Dynamics and morphology of the in vitro polymeric form of elongation factor Tu from Escherichia coli

Helms, Michael K.; Marriott, Gerard; Sawyer, William H.; Jameson, David M.

doi:10.1016/0304-4165(96)00054-2

Cited by 8 publications

(6 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Experiments analogous to those described above at pH 7.5 were carried out at pH 6.5 to 8.5. A broader range of pH values could not be tested, because of EF-Tu aggregation at pH values below 6.5 27 (T.D., H.-J.W. & M.V.R., unpublished results) and significant hydrolysis of Phe-tRNA Phe above pH 8.5, both rendering the ternary complex inactive.…”

Section: Ph Dependence Of Gtp Hydrolysismentioning

confidence: 99%

Essential Role of Histidine 84 in Elongation Factor Tu for the Chemical Step of GTP Hydrolysis on the Ribosome

Daviter

Wieden

Rodnina

2003

Journal of Molecular Biology

133

184

View full text Add to dashboard Cite

Elongation factor Tu (EF-Tu) is a GTP-binding protein that delivers aminoacyl-tRNA to the A site of the ribosome during protein synthesis. The mechanism of GTP hydrolysis in EF-Tu on the ribosome is poorly understood. It is known that mutations of a conserved histidine residue in the switch II region of the factor, His84 in Escherichia coli EF-Tu, impair GTP hydrolysis. However, the partial reaction which is directly affected by mutations of His84 was not identified and the effect on GTP hydrolysis was not quantified. Here, we show that the replacement of His84 with Ala reduces the rate constant of GTP hydrolysis more than 10(6)-fold, whereas the preceding steps of ternary complex binding to the ribosome, codon recognition and, most importantly, the GTPase activation step are affected only slightly. These results show that His84 plays a key role in the chemical step of GTP hydrolysis. Rate constants of GTP hydrolysis by wild-type EF-Tu, measured using the slowly hydrolyzable GTP analog, GTPgammaS, showed no dependence on pH, indicating that His84 does not act as a general base. We propose that the catalytic role of His84 is to stabilize the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or, possibly, the gamma-phosphate group of GTP.

show abstract

Section: Ph Dependence Of Gtp Hydrolysismentioning

confidence: 99%

Essential Role of Histidine 84 in Elongation Factor Tu for the Chemical Step of GTP Hydrolysis on the Ribosome

Daviter

Wieden

Rodnina

2003

Journal of Molecular Biology

133

184

View full text Add to dashboard Cite

show abstract

“…Alternatively, domain III might be required for the bundling of microtubules, possibly via an interaction with another domain III bound to an adjacent microtubule. The prokaryotic form of EF-1␣ polymerizes into sheets in vitro [Helms et al, 1996], and EF-1␣ aggregates are observed in microtubule organizing granules isolated from sea urchin eggs or plant culture cells [Kuriyama and Borisy, 1985;Kumagai et al, 1999]. However, it is not known if the bundling of microtubules observed in vitro is due mainly to charge shielding, bridging of adjacent microtubules by neighboring EF-1␣ molecules, or both.…”

Section: Ef-1␣ Has Two Microtubule-binding Domainsmentioning

confidence: 99%

Association between elongation factor-1? and microtubules in vivo is domain dependent and conditional

Moore

Cyr

2000

Cell Motil. Cytoskeleton

View full text Add to dashboard Cite

Although the precise definition for a microtubule‐associated protein (MAP) has been the subject of debate, elongation factor‐1α (EF‐1α) fits the most basic criteria for a MAP [Durso and Cyr, 1994a]. It binds, bundles, stabilizes, and promotes the assembly of microtubules in vitro, and localizes to plant microtubule arrays in situ. In this study, the in vitro and in vivo association of EF‐1α with microtubules was further investigated. Analysis of the in vitro binding data for EF‐1α and microtubules indicates that EF‐1α binds cooperatively to the microtubule lattice. In order to investigate the interaction of EF‐1α with microtubules in vivo, GFP fusions to EF‐1α or to EF‐1α truncates were transiently expressed in living plant cells. Using this method, two putative microtubule‐binding domains on EF‐1α were identified: one in the N‐terminal domain I and one in the C‐terminal domain III. The binding of domain I to microtubules in vivo, like the binding of full‐length EF‐1α, is conditional, and requires incubation in weak, lipophilic organic acids. The binding of domain III to microtubules in vivo, however, is not conditional, and occurs under normal cellular regimes. Furthermore, domain III stabilizes cortical microtubules as determined by their resistance to the anti‐microtubule herbicide, oryzalin. Because the accumulation of EF‐1α onto microtubules is unconditional in the absence of domain I, we hypothesize that domain I negatively regulates the accumulation of EF‐1α onto microtubules in vivo. This hypothesis is discussed in terms of possible regulatory mechanisms that could affect the accumulation of EF‐1α onto microtubules within living cells. Cell Motil. Cytoskeleton 45:279–292, 2000 © 2000 Wiley‐Liss, Inc.

show abstract

“…13 As discussed in the Materials and Methods, the buffers used in the purification protocol were altered from those previously used 12 to minimize protein aggregation. The known ability of E. coli EF-Tu to aggregate 14,31,32 is a particular problem for the isolation of EF-Tu mutants expressed in E. coli because of the potential for the abundant untagged endogenous cellular protein to associate with the tagged mutant protein and thereby be retained on the Ni-NTA resin and contaminate the final preparation of mutant protein. To evaluate the efficacy of our revised protocol, we used it to purify the H84A mutant protein.…”

Section: Resultsmentioning

confidence: 99%

“…17 Aminoacylated tRNAs were resuspended and stored in 5 mM NaOAc buffer at pH 5 after phenol/chloroform extraction and ethanol precipitation steps. 32 P-labeled T1 tRNA Tyr was prepared to measure rates of dipeptide bond formation of EF-Tu variants. 3 T1 tRNA Tyr was labeled with [α-32 P]-ATP at the 3′ -CCA end and aminoacylated with tyrosine using published methods.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Ternary complex was formed by mixing 0.4 μM EF-Tu with 1.2 μM Tyr-tRNA Tyr or 1.2 μM Tyr-T1 Tyr-tRNA Tyr , and 50 μM γ-32 P-GTP in 50 mM HEPES pH 7, 30 mM KCl, 70 mM NH 4 Cl, 10 mM MgCl 2 , 10 μM GTP, 3 mM phosphoenolpyruvate, 50 μg/mL of pyruvate kinase, and 1 mM DTT according to published protocols. 4,13,23,24 Heat activated ternary complex solution was passed through two Bio-Spin 6 columns (BIO-RAD) to remove excess [γ- 32 were mixed and quenched with 40% formic acid using KinTek quench flow instrument at 20 °C. The hydrolyzed GTP fraction was calculated by analyzing the samples on TLC plates coated with PEI cellulose.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The Interface between Escherichia coli Elongation Factor Tu and Aminoacyl-tRNA

et al. 2014

View full text Add to dashboard Cite

Nineteen of the highly conserved residues of Escherichia coli (E. coli) Elongation factor Tu (EF-Tu) that form the binding interface with aa-tRNA were mutated to alanine to better understand how modifying the thermodynamic properties of EF-Tu–tRNA interaction can affect the decoding properties of the ribosome. Comparison of ΔΔGo values for binding EF-Tu to aa-tRNA show that the majority of the interface residues stabilize the ternary complex and their thermodynamic contribution can depend on the tRNA species that is used. Experiments with a very tight binding mutation of tRNATyr indicate that interface amino acids distant from the tRNA mutation can contribute to the specificity. For nearly all of the mutations, the values of ΔΔGo were identical to those previously determined at the orthologous positions of Thermus thermophilus (T. thermophilus) EF-Tu indicating that the thermodynamic properties of the interface were conserved between distantly related bacteria. Measurement of the rate of GTP hydrolysis on programmed ribosomes revealed that nearly all of the interface mutations were able to function in ribosomal decoding. The only interface mutation with greatly impaired GTPase activity was R223A which is the only one that also forms a direct contact with the ribosome. Finally, the ability of the EF-Tu interface mutants to destabilize the EF-Tu–aa-tRNA interaction on the ribosome after GTP hydrolysis were evaluated by their ability to suppress the hyperstable T1 tRNATyr variant where EF-Tu release is sufficiently slow to limit the rate of peptide bond formation (kpep) . In general, interface mutations that destabilize EF-Tu binding are also able to stimulate kpep of T1 tRNATyr, suggesting that the thermodynamic properties of the EF-Tu–aa-tRNA interaction on the ribosome are quite similar to those found in the free ternary complex.

show abstract

Dynamics and morphology of the in vitro polymeric form of elongation factor Tu from Escherichia coli

Cited by 8 publications

References 32 publications

Essential Role of Histidine 84 in Elongation Factor Tu for the Chemical Step of GTP Hydrolysis on the Ribosome

Essential Role of Histidine 84 in Elongation Factor Tu for the Chemical Step of GTP Hydrolysis on the Ribosome

Association between elongation factor-1? and microtubules in vivo is domain dependent and conditional

The Interface between Escherichia coli Elongation Factor Tu and Aminoacyl-tRNA

Contact Info

Product

Resources

About