Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA

The 2′‐OH group of the peptidyl‐tRNA stabilizes an active conformation of the ribosomal PTC

“…These findings reconcile our initial observations of the inability of EFTu to stimulate the acceptor activity of the dA76 aa‐tRNA (Figures 3B and 5A). These results are in direct conflict with earlier studies where no differences in the dissociation rates of the rA76 and dA76 aa‐tRNAs from EFTu were observed (Pleiss and Uhlenbeck, 2001). The discrepancy between these earlier observations and our own might be reconciled by the different aa‐tRNAs and EFTu factors utilized; Uhlenbeck and colleagues used in vitro transcribed variants of the yeast tRNA Phe and Thermus thermophilus EFTu, while we used native tRNA Lys and EFTu, both from E. coli .…”

The 2′‐OH group of the peptidyl‐tRNA stabilizes an active conformation of the ribosomal PTC

“…These findings reconcile our initial observations of the inability of EFTu to stimulate the acceptor activity of the dA76 aa‐tRNA (Figures 3B and 5A). These results are in direct conflict with earlier studies where no differences in the dissociation rates of the rA76 and dA76 aa‐tRNAs from EFTu were observed (Pleiss and Uhlenbeck, 2001). The discrepancy between these earlier observations and our own might be reconciled by the different aa‐tRNAs and EFTu factors utilized; Uhlenbeck and colleagues used in vitro transcribed variants of the yeast tRNA Phe and Thermus thermophilus EFTu, while we used native tRNA Lys and EFTu, both from E. coli .…”

Perturbation of the tRNA Tertiary Core Differentially Affects Specific Steps of the Elongation Cycle

“…Overall, these results demonstrate the importance of the 18 -55 and 19 -56 bp for functional tRNA interaction with the ribosome. However, it must be acknowledged that the validity of our results for what might be observed in vivo, along with the results of others who also study tRNA variants produced by in vitro transcription (7,15,22), is subject to the caveat that the quantitative effects of mutation could be different in the context of native tRNA, which contains modified bases.…”

Hierarchical mechanism of amino acid sensing by the T-box riboswitch

“…7a, b ), confirming the specificity of EF-Tu:GTP for aminoacylated tRNA. Measuring instead the affinity through the protection of Cy5-Gly-tRNA Gly by EF-Tu:GTP against RNase degradation followed by nitrocellulose binding 32 , 33 yielded a more realistic, higher-affinity apparent K d of 310 ± 40 nM (Fig. 7c ), establishing the fraction of Cy5-Gly-tRNA Gly bound to EF-Tu:GTP as >80% under the conditions of our single-molecule fluorescence coincidence assays.…”

The 2′‐OH group of the peptidyl‐tRNA stabilizes an active conformation of the ribosomal PTC

“…These findings reconcile our initial observations of the inability of EFTu to stimulate the acceptor activity of the dA76 aa‐tRNA (Figures 3B and 5A). These results are in direct conflict with earlier studies where no differences in the dissociation rates of the rA76 and dA76 aa‐tRNAs from EFTu were observed (Pleiss and Uhlenbeck, 2001). The discrepancy between these earlier observations and our own might be reconciled by the different aa‐tRNAs and EFTu factors utilized; Uhlenbeck and colleagues used in vitro transcribed variants of the yeast tRNA Phe and Thermus thermophilus EFTu, while we used native tRNA Lys and EFTu, both from E. coli .…”

Perturbation of the tRNA Tertiary Core Differentially Affects Specific Steps of the Elongation Cycle

“…Overall, these results demonstrate the importance of the 18 -55 and 19 -56 bp for functional tRNA interaction with the ribosome. However, it must be acknowledged that the validity of our results for what might be observed in vivo, along with the results of others who also study tRNA variants produced by in vitro transcription (7,15,22), is subject to the caveat that the quantitative effects of mutation could be different in the context of native tRNA, which contains modified bases.…”

Hierarchical mechanism of amino acid sensing by the T-box riboswitch

“…7a, b ), confirming the specificity of EF-Tu:GTP for aminoacylated tRNA. Measuring instead the affinity through the protection of Cy5-Gly-tRNA Gly by EF-Tu:GTP against RNase degradation followed by nitrocellulose binding 32 , 33 yielded a more realistic, higher-affinity apparent K d of 310 ± 40 nM (Fig. 7c ), establishing the fraction of Cy5-Gly-tRNA Gly bound to EF-Tu:GTP as >80% under the conditions of our single-molecule fluorescence coincidence assays.…”

The 2′‐OH group of the peptidyl‐tRNA stabilizes an active conformation of the ribosomal PTC

“…These findings reconcile our initial observations of the inability of EFTu to stimulate the acceptor activity of the dA76 aa‐tRNA (Figures 3B and 5A). These results are in direct conflict with earlier studies where no differences in the dissociation rates of the rA76 and dA76 aa‐tRNAs from EFTu were observed (Pleiss and Uhlenbeck, 2001). The discrepancy between these earlier observations and our own might be reconciled by the different aa‐tRNAs and EFTu factors utilized; Uhlenbeck and colleagues used in vitro transcribed variants of the yeast tRNA Phe and Thermus thermophilus EFTu, while we used native tRNA Lys and EFTu, both from E. coli .…”

Perturbation of the tRNA Tertiary Core Differentially Affects Specific Steps of the Elongation Cycle

“…Overall, these results demonstrate the importance of the 18 -55 and 19 -56 bp for functional tRNA interaction with the ribosome. However, it must be acknowledged that the validity of our results for what might be observed in vivo, along with the results of others who also study tRNA variants produced by in vitro transcription (7,15,22), is subject to the caveat that the quantitative effects of mutation could be different in the context of native tRNA, which contains modified bases.…”

Hierarchical mechanism of amino acid sensing by the T-box riboswitch

“…7a, b ), confirming the specificity of EF-Tu:GTP for aminoacylated tRNA. Measuring instead the affinity through the protection of Cy5-Gly-tRNA Gly by EF-Tu:GTP against RNase degradation followed by nitrocellulose binding 32 , 33 yielded a more realistic, higher-affinity apparent K d of 310 ± 40 nM (Fig. 7c ), establishing the fraction of Cy5-Gly-tRNA Gly bound to EF-Tu:GTP as >80% under the conditions of our single-molecule fluorescence coincidence assays.…”