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Specificity of the ribosomal A site for aminoacyl-tRNAs.

Dale T, Fahlman RP, Olejniczak M, Uhlenbeck OC - Nucleic Acids Res. (2009)

Bottom Line: When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed.However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold.We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.

View Article: PubMed Central - PubMed

Affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.

ABSTRACT
Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2'-deoxynucleotide-substituted mRNAs are used to demonstrate the presence of a lower threshold in k(off) values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed. However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold. We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.

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Effects of anticodon changes, different tRNA bodies and phenylalanylation on koff from the ribosomal A site (data from Table 1). (A) Deacylated and phenylalanylated tRNAs containing the GAC (valine) anticodon. (B) Deacylated and phenylalanylated tRNAs containing the GAA (phenylalanine) anticodon. The gray horizontal band represents the range of stabilities of cognate, purified aa-tRNAs in the A site: (2.7 × 10−3 to 6.4 × 10−3 min−1 (1)) and defines the threshold. 0.5 min−1 is the upper limit of the assay.
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Figure 2: Effects of anticodon changes, different tRNA bodies and phenylalanylation on koff from the ribosomal A site (data from Table 1). (A) Deacylated and phenylalanylated tRNAs containing the GAC (valine) anticodon. (B) Deacylated and phenylalanylated tRNAs containing the GAA (phenylalanine) anticodon. The gray horizontal band represents the range of stabilities of cognate, purified aa-tRNAs in the A site: (2.7 × 10−3 to 6.4 × 10−3 min−1 (1)) and defines the threshold. 0.5 min−1 is the upper limit of the assay.

Mentions: Two major conclusions may be drawn from the comparison of the stabilities of the deacylated tRNAs in the A site (Table 1, Figure 2). First, each tRNA displays a slower koff when it contains the GAC anticodon than when it contains the GAA anticodon, clearly demonstrating the non-equivalent binding contributions of different codon•anticodon pairs (3). Interestingly, however, the difference in stability between each GAC/GAA tRNA pair varies in a manner that depends on the tRNA body. For example, the difference in koff values between tRNATrp(GAC) and tRNATrp(GAA) is only 3-fold, but the difference between tRNAPhe(GAC) and tRNAPhe(GAA) is 27-fold. This suggests that the relative contribution of the anticodon is different for different tRNA bodies. Second, the variation in koff among the different tRNA bodies is much broader in the GAA anticodon background than in the GAC anticodon background. The deacyl-tRNA(GAA)s show about a 40-fold range of koff values, with some dissociating faster than the cognate tRNAPhe(GAA) and others actually dissociating more slowly. For example, the very weak tRNAAla(GAA) has a koff at least 4-fold faster than tRNAPhe(GAA), but the koff for tight tRNATrp(GAA) is more than 8-fold slower than tRNAPhe(GAA). In contrast, except for tRNAAla(GAC), all of the deacyl-tRNA(GAC)s display koff values within 2-fold of the cognate deacyl-tRNAVal(GAC). Taken together, these results indicate that different tRNA bodies do interact differently with the ribosome; however, these effects are only observed in the weaker GAA anticodon background. Presumably, the tRNA body effects still exist in the GAC anticodon-substituted tRNAs but are somehow masked by the very stable GAC anticodon•codon interaction.Figure 2.


Specificity of the ribosomal A site for aminoacyl-tRNAs.

Dale T, Fahlman RP, Olejniczak M, Uhlenbeck OC - Nucleic Acids Res. (2009)

Effects of anticodon changes, different tRNA bodies and phenylalanylation on koff from the ribosomal A site (data from Table 1). (A) Deacylated and phenylalanylated tRNAs containing the GAC (valine) anticodon. (B) Deacylated and phenylalanylated tRNAs containing the GAA (phenylalanine) anticodon. The gray horizontal band represents the range of stabilities of cognate, purified aa-tRNAs in the A site: (2.7 × 10−3 to 6.4 × 10−3 min−1 (1)) and defines the threshold. 0.5 min−1 is the upper limit of the assay.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651786&req=5

Figure 2: Effects of anticodon changes, different tRNA bodies and phenylalanylation on koff from the ribosomal A site (data from Table 1). (A) Deacylated and phenylalanylated tRNAs containing the GAC (valine) anticodon. (B) Deacylated and phenylalanylated tRNAs containing the GAA (phenylalanine) anticodon. The gray horizontal band represents the range of stabilities of cognate, purified aa-tRNAs in the A site: (2.7 × 10−3 to 6.4 × 10−3 min−1 (1)) and defines the threshold. 0.5 min−1 is the upper limit of the assay.
Mentions: Two major conclusions may be drawn from the comparison of the stabilities of the deacylated tRNAs in the A site (Table 1, Figure 2). First, each tRNA displays a slower koff when it contains the GAC anticodon than when it contains the GAA anticodon, clearly demonstrating the non-equivalent binding contributions of different codon•anticodon pairs (3). Interestingly, however, the difference in stability between each GAC/GAA tRNA pair varies in a manner that depends on the tRNA body. For example, the difference in koff values between tRNATrp(GAC) and tRNATrp(GAA) is only 3-fold, but the difference between tRNAPhe(GAC) and tRNAPhe(GAA) is 27-fold. This suggests that the relative contribution of the anticodon is different for different tRNA bodies. Second, the variation in koff among the different tRNA bodies is much broader in the GAA anticodon background than in the GAC anticodon background. The deacyl-tRNA(GAA)s show about a 40-fold range of koff values, with some dissociating faster than the cognate tRNAPhe(GAA) and others actually dissociating more slowly. For example, the very weak tRNAAla(GAA) has a koff at least 4-fold faster than tRNAPhe(GAA), but the koff for tight tRNATrp(GAA) is more than 8-fold slower than tRNAPhe(GAA). In contrast, except for tRNAAla(GAC), all of the deacyl-tRNA(GAC)s display koff values within 2-fold of the cognate deacyl-tRNAVal(GAC). Taken together, these results indicate that different tRNA bodies do interact differently with the ribosome; however, these effects are only observed in the weaker GAA anticodon background. Presumably, the tRNA body effects still exist in the GAC anticodon-substituted tRNAs but are somehow masked by the very stable GAC anticodon•codon interaction.Figure 2.

Bottom Line: When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed.However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold.We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.

View Article: PubMed Central - PubMed

Affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.

ABSTRACT
Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2'-deoxynucleotide-substituted mRNAs are used to demonstrate the presence of a lower threshold in k(off) values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed. However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold. We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.

Show MeSH
Related in: MedlinePlus