott et al 1998;Serebrov et al 1998). We illustrate here the interchangeable stabilizing role of metal binding and post-transcriptional modification on a specific tRNA tertiary interaction, the G15-C48 Levitt base pair, provided with a physicochemical explanation of the phenomenon.…”

Section: Discussionmentioning

confidence: 78%

Mg²⁺ binding and archaeosine modification stabilize the G15–C48 Levitt base pair in tRNAs

Oliva

Tramontano²,

Cavallo

2007

RNA

101

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The G15-C48 Levitt base pair, located at a crucial position in the core of canonical tRNAs, assumes a reverse Watson-Crick (RWC) geometry. By means of bioinformatics analysis and quantum mechanics calculations we show here that such a geometry is moderately more stable than an alternative bifurcated trans geometry, involving the guanine Watson-Crick face and the cytosine keto group, which we have also found in known RNA structures. However we also demonstrate that the RWC geometry can take advantage of additional stabilizing effects such as metal binding or post-transcriptional chemical modification. One of the few strong metal binding sites characterized for cytosolic tRNAs is localized on G15, and a domain-specific complex modification known as archaeosine is widespread at position 15 in archaeal tRNAs. We have found that both the bound Mg 2+ ion and the archaeosine modification induce an analogous electron density redistribution, which results in an effective stabilization of the RWC geometry. Metal binding and chemical modification thus play an interchangeable role in stabilizing the G15-C48 correct geometry. Interestingly, these different but convergent strategies are selectively adopted in the different life domains.

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Section: Discussionmentioning

confidence: 78%

Mg²⁺ binding and archaeosine modification stabilize the G15–C48 Levitt base pair in tRNAs

Oliva

Tramontano²,

Cavallo

2007

RNA

101

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“…2, Table 2). The experiments were performed at 20°C in a buffer containing 10 mM MgCl 2 to ensure that the unmodified tRNAs were fully folded (23)(24)(25). The first assay measures the equilibrium binding constant of the ternary complex to ribosomes by using the active site mutant of EF-Tu(H84A) to prevent GTP hydrolysis (26,27).…”

Section: Resultsmentioning

confidence: 99%

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

Schrader

Chapman

Uhlenbeck

2011

Proc. Natl. Acad. Sci. U.S.A.

123

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To better understand why aminoacyl-tRNAs (aa-tRNAs) have evolved to bind bacterial elongation factor Tu (EF-Tu) with uniform affinities, mutant tRNAs with differing affinities for EF-Tu were assayed for decoding on Escherichia coli ribosomes. At saturating EF-Tu concentrations, weaker-binding aa-tRNAs decode their cognate codons similarly to wild-type tRNAs. However, tighter-binding aa-tRNAs show reduced rates of peptide bond formation due to slow release from EF-Tu•GDP. Thus, the affinities of aa-tRNAs for EF-Tu are constrained to be uniform by their need to bind tightly enough to form the ternary complex but weakly enough to release from EF-Tu during decoding. Consistent with available crystal structures, the identity of the esterified amino acid and three base pairs in the T stem of tRNA combine to define the affinity of each aa-tRNA for EF-Tu, both off and on the ribosome.T he ternary complex of bacterial elongation factor Tu (EF-Tu), GTP and aminoacyl-tRNA (aa-tRNA) binds to the ribosome and participates in a multistep decoding pathway in which GTP is hydrolyzed, EF-Tu•GDP is released, and the aa-tRNA enters the ribosomal A site (1-6). Although all elongator aa-tRNAs bind EF-Tu•GTP with similar affinities (7-9), studies with misacylated tRNAs reveal that the protein shows substantial specificity for both the esterified amino acid and the tRNA body (10-12). The nearly uniform EF-Tu binding affinity observed for tRNAs acylated with their correct (cognate) amino acid occurs because the sequence of each tRNA has evolved to compensate for the variable thermodynamic contribution of the esterified amino acid. Thus, weak-binding esterified amino acids such as glycine and alanine have corresponding tRNAs that bind the protein tightly, while tight-binding amino acids such as tyrosine or glutamine have corresponding tRNAs that bind poorly. The crystal structure of Thermus aquaticus EF-Tu•GTP bound to Saccharomyces cerevisiae Phe-tRNA Phe (13) reveals that the protein primarily forms extensive interactions with the helical phosphodiester backbone of the acceptor and T stems of tRNA Phe . Recent protein (14) and tRNA (15, 16) mutagenesis experiments indicate that much of the specificity is the result of interactions made between three amino acids of EF-Tu and three adjacent base pairs in the T stem (16). Additional mutagenesis experiments indicate that the thermodynamic contribution of each of the three base pairs is independent of the others, making it possible to adjust the affinity of aa-tRNAs to EF-Tu in a predictable manner.Although a detailed structural and thermodynamic understanding of how EF-Tu achieves uniform binding with different aa-tRNAs is beginning to emerge, the underlying selective pressures that lead to uniform binding are less clear. While aa-tRNAs must bind EF-Tu tightly enough to participate in translation, the high intracellular concentration of EF-Tu (17) ensures that they do not significantly compete with one another for the protein.It therefore seems unlikely that a minimum threshold binding af...

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“…Plasmid pUTL4 encoding wild-type E. coli tRNA CAG Leu and pUTL66 encoding the tRNA CAG Leu minihelix have been previously described (19). Plasmids encoding wild-type yeast tRNA Asp (29) as well as mutants G36 and A37 were a gift from Catherine Florentz. All these pTFMa constructs differ from the true wild type in that the first base pair, U1-A72, has been replaced by G1-C72 for improving in vitro transcription.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Streptococcus pneumoniae TrmD, a tRNA Methyltransferase Essential for Growth

et al. 2004

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Down-regulation of expression of trmD, encoding the enzyme tRNA (guanosine-1)-methyltransferase, has shown that this gene is essential for growth of Streptococcus pneumoniae. The S. pneumoniae trmD gene has been isolated and expressed in Escherichia coli by using a His-tagged T7 expression vector. Recombinant protein has been purified, and its catalytic and physical properties have been characterized. The native enzyme displays a molecular mass of approximately 65,000 Da, suggesting that streptococcal TrmD is a dimer of two identical subunits. In fact, this characteristic can be extended to several other TrmD orthologs, including E. coli TrmD. Kinetic studies show that the streptococcal enzyme utilizes a sequential mechanism. Binding of tRNA by gel mobility shift assays gives a dissociation constant of 22 nM for one of its substrates, tRNA CAG Leu . Other heterologous nonsubstrate tRNA species, like tRNA GGT Thr , tRNA Phe , and tRNA TGC Ala , bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).Proteins that methylate tRNA at position G37 are ubiquitous in nature, and no cellular form has been found which does not possess this important activity. In bacteria, the enzyme tRNA (guanosine-1)-methyltransferase (1MGT or TrmD) modifies the subset of tRNAs that have a G at position 36 and recognize codons beginning with C, including those for leucine, proline, and arginine. It has been shown that methylation at G37 prevents frameshifting (7), and in strains of Salmonella enterica serovar Typhimurium lacking this activity, frameshifting occurs at selected proline codons (32,33). The deficiency of m 1 G in these strains also appears to have a more global effect on cell physiology as evidenced by changes in carbon source metabolism and sensitivity to amino acid analogs (24). In particular, lack of m 1 G is found to affect metabolism of thiamine and pantothenate (4, 6). TrmD activity has been shown to be important for growth of the gram-negative bacteria S. enterica serovar Typhimurium (5) and Escherichia coli (30), and recent studies suggest that TrmD is essential for growth of the grampositive organisms Streptococcus pneumoniae (36) and Bacillus subtilis (22).It has been shown that the entire tRNA structure is required for maximal catalytic activity of E. coli TrmD (18). For example, a derivative of tRNA CAG Leu with the D and T loops deleted is a very poor substrate for this enzyme. Subsequent studies have shown that the sequence G36pG37 is the key recognition element in tRNA (19) and that G37 must be in the correct position in space relative to important interactions elsewhere in the molecule (19). Enzymatic and chemical probing experiments have shown that the enzyme makes contacts in key sites in the anticodon loop (13).Although tRNA-modifying enzymes from E. coli have been well studied, little is known concerning modification enzymes from gram-positive bacteria. It is not clear, for instance, if gram-negative and gram-positive TrmD orthologs will display si...

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Conformation in solution of yeast tRNAAsp transcripts deprived of modified nucleotides

Cited by 127 publications

References 39 publications

Mg²⁺ binding and archaeosine modification stabilize the G15–C48 Levitt base pair in tRNAs

Mg²⁺ binding and archaeosine modification stabilize the G15–C48 Levitt base pair in tRNAs

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

Characterization of Streptococcus pneumoniae TrmD, a tRNA Methyltransferase Essential for Growth

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Conformation in solution of yeast tRNAAsp transcripts deprived of modified nucleotides

Cited by 127 publications

References 39 publications

Mg2+ binding and archaeosine modification stabilize the G15–C48 Levitt base pair in tRNAs

Mg2+ binding and archaeosine modification stabilize the G15–C48 Levitt base pair in tRNAs

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

Characterization of Streptococcus pneumoniae TrmD, a tRNA Methyltransferase Essential for Growth

Contact Info

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Mg²⁺ binding and archaeosine modification stabilize the G15–C48 Levitt base pair in tRNAs

Mg²⁺ binding and archaeosine modification stabilize the G15–C48 Levitt base pair in tRNAs