The amino acid sequence of elongation factor Tu of Escherichia coli. The complete sequence.

Laursen, Richard A.; L’Italien, James; Nagarkatti, S; Miller, David L.

doi:10.1016/s0021-9258(18)43394-7

Cited by 75 publications

(9 citation statements)

References 43 publications

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“…Considering the structural homology of both proteins, the region around amino acid residues 45-60 of T. thermophilus EF-Tu would correspond to this "effector" region in p21 (Halliday, 1984;de Vos et al, 1988). Indeed, this part of E. coli EF-Tu was implicated in interaction with aa-tRNA (Laursen et al, 1981) which is the effector in the case of the EF-Tu cycle (Bourne, 1986). The basic amino acid residues in this loop are protected from proteolysis by the binding of aa-tRNA (Jacobson & Rosenbusch, 1977).…”

Section: Discussionmentioning

confidence: 99%

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Peter¹,

Wittmann‐Liebold²,

Sprinzl³

1988

Biochemistry

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Elongation factor Tu from Thermus thermophilus was treated successively with periodate-oxidized GDP or GTP and cyanoborohydride. Covalently modified cyanogen bromide or trypsin fragments of the protein were isolated, and the position of their modification was determined. Lysine residues 52 and 137 were heavily labeled, lysine-137 being considerably more reactive in the GTP form as compared to the GDP form of the protein. These residues are in the proximity of the GDP/GTP binding site. Lys-325 was also labeled, but to a lower extent. The part of the EF-Tu containing residue 52 is missing in crystallized EF-Tu.GDP from Escherichia coli [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36]. These results place the part of T. thermophilus EF-Tu corresponding to the missing fragment in E. coli EF-Tu in the vicinity of the nucleotide binding site and allow its role in the interaction with aminoacyl-tRNA and elongation factor Ts to be evaluated. Cross-linking of EF-Tu.GDP by irradiation at 257 nm showed that a sequence of 10 amino acids residues which is found in the Thermus thermophilus elongation factor Tu but not in other homologous bacterial proteins is located in the vicinity of the GDP/GTP binding site.

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Section: Discussionmentioning

confidence: 99%

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Peter¹,

Wittmann‐Liebold²,

Sprinzl³

1988

Biochemistry

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“…When EF-Tu was unfolded in 6 M guanidine hydrochloride, less than a 1% change in the absorption at 280 nm was observed. Hence, none of the absorbing residues [3 cysteines, 1 tryptophan, 10 tyrosines, and 1 GDP (Arai et al, 1980;Laursen et al, 1981)] were in environments that significantly altered their molar extinction coefficients from the values observed in nondenaturing conditions. This newly determined extinction coefficient for EF-Tu-GDP is substantially closer to the theoretical value than the extinction coefficients previously reported (Arai et al, 1973;Miller & Weissbach, 1977a).…”

Section: Methodsmentioning

confidence: 99%

“…where V is the molecular volume, the partial specific volume associated with the water of hydration, the viscosity, the Boltzmann constant, and T the absolute temperature. The molecular volume for EF-Tu-GTP was calculated by using a molecular weight of 43 200 (Arai et al, 1980) and a specific volume of 0.74 mL/g [determined from the sequence (Arai et al, 1980;Laursen et al, 1981) by summing the volume of the constituent amino acids (Cohn & Edsall, 1943;McMeekin et al, 1949)]. The molecular volume for E. coli tRNAPhe was determined from the specific volume, 0.503 mL/g (Tao et al, 1970), and a molecular weight of 25 000 (Barrell & Sanger, 1969; Uziel & Gassen, 1969).…”

Section: Methodsmentioning

confidence: 99%

Time-resolved fluorescence studies on the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and phenylalanyl-tRNAPhe

Hazlett

Johnson²,

Jameson³

1989

Biochemistry

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Time-resolved fluorescence spectroscopy was used to investigate the solution dynamics of Escherichia coli tRNAPhe, Phe-tRNAPhe, and Phe-tRNAPhe associated with GTP and elongation factor Tu (EF-Tu) in a ternary complex. Two fluorescence probes were employed: fluorescein, covalently bound to Phe-tRNAPhe at the s4U8 base (Phe-tRNAPhe-Fl8), and ethidium bromide, noncovalently associated with the tRNA (EB.Phe-tRNAPhe). The lifetimes observed for ethidium bromide were 1.89 ns, free in solution, and 26.3 ns, bound to its tight binding site on tRNA. Fluorescein-labeled tRNA had a lifetime of 4.3 ns, with no significant difference among the values for aminoacylated, unacylated, and EF-Tu-bound Phe-tRNAPhe-Fl8. Differential phase and modulation data for each fluorophore-tRNA system were fit with local and global Debye rotational relaxation times. Local motion of the labeled fluorescein in Phe-tRNAPhe-Fl8, tRNAPhe-Fl8, and Phe-tRNAPhe-Fl8.EF-Tu.GTP was characterized by rotational relaxation times of 2.7 +/- 0.5, 2.4 +/- 0.4, and 2.4 +/- 0.1 ns, respectively. These values are equal, within experimental error, and suggest that the rotational mobility of the s4U8-conjugated dye is unaffected by either tRNAPhe aminoacylation or ternary complex formation. Global rotational relaxation times for Phe-tRNAPhe-Fl8, 97 ns, and EB.Phe-tRNAPhe, 140 ns, were equivalent to those determined for the unacylated species, denoting little change in the overall size or shape of the tRNA molecule upon aminoacylation. These values for (Phe-)tRNA were larger than expected for a hydrated sphere of equivalent volume, 83 ns, and therefore confirm the asymmetric nature of the tRNA structure in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Gene replacement was "+" designates clones with complementing activity; " -" designates clones lacking complementing activity, and " -" designates a clone that could complement the budding defect of bud4-1 when present in YEp24 but not in YCp50. (B) Comparison of Bud4p to conserved sequences in a variety of known GTPases (Bobak et al, 1989;Gallwitz et al, 1983;Laursen et al, 1981;Madaule et al, 1987;Capon et al, 1983;Robishaw et al, 1986) and to the consensus sequence (Bourne et al, 1991). (C) Comparison of upstream nucleotide sequences of B UD4 to the upstream regulatory sequences of SWI5 and CLB2 that are necessary and sufficient for proper cell cycle-regulated transcription (Lydall et al, 1991;Maher et al, 1995) and to the upstream sequences of BUD3 .…”

Section: B U D 4 Null Phenotypementioning

confidence: 99%

The BUD4 protein of yeast, required for axial budding, is localized to the mother/BUD neck in a cell cycle-dependent manner.

Sanders

Herskowitz

1996

The Journal of Cell Biology

163

230

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A and alpha cells of the yeast Saccharomyces cerevisiae exhibit an axial budding pattern, whereas a/alpha diploid cells exhibit a bipolar pattern. Mutations in BUD3, BUD4, and AXL1 cause a and alpha cells to exhibit the bipolar pattern, indicating that these genes are necessary to specify the axial budding pattern (Chant, J., and I. Herskowitz. 1991. Cell. 65:1203-1212; Fujita, A., C. Oka, Y. Arikawa, T. Katagi, A. Tonouchi, S. Kuhara, and Y. Misumi. 1994. Nature (Lond.). 372:567-570). We cloned and sequenced BUD4, which codes for a large, novel protein (Bud4p) with a potential GTP-binding motif. Bud4p is expressed and localized to the mother/bud neck in all cell types. Most mitotic cells contain two apparent rings of Bud4 immunoreactive staining, as observed for Bud3p (Chant, J., M. Mischke, E. Mitchell, I. Herskowitz, and J.R. Pringle. 1995. J. Cell Biol. 129: 767-778). Early G1 cells contain a single ring of Bud4p immunoreactive staining, whereas cells at START and in S phase lack these rings. The level of Bud4p is also regulated in a cell cycle-dependent manner. Bud4p is inefficiently localized in bud3 mutants and after a temperature shift of a temperature-sensitive mutant, cdc12, defective in the neck filaments. These observations suggest that Bud4p and Bud3p cooperate to recognize a spatial landmark (the neck filaments) during mitosis and support the hypothesis that they subsequently become a landmark for establishing the axial budding pattern in G1.

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The amino acid sequence of elongation factor Tu of Escherichia coli. The complete sequence.

Cited by 75 publications

References 43 publications

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Time-resolved fluorescence studies on the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and phenylalanyl-tRNAPhe

The BUD4 protein of yeast, required for axial budding, is localized to the mother/BUD neck in a cell cycle-dependent manner.

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