Toward a Model for the Interaction Between Elongation Factor Tu and the Ribosome

Abstract

In the elongation cycle of bacterial protein synthesis the interaction between elongation factor-Tu (EF-Tu).guanosine triphosphate (GTP), aminoacyl-transfer RNA (aa-tRNA), and messenger RNA-programmed ribosomes is associated with the hydrolysis of GTP. This interaction determines the selection of the proper aa-tRNA for incorporation into the polypeptide. In the canonical scheme, one molecule of GTP is hydrolyzed in the EF-Tu-dependent binding of aa-tRNA to the ribosome, and a second molecule is hydrolyzed in the elongation factor-G (EF-G)-mediated translocation of the polypeptide from the ribosomal A site to the P site. Substitution of Asp138 with Asn in EF-Tu changed the substrate specificity from GTP to xanthosine triphosphate and demonstrated that the EF-Tu-mediated reactions involved the hydrolysis of two nucleotide triphosphates for each Phe incorporated. This stoichiometry of two is associated with the binding of the correct aa-tRNA to the ribosome.

Why does Escherichia coli have two primary pathways for synthesis of glutamate?

“…The formation of a gram (dry weight) of cells of E. coli has been calculated to require the conversion of 43 mmol of ATP to ADP (24). This calculation underestimates the cost of protein synthesis (35) and ignores the cost of transport of materials other than glucose. Adjusting to include those costs, I estimate the overall cost of synthesis to be 50 to 60 mmol of ATP per g (dry weight), or (for purposes of calculation) about 55 mmol.…”

Asparagine‐135 of elongation factor Tu is a crucial residue for the folding of the guanine nucleotide binding pocket

“…similarly to the corresponding residue (Asp'19) in the 3D model of c-H-ras p21 at 1.35 A resolution [9]. Raman spectroscopy confirmed the existence of this hydrogen bond [lo] [12] observed that EF-TuD138N recognizes XTP with an affinity comparable to that of wild-type EF-Tu (EF-Tu wt) for GTP. Less clear is the situation concerning the exocyclic O(6) of the base.…”

“…The ohgodeoxynucleotide GTGTTCCTGGACAAATG was used to substitute Asn"' with Asp in EF-TuDl38N [12]. The modified tufA was overexpressed via pTTQl8 under control of the tuc promotor m the RecA.…”

“…The modified tufA was overexpressed via pTTQl8 under control of the tuc promotor m the RecA. E. cd stram PM1455, containing only one active rufA gene encoding a kirromycinresistant product [15] EF-Tu Nl35D/Dl38N was purified according to the method utilizmg the antibiotic ktrromycm [12]. The reactivities of EF-Tu wt and Nl35DIDl38N to GTP.…”

Two GTPs are consumed on EF‐Tu per peptide bond in poly(Phe) synthesis, in spite of switching stoichiometry of the EF‐Tu·aminoacyl‐tRNA complex with temperature

“…The 2:1 stoichiometry between EF-Tu and aa-tRNA, observed at 37°C, has been questioned [7,8,11,12]. However, this controversy about the extended ternary complex has recently been resolved [10,13]: RNAse A protection assays in combination with the quench-flow technique show 1 : 1 stoichiometry at 0°C and 2 : 1 at 37°C, which demonstrates that the stoichiometry of the ternary complex changes with temperature.…”