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Biomedical subjects

M Sprinzl

Publications and source records attributed to M Sprinzl.

At least 55 records · Page 3Linked to original sources
PubMed1995

Overexpression and purification of Thermus thermophilus elongation factors G, Tu, and Ts from Escherichia coli.

The translation elongation factors G (EF-G), Tu (EF-Tu), and Ts (EF-Ts) from the extreme thermophilic bacterium Thermus thermophilus were overproduced in Escherichia coli. The fus gene coding for EF-G and the tufA gene coding for EF-Tu were expressed under the control of a tac promoter, whereas EF-Ts was overproduced with the T7 RNA polymerase system. A detailed description for the purification of the three elongation factors from E. coli is presented. EF-G and EF-Tu are isolated by Q-Sepharose FF chromatography, heat treatment at 65 or 60 degrees C, respectively, and Sephacryl S200 gel permeation chromatography. For the purification of EF-Ts, a heat denaturation step is followed by DEAE-cellulose chromatography and a cation exchange EMD-SO-3 650 column. The overproduced factors show the same properties as those purified from T. thermophilus. As the crystal structures of T. thermophilus EF-Tu and EF-G have been solved recently, many questions concerning the function of particular residues or domains arise, which may be best addressed by studying the in vitro behavior and structure of altered recombinant constructs. The methods presented here should facilitate such studies.

Bacteriophage T7↗
PubMed1995

Mass spectrometric approaches to molecular characterization of protein-nucleic acid interactions.

The recent development of 'soft' ionization-desorption methods has lead to a breakthrough for the mass spectrometric analysis of biomacromolecules such as proteins and nucleic acids. In particular, the feasibility of electrospray-ionization mass spectrometry (ESI-MS) for the direct characterization of non-covalent supramolecular complexes is opening new analytical perspectives. Examples hitherto analyzed by ESI-MS include enzyme-substrate and -inhibitor complexes, homo- and heterodimers/trimers of leucine zipper polypeptides, and several other DNA- and RNA-binding proteins. Furthermore, the characterization of double-stranded and higher-order oligo- and polynucleotide complexes by negative-ion ESI has been demonstrated. Ions specific of non-covalent protein and oligonucleotide complexes can be selectively dissociated by changing the solution conditions and by increasing the desolvation potential. These results form the basis for the molecular characterization of protein-nucleotide interactions, thus complementing protein-chemical approaches, and other methods of structure determination.

Amino Acid Sequence↗
PubMed1995

Crystal structure of NADH oxidase from Thermus thermophilus.

The crystal structures of the flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) containing isoforms of NADH oxidase from Thermus thermophilus have been determined by isomorphous and molecular replacement and refined to 2.3 A and 1.6 A resolution with R-values of 18.5% and 18.6% respectively. The structure of the homodimeric enzyme consists of a central 4-stranded antiparallel beta-sheet covered by helices, a more flexible domain formed by two helices, and a C-terminal excursion connecting the subunits. The active sites are located in a deep cleft between the subunits. The binding site of the flavin cofactor lacks the common nucleotide binding fold and is different from the FMN binding site found in flavodoxins.

Binding Sites↗
PubMed1995

Interaction of mitochondrial elongation factors Tu.Ts with aminoacyl-tRNA.

The interaction between the bovine mitochondrial translational elongation factor Tu.Ts complex (EF-Tu.Tsmt) and aminoacyl-tRNA has been investigated using a nuclease protection assay and fluorescence enhancement of [AEDANS-s2C]Tyr-tRNA(Tyr). The equilibrium dissociation constant, Kd, for the EF-Tu.Tsmt:GTP:E. coli Phe-tRNA complex is approximately 50 nM. A similar binding constant (30 nM) is obtained using bovine mitochondrial Phe-tRNA. The equilibrium binding constant for the EF-Tu.Tsmt:GTP:yeast [AEDANS-s2C]Tyr-tRNA(Tyr) complex is approximately 4 nM when determined using the fluorescence enhancement assay.

Animals↗
PubMed1994

Minimalist aminoacylated RNAs as efficient substrates for elongation factor Tu.

We demonstrate here, using RNA variants derived from tRNAAsp, that the minimalist aminoacylated structure able to interact efficiently with elongation factor Tu comprises a 10 base-pair helix linked to the 3'-terminal NCCA sequence. Shorter structures can interact with the elongation factor, but with significantly decreased affinity. Conserved features in the aminoacyl acceptor branch of tRNAs, such as base pair G53-C61 and the T-loop architecture, could be replaced respectively by the inverted base pair C53-G61 and by unusual anticodon loop or tetraloop sequences. Variants of whole tRNAAsp or of the 12 base-pair aspartate minihelix, with enlarged 13 base-pair long aminoacyl acceptor branches, as in selenocysteine-inserting tRNAs that are not recognized by elongation factor Tu, keep their binding ability to this factor. These functional results are well accounted for by the crystallographic structure of the Thermus thermophilus binary EF-Tu.GTP complex, which possesses a binding cleft accommodating the minimalist 10 base-pair domain of the tRNA aminoacyl acceptor branch.

Acylation↗
PubMed1994

Effector region of the translation elongation factor EF-Tu.GTP complex stabilizes an orthoester acid intermediate structure of aminoacyl-tRNA in a ternary complex.

tRNA(Val) from Escherichia coli was aminoacylated with [1-13C]valine and its complex with Thermus thermophilus elongation factor EF-Tu.GTP was analyzed by 13C NMR spectroscopy. The results suggest that the aminoacyl residue of the valyl-tRNA in ternary complex with bacterial EF-Tu and GTP is not attached to tRNA by a regular ester bond to either a 2'- or 3'-hydroxyl group; instead, an intermediate orthoester acid structure with covalent linkage to both vicinal hydroxyls of the terminal adenosine-76 is formed. Mutation of arginine-59 located in the effector region of EF-Tu, a conserved residue in protein elongation factors and the alpha subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), abolishes the stabilization of the orthoester acid structure of aminoacyl-tRNA.

Binding Sites↗
PubMed1994

Undecagold cluster modified tRNA(Phe) from Escherichia coli and its activity in the protein elongation cycle.

An undecagold cluster (Au11) of molecular mass 6200Da was attached to the 3-(3-amino-3-carboxypropyl)uridine at position 47 of tRNA(Phe) from Escherichia coli. This modified tRNA can be enzymically aminoacylated with phenylalanine in the reaction catalyzed by phenylalanyl-tRNA synthetase. Au11-labeled Phe-tRNA(Phe) forms a ternary complex with the elongation factor Tu.GTP and is active in poly(U)-dependent poly(phe) synthesis. The Au11 modification does not hinder the specific binding of tRNA to distinct ribosomal binding sites or the precise positioning of the aminoacyl and peptidyl residues in the peptidyltransferase center, and does not impair the translocation. The modified tRNA is suitable for the identification of ribosomal binding sites by scanning transmission electron microscopy and for crystallographic studies of the 70S ribosome at different states of the protein-elongation cycle.

Binding Sites↗
PubMed1994

Discrimination against misacylated tRNA by chloroplast elongation factor Tu.

Chloroplast elongation factor Tu was purified from Pisum sativum and the binding properties of glutamylated chloroplast tRNAs were studied by gel-permeation chromatography. Whereas chloroplast Glu-tRNA(Glu) is efficiently bound by this factor, the misacylated Glu-tRNA(Gln) does not interact with chloroplast elongation factor Tu.GTP and is thus efficiently excluded from protein synthesis. Comparison with the behaviour of Escherichia coli elongation factor Tu.GTP shows that this factor, which is not confronted with the in vivo misacylation phenomenon of organelles, binds both Glu-tRNA(Glu) and Glu-tRNA(Gln) from chloroplasts with approximately equal efficiency.

Chloroplasts↗
PubMed1994

Elongation factor Tu: a regulatory GTPase with an integrated effector.

Several elongation factors involved in protein synthesis are GTPases that share structural and mechanistic homology with the large family of proteins including Ras and heterotrimeric receptor-coupled G proteins. The structure of elongation factor Tu (EF-Tu) from thermophilic bacteria, in its 'active' GTP-bound form, has recently been solved by X-ray crystallography. Comparison of this structure with the structure of Escherichia coli EF-Tu bound to GDP reveals a dramatic conformational change that is dependent on GTPase activity. The mechanism of this conformational change and of GTPase activation are discussed, and a model for the EF-Tu-GTP complex with aminoacyl-tRNA is presented.

Amino Acid Sequence↗
PubMed1993

Stability of triple helices containing RNA and DNA strands: experimental and molecular modeling studies.

UV-absorption spectrophotometry and molecular modeling have been used to study the influence of the chemical nature of sugars (ribose or deoxyribose) on triple helix stability. For the Pyrimidine.purine* Pyrimidine motif, all eight combinations were tested with each of the three strands composed of either DNA or RNA. The chemical nature of sugars has a dramatic influence on triple helix stability. For each double helix composition, a more stable triple helix was formed when the third strand was RNA rather than DNA. No stable triple helix was detected when the polypurine sequence was made of RNA with a third strand made of DNA. Energy minimization studies using the JUMNA program suggested that interactions between the 2'-hydroxyl group of the third strand and the phosphates of the polypurine strand play an important role in determining the relative stabilities of triple-helical structures in which the polypyrimidine third strand is oriented parallel to the polypurine sequence. These interactions are not allowed when the third strand adopts an antiparallel orientation with respect to the target polypurine sequence, as observed when the third strand contains G and A or G and T/U. We show by footprinting and gel retardation experiments that an oligoribonucleotide containing G and A or G and U fails to bind double helical DNA, while the corresponding DNA oligomers form stable triple-helical complexes.

Base Sequence↗
PubMed1993

Discrimination between initiation and elongation of protein biosynthesis in yeast: identity assured by a nucleotide modification in the initiator tRNA.

Cytoplasmic initiator tRNAs from plants and fungi possess an unique 2'-phosphoribosyl residue at position 64 of their sequence. In yeast tRNA(iMet), this modified nucleotide located in the T-stem of the tRNA is a 2'-1''-(beta-O-ribofuranosyl-5''-phosphoryl)-adenosine. The phosphoribosyl residue of this modified nucleoside was removed chemically by treatment involving periodate oxidation of tRNA(iMet) and regeneration of the 3'-terminal adenosine with ATP (CTP):tRNA nucleotidyl transferase. The role of phosphoribosylation at position 64 for interaction with elongation factor eEF-1 alpha and initiation factor 2 (eIF-2) was investigated in the homologous yeast system. Whereas the 5'-phosphoribosyl residue prevents the binding of Met-tRNA(iMet) to eEF-1 alpha, it does not influence the interaction with eIF-2. After removal of the ribosyl group, the demodified initiator tRNA showed binding to eEF-1 alpha, but no change was detected with respect to the interaction with the initiation factor eIF-2. This observation is interpreted to mean that a single modification of an eucaryotic initiator tRNA in yeast serves as a negative discriminant for eEF-1 alpha, thus preventing the initiator tRNA(iMet) from entering the elongation cycle of protein biosynthesis.

Chromatography, Affinity↗
PubMed1993

Crystal structure of active elongation factor Tu reveals major domain rearrangements.

The crystal structure of intact elongation factor Tu (EF-Tu) from Thermus thermophilus has been determined and refined at an effective resolution of 1.7 A, with incorporation of data extending to 1.45 A. The effector region, including interaction sites for the ribosome and for transfer RNA, is well defined. Molecular mechanisms are proposed for transduction and amplification of the signal induced by GTP binding as well as for the intrinsic and effector-enhanced GTPase activity of EF-Tu. Comparison of the structure with that of EF-Tu-GDP reveals major mutual rearrangements of the three domains of the molecule.

Computer Graphics↗
PubMed1993

Affinity labeling of c-H-ras p21 consensus elements with periodate-oxidized GDP and GTP.

The amino acid sequence motifs of human c-H-ras p21 involved in the interaction with guanosine nucleotides were cross-linked to in situ periodate-oxidized [alpha-32P]GDP or [alpha-32P]GTP. Site-specific reaction was achieved by cross-linking conserved lysine residues close to the G-nucleotide binding site of p21 with the 2',3'-dialdehyde derivatives of GDP or GTP under kinetically controlled conditions. After endoproteinase Asp-N digestion, HPLC separation of 32P-labeled peptides and N-terminal microsequence analysis, two single lysine residues, namely, K117 and K147, which are parts of the N-K-X-D and S-A-K/L consensus elements of ras proteins, respectively, were identified. No significant divergences in the position and extent of covalent modification could be detected between p21.GDP and p21.GTP. This is in contrast to Thermus thermophilus EF-Tu.GDP and EF-Tu.GTP, which were investigated with the same technique [Peter, M. E., Wittmann-Liebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132-9139] and which exhibited considerable differences in cross-linking efficiency in the GTP form as compared to the GDP form of the protein. The described affinity labeling technique of cross-linking [alpha-32P]GTP with GTP-binding proteins can be used as a general analytical method for the detection and identification of consensus elements in GTPases from different organisms.

Affinity Labels↗
PubMed1993

The 3'-terminal end (NCCA) of tRNA determines the structure and stability of the aminoacyl acceptor stem.

We have done a systematic study on the contribution of the single-stranded NCCA end (where N is any nucleotide) to the stability of the aminoacyl stem of tRNA. A 7-bp RNA duplex with the single-strand ACCA 3' terminus derived from the aminoacyl stem of Escherichia coli tRNA(Ala) and several chemically synthesized sequence variants are characterized by proton NMR and thermodynamic parameters. The single-stranded 3' terminus noticeably stabilizes the duplex in a sequence-dependent manner. Though the largest contribution to the stability gain due to the ACCA end is provided by the first dangling 3' nucleotide, the influence of even the fourth nucleotide is measurable. The nature of the N73 discriminator base influences the stem structure and stability, which may be important for the recognition of tRNA by aminoacyl-tRNA synthetase. The stepwise attachment of the nucleotides to the 3' tail improves the stacking of the unpaired bases over the helix stem. Hence, the ACCA end appears to be structured. Replacing Mg2+ with Mn2+ causes broadening of certain imino proton peaks in the NMR spectrum, indicating a specific divalent metal ion binding site in the vicinity of the major identity element of the duplex (G3-U70) that is required for its recognition by the Ala-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Nucleotide binding and GTP hydrolysis by the 21-kDa product of the c-H-ras gene as monitored by proton-NMR spectroscopy.

Proton-NMR signals in the downfield region (below approximately 10 ppm) have been shown to provide a useful spectroscopic window to monitor the binding of guanine nucleotides to the active site of GTP/GDP-binding proteins via H-bonds, as specified here by the 21-kDa product of the c-H-ras gene (p21). The time course of the intensity change of certain peaks upon addition of GTP to nucleotide-free p21 corresponds to the GTP hydrolysis rate as determined by HPLC. Though there are fewer potential H-bond acceptors in the GDP-bound protein than in the GTP complex, more downfield peaks are found in the former complex, suggesting tighter binding of GDP. Moreover, inspection of the downfield proton-NMR spectra permits rapid detection of subtle changes of the active site induced by complexation with slowly hydrolyzing GTP analogues resulting from mutations of the amino acid sequence, especially in the phosphate binding loop. Our studies strongly suggest that no major conformational change of the phosphate-binding region occurs upon nucleotide complexation that precedes the catalytic step. Besides, it is suspected that the Ser17 hydroxyl group is involved in nucleotide binding and GTP hydrolysis.

Binding Sites↗