Functional and structural studies on a tryptic fragment of eucaryotic elongation factor Tu from rabbit reticulocytes

Slobin, Lawrence I.; Clark, R. V.; Olson, Matthew S.

doi:10.1021/bi00523a019

Cited by 32 publications

(29 citation statements)

References 36 publications

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“…The partial amino acid sequence of EF-la from rabbit reticulocytes (14) and A. salina (15) was determined, and sequence conservation between A. salina EF-la and E. coli EF-Tu has been reported (15).…”

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confidence: 99%

Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae.

Nagata¹,

Tsunetsugu-Yokota²,

Naito³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

131

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A 3. 1-kilobase Bgl H fragment of Saccharomyces cerevisiae carrying the nuclear gene encoding the mitochondrial polypeptide chain elongation factor (EF) Tu has been cloned on pBR327 to yield a chimeric plasmid pYYB. The identification of the gene designated as tufM was based on the cross-hybridization with the Escherichia coli tufB gene, under low stringency conditions. The complete nucleotide sequence of the yeast tufM gene was established together with its 5'-and 3'-flankdng regions. The sequence contained 1,311 nucleotides coding for a protein of 437 amino acids with a calculated Mr of 47,980. The nucleotide sequence and the deduced amino acid sequence of tufM were 60% and 66% homologous, respectively, to the corresponding sequences of E. coli tufA, when aligned to obtain the maximal homology. Plasmid YRpYB was then constructed by cloning the 2.5-kilobase EcoRI fragment of pYYB carrying tufM into a yeast cloning vector YRp-7. A mRNA hybridizable with tufM was isolated from the total mRNA of S. cerevisiae D13-IA transformed with YRpYB and translated in the reticulocyte lysate. The mRNA could direct the synthesis of a protein with Mr 48,000, which was immunoprecipitated with an anti-E. coli EF-Tu antibody but not with an antibody against yeast cytoplasmic EF-la. The results indicate that the tufM gene is a nuclear gene coding for the yeast mitochondrial EF-Tu.The polypeptide chain elongation factor Tu (EF-Tu) promotes a GTP-dependent binding of an aminoacyl-tRNA to the A site of ribosomes (1). EF-Tu from Escherichia coli consists of a single polypeptide chain with Mr 43,000, and the primary structure comprised of 393 amino acid residues has been determined (2). The protein is encoded by two nearly identical genes on the E. coli chromosome (3), tufA at 73 min and tufB at 89 min (4). Both tufA (5) and tufB (6) have been cloned and their nucleotide sequences determined. The sequences of tufA (7) and tufB (8) are nearly homologous and differ only in 13 positions but the gene products, EF-TuA and EF-TuB, are identical except for the COOH-terminal amino acid (2).In eukaryotes, the counterpart of prokaryotic EF-Tu, designated as EF-la, has been purified from various sources, including pig liver (9), rabbit reticulocytes (10), Artemia salina (11), wheat germ (12), and yeast (13), and was shown to consist of a single polypeptide chain, Mr 47,000-53,000. The partial amino acid sequence of EF-la from rabbit reticulocytes (14) and A. salina (15) was determined, and sequence conservation between A. salina EF-la and E. coli EF-Tu has been reported (15).In addition to EF-la, which functions in the cytoplasmic fraction in conjunction with 80S ribosomes, eukaryotic cells possess mitochondrial EF-Tu (designated as mEF-Tu) that functions in the mitochondrial translational apparatus (16,17). Translational factors as well as ribosomal proteins in the mitochondria are encoded by nuclear genes, synthesized in cytoplasmic fractions, and transported into the mitochondria (18). The translational machineries of mitochondria have...

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mentioning

confidence: 99%

Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae.

Nagata¹,

Tsunetsugu-Yokota²,

Naito³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

131

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“…From our experimens with the 37/43-kDa tryptic fragments, it can be concluded that the proteolytic removal of the region spanning the residues 37 -68 and 37 -128 completely abolished the transport of aminoacyl tRNA to the ribosome. However, in two other reports, tryptic removal of residues 37 -68 in EF-la solely reduces the aminoacyl tRNA binding to only 30% and 6O%, respectively [16,171. The larger reduction in our experiments (to lo%), obtained on a mixture of the 37-kDa and 43-kDa fragments, could be due to the additional presence of the 37-kDa fragment lacking the residues 37 -128 and having no tRNA binding activity at all.…”

Section: Discussionmentioning

confidence: 81%

“…Limited digestion experiments have already been described with eukaryotic EF-la [16,171, which revealed digestion patterns reminiscent of prokaryotic EF-Tu from Escherichiu coli [18,191 and Thermus thermophilus [20]. A major difference between EF-Tu and EF-la concerns the exchange of proteinbound guanine nucleotides.…”

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confidence: 99%

Identification of the sites in the eukaryotic elongation factor 1α involved in the binding of elongation factor 1β and aminoacyl‐tRNA

Damme¹,

Amons²,

Möller³

1992

European Journal of Biochemistry

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In this article we report the identification of the sites which are involved in the binding of the GDP-exchange factor EF-1P and aminoacyl tRNA to the a-subunit of the eukaryotic elongation factor 1 (EF-1) from Artemiu. For this purpose the polypeptide chain of EF-la, having 461 amino acid residues, was proteolytically cleaved into large fragments by distinct proteases. Under well defined conditions, a mixture of two large fragments, free from intact EF-la and with molecular masses of 37 kDa and 43 kDa, was obtained. The 37-kDa and 43-kDa fragments comprise the residues 129 -461 and 69 -461, respectively. However, in aqueous solution and under non-denaturing conditions, the mixture still contained a short amino-terminal peptide, encompassing the residues 1 -36, that remained tightly bound. The ability of the mixture of the 37 + 43-kDa fragments, including this amino-terminal peptide 1 -36, to bind GDP or to facilitate aminoacyl tRNA binding to saltwashed ribosomes was severely reduced, compared to intact EF-la. However, both of these complexes were able to bind to the GDP-exchange-stimulating subunit EF-1P . A 30-kDa fragment, comprising the residues 1 -287, was generated after treatment of the protein with endoproteinase Glu-C. This fragment contained the complete guanine nucleotide binding pocket. Although it was able to bind GDP and to transport aminoacyl tRNA to the ribosome, no affinity towards EF-1P was observed. We propose that the guanine-nucleotide-exchange stimulation by EF-1P is induced through binding of this factor to the carboxy-terminal part of EF-la. As a result, a decreased susceptibility towards trypsin of the guanine-nucleotide-binding pocket of EF-la, especially in the region of its presumed effector loop is induced.The most abundant eukaryotic translational factor is elongation factor 1 (EF-I), which catalyzes, together with the translocation factor EF-2, the elongation cycle in the eukaryotic protein synthesis [I, 21. EF-1 contains four subunits (a, /J, y and 6). The elongation starts with the formation of a ternary complex between EF-la, aminoacyl tRNA and GTP. Subsequently, aminoacyl tRNA is attached to the mRNAribosome complex. This latter step requires hydrolysis of GTP [2]. The elongation rate is increased by two other subunits, EF-1P and EF-16, which both accelerate the exchange of GDP, bound to EF-la, for GTP [2, 31. EF-1P and EF-16 have been isolated in complex with EF-1y as heterodimers or heterotrimers (EF-Ifiy or EF-lPyG, respectively). Together with EF-1 a, these EF-IPy(6) complexes form high-moleclarmass aggregates, which have been described in many eukaryotic species [4 -81. Furthermore, these aggregates have been found associated with valyl tRNA synthetase [9, 101.

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“…On removal of heme, the region around Asp-47 also shows increased accessibility to proteolysis. We have observed that HbA is very resistant to V8 protease digestion (17). In the tetrameric Hb none of the glutamyl/ aspartyl peptide bonds of a-or P-chain is accessible to V8 protease digestion.…”

Section: Discussionmentioning

confidence: 84%

“…Polyhydric alcohols, like ethylene glycol and glycerol, have been reported to stabilize the protein structure (cryoprotectants; ref. 16), and in some cases these have been shown to limit the proteolytic digestion of proteins (17). Furthermore, glycerol is the widely used solvent to induce proteases to reform peptide bonds in the fragment-complementing systems (7).…”

Section: Methodsmentioning

confidence: 99%

Conformational studies of alpha-globin in 1-propanol: propensity of the alcohol to limit the sites of proteolytic cleavage.

Iyer

Acharya

1987

Proc. Natl. Acad. Sci. U.S.A.

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Selective condensation of the unprotected fragments of a-globin-namely, a1..30 and a31.141-is catalyzed by Staphylococcus aureus V8 protease in the presence of 25% 1-propanol. The propensity of 1-propanol to induce the ahelical conformation and to generate a "native-like" topology for the polypeptide chain has been now investigated in an attempt to understand the molecular basis of this enzymecatalyzed stereospecific condensation. Removal of heme from the a-chain decreases the overall a-helical conformation of the protein considerably. A significant amount of the a-helical conformation is restored in the presence of25% 1-propanol and the digestion of a-globin by V8 protease becomes more selective concomitant with the increase in helicity. V8 protease digestion of a-globin at pH 6.0 and 40C occurs at Glu-30, Asp47, Glu-27, and Glu-23 in the absence of 1-propanol. In the presence of 25% 1-propanol, the digestion is selective to the peptide bond of Glu-30. This selectivity appears to be a characteristic feature of the native conformation of a-chain (polypeptide chain with bound heme). 1-Propanol induces the a-helical conformation into RNase S. peptide also. However, this increased helical conformation did not protect the RNase S peptide from V8 protease digestion at the Glu-9-Arg-10 peptide bond. RNase S peptide is in an a-helical conformation in RNase S, an interacting fragment-complementing system of S protein and S peptide. S peptide is resistant to V8 protease hydrolysis in this conformation. Thus, the resistance of a peptide bond in a segment of a protein to protease digestion appears to be a consequence of the secondary structure as well as the tertiary interactions of this segment with the rest of the molecule. The results suggest that the 1-propanol induces a-helical conformation into segments of a-globin as well as packing of these helices in a native-like topology.There has been considerable interest in recent years in the structure of proteins in organic solvents (1). The lowtemperature studies of enzymes, cryoenzymology, as well as low-temperature crystallography have necessitated the use of organic solvents in these protein structural studies (2). In addition, many enzyme-catalyzed reactions are also being investigated in organic media to enhance the industrial applications, specifically to take advantage of the high selectivity and specificity of the enzyme (3). With proteases, it has been possible to achieve "reverse proteolysis"-i.e., use proteases to catalyze the synthesis of peptide bonds by incorporating large amounts of organic solvents (i.e., like ethylene glycol, glycerol, etc.) in the reaction mixtures (4). The reformation of ribonuclease A from RNase S (5) and nuclease A from nuclease T (6) are the classical examples of this reverse proteolysis approach for semisynthesis of proteins (7).The protease-catalyzed reverse proteolysis for the semisynthesis of noncovalent analogues of proteins has been emerging as a useful procedure to prepare covalent variants of proteins (7). The presence...

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Functional and structural studies on a tryptic fragment of eucaryotic elongation factor Tu from rabbit reticulocytes

Cited by 32 publications

References 36 publications

Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae.

Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae.

Identification of the sites in the eukaryotic elongation factor 1α involved in the binding of elongation factor 1β and aminoacyl‐tRNA

Conformational studies of alpha-globin in 1-propanol: propensity of the alcohol to limit the sites of proteolytic cleavage.

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