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Interchanging functionality among homologous elongation factors using signatures of heterotachy.

Cacan E, Kratzer JT, Cole MF, Gaucher EA - J. Mol. Evol. (2013)

Bottom Line: These EFs are GTPases that participate in protein translation by presenting aminoacylated-tRNAs to the ribosome.The two nucleotide exchange factors perform analogous functions despite not being homologous proteins.We further demonstrate that eEF1A variants, unlike yeast wild-type, can function in a reconstituted in vitro bacterial translation system.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Georgia Institute of Technology, Atlanta, GA, USA.

ABSTRACT
Numerous models of molecular evolution have been formulated to describe the forces that shape sequence divergence among homologous proteins. These models have greatly enhanced our understanding of evolutionary processes. Rarely are such models empirically tested in the laboratory, and even more rare, are such models exploited to generate novel molecules useful for synthetic biology. Here, we experimentally demonstrate that the heterotachy model of evolution captures signatures of functional divergence among homologous elongation factors (EFs) between bacterial EF-Tu and eukaryotic eEF1A. These EFs are GTPases that participate in protein translation by presenting aminoacylated-tRNAs to the ribosome. Upon release from the ribosome, the EFs are recharged by nucleotide exchange factors EF-Ts in bacteria or eEF1B in eukaryotes. The two nucleotide exchange factors perform analogous functions despite not being homologous proteins. The heterotachy model was used to identify a set of sites in eEF1A/EF-Tu associated with eEF1B binding in eukaryotes and another reciprocal set associated with EF-Ts binding in bacteria. Introduction of bacterial EF-Tu residues at these sites into eEF1A protein efficiently disrupted binding of cognate eEF1B as well as endowed eEF1A with the novel ability to bind bacterial EF-Ts. We further demonstrate that eEF1A variants, unlike yeast wild-type, can function in a reconstituted in vitro bacterial translation system.

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Related in: MedlinePlus

In vitro translation assay using wild-type eEF1A and the KOKI variants. A total of 15 replicate datasets were used from two independent protein purifications. Due to differences in protein refolding among the eEF1A proteins, a final buffer with 500 mM urea was used for the first round (with 1.3 mg of protein per reaction) and 125 mM urea for the second round (with 1 mg of protein per reaction). eEF1A variant KO3 was only active in the higher of the urea buffers, while KIKO3 was only active in the lower of the urea buffers. Data were collected using only the appropriate buffers for each of these two variants (6 replicates for KI3 and 9 replicates for KOKI3). All other analyses included data from both buffers to determine what, if any, affect the buffers have on the other variants (giving a total of 15 replicates). CPM were first normalized to the control reactions lacking any eEF1A protein and two data points were removed as statistical outliers (p < 0.05, Grubb’s test). Averages and standard errors of the mean are shown in the graph for each variant and * indicates significantly different from wild-type eEF1A at p < 0.05 while ** indicates significantly different at p < 0.01 using a student’s one-tailed t test. Results are shown relative to a background reaction lacking EF protein
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Fig3: In vitro translation assay using wild-type eEF1A and the KOKI variants. A total of 15 replicate datasets were used from two independent protein purifications. Due to differences in protein refolding among the eEF1A proteins, a final buffer with 500 mM urea was used for the first round (with 1.3 mg of protein per reaction) and 125 mM urea for the second round (with 1 mg of protein per reaction). eEF1A variant KO3 was only active in the higher of the urea buffers, while KIKO3 was only active in the lower of the urea buffers. Data were collected using only the appropriate buffers for each of these two variants (6 replicates for KI3 and 9 replicates for KOKI3). All other analyses included data from both buffers to determine what, if any, affect the buffers have on the other variants (giving a total of 15 replicates). CPM were first normalized to the control reactions lacking any eEF1A protein and two data points were removed as statistical outliers (p < 0.05, Grubb’s test). Averages and standard errors of the mean are shown in the graph for each variant and * indicates significantly different from wild-type eEF1A at p < 0.05 while ** indicates significantly different at p < 0.01 using a student’s one-tailed t test. Results are shown relative to a background reaction lacking EF protein

Mentions: We exploited a reconstituted in vitro protein translation system to address the functionality of these two eEF1A variants. This system is composed of recombinant E. coli biomolecules sufficient for in vitro translation (Shimizu et al. 2001). Such control of the system allows us to add/omit particular components. As such, the eEF1A variants could be added to the system in lieu of E. coli EF-Tu. Figure 3 demonstrates that both eEF1A variants KI3 and KOKI3 were able to participate significantly better in translation than the KO3 variant and the yeast wild-type eEF1A. Both KI3 and KOKI3 were able to bind EF-Ts (Fig. 2b, c) but the translation assay demonstrates that these variants have been engineered with the ability to actually participate in translation with bacterial components. It is curious that KOKI3 does not participate in translation as well as KI3 although the latter binds EF-Ts more efficiently. We suspect this may be due, in part, to the fact that these variants were soluble in different buffer systems. This may have affected the assays but additional studies will be required to dissect the differences as well as determine exactly how KI3 and KOKI3 are able to participate in translation while the other eEF1A proteins cannot.Fig. 3


Interchanging functionality among homologous elongation factors using signatures of heterotachy.

Cacan E, Kratzer JT, Cole MF, Gaucher EA - J. Mol. Evol. (2013)

In vitro translation assay using wild-type eEF1A and the KOKI variants. A total of 15 replicate datasets were used from two independent protein purifications. Due to differences in protein refolding among the eEF1A proteins, a final buffer with 500 mM urea was used for the first round (with 1.3 mg of protein per reaction) and 125 mM urea for the second round (with 1 mg of protein per reaction). eEF1A variant KO3 was only active in the higher of the urea buffers, while KIKO3 was only active in the lower of the urea buffers. Data were collected using only the appropriate buffers for each of these two variants (6 replicates for KI3 and 9 replicates for KOKI3). All other analyses included data from both buffers to determine what, if any, affect the buffers have on the other variants (giving a total of 15 replicates). CPM were first normalized to the control reactions lacking any eEF1A protein and two data points were removed as statistical outliers (p < 0.05, Grubb’s test). Averages and standard errors of the mean are shown in the graph for each variant and * indicates significantly different from wild-type eEF1A at p < 0.05 while ** indicates significantly different at p < 0.01 using a student’s one-tailed t test. Results are shown relative to a background reaction lacking EF protein
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: In vitro translation assay using wild-type eEF1A and the KOKI variants. A total of 15 replicate datasets were used from two independent protein purifications. Due to differences in protein refolding among the eEF1A proteins, a final buffer with 500 mM urea was used for the first round (with 1.3 mg of protein per reaction) and 125 mM urea for the second round (with 1 mg of protein per reaction). eEF1A variant KO3 was only active in the higher of the urea buffers, while KIKO3 was only active in the lower of the urea buffers. Data were collected using only the appropriate buffers for each of these two variants (6 replicates for KI3 and 9 replicates for KOKI3). All other analyses included data from both buffers to determine what, if any, affect the buffers have on the other variants (giving a total of 15 replicates). CPM were first normalized to the control reactions lacking any eEF1A protein and two data points were removed as statistical outliers (p < 0.05, Grubb’s test). Averages and standard errors of the mean are shown in the graph for each variant and * indicates significantly different from wild-type eEF1A at p < 0.05 while ** indicates significantly different at p < 0.01 using a student’s one-tailed t test. Results are shown relative to a background reaction lacking EF protein
Mentions: We exploited a reconstituted in vitro protein translation system to address the functionality of these two eEF1A variants. This system is composed of recombinant E. coli biomolecules sufficient for in vitro translation (Shimizu et al. 2001). Such control of the system allows us to add/omit particular components. As such, the eEF1A variants could be added to the system in lieu of E. coli EF-Tu. Figure 3 demonstrates that both eEF1A variants KI3 and KOKI3 were able to participate significantly better in translation than the KO3 variant and the yeast wild-type eEF1A. Both KI3 and KOKI3 were able to bind EF-Ts (Fig. 2b, c) but the translation assay demonstrates that these variants have been engineered with the ability to actually participate in translation with bacterial components. It is curious that KOKI3 does not participate in translation as well as KI3 although the latter binds EF-Ts more efficiently. We suspect this may be due, in part, to the fact that these variants were soluble in different buffer systems. This may have affected the assays but additional studies will be required to dissect the differences as well as determine exactly how KI3 and KOKI3 are able to participate in translation while the other eEF1A proteins cannot.Fig. 3

Bottom Line: These EFs are GTPases that participate in protein translation by presenting aminoacylated-tRNAs to the ribosome.The two nucleotide exchange factors perform analogous functions despite not being homologous proteins.We further demonstrate that eEF1A variants, unlike yeast wild-type, can function in a reconstituted in vitro bacterial translation system.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Georgia Institute of Technology, Atlanta, GA, USA.

ABSTRACT
Numerous models of molecular evolution have been formulated to describe the forces that shape sequence divergence among homologous proteins. These models have greatly enhanced our understanding of evolutionary processes. Rarely are such models empirically tested in the laboratory, and even more rare, are such models exploited to generate novel molecules useful for synthetic biology. Here, we experimentally demonstrate that the heterotachy model of evolution captures signatures of functional divergence among homologous elongation factors (EFs) between bacterial EF-Tu and eukaryotic eEF1A. These EFs are GTPases that participate in protein translation by presenting aminoacylated-tRNAs to the ribosome. Upon release from the ribosome, the EFs are recharged by nucleotide exchange factors EF-Ts in bacteria or eEF1B in eukaryotes. The two nucleotide exchange factors perform analogous functions despite not being homologous proteins. The heterotachy model was used to identify a set of sites in eEF1A/EF-Tu associated with eEF1B binding in eukaryotes and another reciprocal set associated with EF-Ts binding in bacteria. Introduction of bacterial EF-Tu residues at these sites into eEF1A protein efficiently disrupted binding of cognate eEF1B as well as endowed eEF1A with the novel ability to bind bacterial EF-Ts. We further demonstrate that eEF1A variants, unlike yeast wild-type, can function in a reconstituted in vitro bacterial translation system.

Show MeSH
Related in: MedlinePlus