PubMed Health⌕ Search

Biomedical subjects

M Sprinzl

Publications and source records attributed to M Sprinzl.

At least 19 recordsLinked to original sources
PubMed1998

In vitro selected RNA molecules that bind to elongation factor Tu.

RNA molecules which bind to elongation factor Tu from T. thermophilus were isolated from a pool of ribooligonucleotides with a randomized sequence region. These RNAs interact with elongation factor Tu in both the GTP and the GDP form. A slight preference for the GTP form of the protein was observed. The isolated RNA aptamers compete with each other for a common binding site on elongation factor Tu. This binding site is different from the binding site for aminoacyl-tRNA or the binding site for elongation factor Ts and is located on domain II of elongation factor Tu. The selected RNAs do not bind to elongation factor G. The EF-Tu binding RNAs share a short consensus sequence, 5'-ACCGAAG-3', which was also found in the alpha-sarcin domain of T. thermophilus23S rRNA. The isolated RNAs have a hairpin structure with the 5'-ACCGAAG-3' sequence located in non-base-paired regions. Chemical probing and deletion experiments indicate that the consensus sequence is required for the interaction with elongation factor Tu.

Base Sequence↗
PubMed1998

Compilation of tRNA sequences and sequences of tRNA genes.

Sequences of 3279 sequences of tRNA genes and tRNAs published up to December 1996 are included in the compilation. Alignment of the sequences, which is most compatible with the tRNA phylogeny and known three-dimensional structures of tRNA, is used. Sequences and references are available under http://www.uni-bayreuth. de/departments/biochemie/trna/

Animals↗
PubMed1997

Interaction of N-tosyl-L-phenylalanylchloromethane with Thermus thermophilus elongation factor Tu.

The interaction of N-tosyl-L-phenylalanylchloromethane (TosPheCH2Cl) with Thermus thermophilus elongation factor Tu (EF-Tu) was studied by affinity labelling and NMR spectroscopy. TosPheCH2Cl binds to GDP and GTP conformers of EF-Tu. The interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of Cys82, while interaction of TosPheCH2Cl with EF-Tu x GTP does not lead to covalent labelling. [A82]EF-Tu, in which the Cys82 is replaced by Ala, has similar properties to wild-type EF-Tu with respect to GTPase activity, binding of guanine nucleotides, interaction with elongation factor Ts (EF-Ts) and interaction with ribosomes. This structural change did not lead to changes, compared with wild-type EF-Tu in the functionality of [A82]EF-Tu, either in the GTP or in the GDP conformation. TosPheCH2Cl binds to EF-Tu x GTP with a dissociation constant of 10 microM. The interaction of TosPheCH2Cl with EF-Tu promotes the hydration of the carbonyl group of TosPheCH2Cl. TosPheCH2Cl competes with aminoacyl-tRNA for its binding site on EF-Tu x GTP. Covalent modification of Cys82 by TosPheCH2Cl does not prevent nucleotide binding and GTPase activity, but interferes with the interaction with aminoacyl-tRNA. TosPheCH2Cl probably mimics the aminoacyl residue of the aminoacyl-tRNA and binds to its binding site on EF-Tu x GTP. This rather specific interaction with EF-Tu x GTP does not allow the modification of Cys82, whereas the loose interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of this residue.

Affinity Labels↗
PubMed1997

The structure of 3'-O-anthraniloyladenosine, an analogue of the 3'-end of aminoacyl-tRNA.

3'-O-Anthraniloyladenosine, an analogue of the 3'- terminal aminoacyladenosine residue in aminoacyl-tRNAs, was prepared by chemical synthesis, and its crystal structure was determined. The sugar pucker of 3'-O-anthraniloyladenosine is 2'-endo resulting in a 3'-axial position of the anthraniloyl residue. The nucleoside is insynconformation, which is stabilized by alternating stacking of adenine and benzoyl residues of the neighboring molecules in the crystal lattice. The conformation of the 5'-hydroxymethylene in 3'-O- anthraniloyladenosine is gauche-gauche. There are two intramolecular and two intermolecular hydrogen bonds and several H-bridges with surrounding water molecules. The predominant structure of 3'-O-anthraniloyladenosine in solution, as determined by NMR spectroscopy, is 2'-endo,gauche-gauche and anti for the sugar ring pucker, the torsion angle around the C4'-C5'bond and the torsion angle around the C1'-N9 bond, respectively. The 2'-endo conformation of the ribose in 2'(3')-O-aminoacyladenosine, which places the adenine and aminoacyl residues in equatorial and axial positions, respectively, could serve as a structural element that is recognized by enzymes that interact with aminoacyl-tRNA or by ribosomes to differentiate between aminoacylated and non-aminoacylated tRNA.

Acylation↗
PubMed1997

Identification and purification of translation initiation factor 2 (IF2) from Thermus thermophilus.

Translation initiation factor 2 (IF2) is one of three protein factors required for initiation of protein synthesis in eubacteria. The protein is responsible for binding the initiator RNA to the ribosomal P site. IF2 is a member of the GTP GDP-binding protein superfamily. In the extreme thermophilic bacterium Thermus thermophilus, IF2 was identified as a 66-kDa protein by affinity labeling and immunoblotting. The protein was purified to homogeneity. The specific activity indicates a stoichiometric IF2-mediated binding of formylmethionine-tRNA to 70S ribosomes. The N-terminal amino acid sequences of the intact protein and of two proteolytic fragments of 25 kDa and 40 kDa were determined. Comparison with other bacterial IF2 sequences indicates a similar domain architecture in all bacterial IF2 proteins.

Amino Acid Sequence↗
PubMed1997

Organization of the Thermus thermophilus nusA/infB operon and overexpression of the infB gene in Escherichia coli.

The structural gene for translation initiation factor IF2 from Thermus thermophilus was identified on the basis of the N-terminal amino acid sequence of intact T thermophilus IF2 and an internal 25 kDa IF2 fragment. A total of 5135 bp was cloned and sequenced, comprising the open reading frames for p15A, NusA, p10A, IF2, p10B and SecD, which may form an operon. There are pronounced similarities between the operon arrangement and primary sequence of the T thermophilus genes and proteins, respectively, and their counterparts from other organisms. The T thermophilus infB gene was expressed to a high level in E coli. Four hundred milligrams of homogenous T thermophilus IF2 were prepared from 60 g of overproducing cells.

Amino Acid Sequence↗
PubMed1997

Crystal structure of the EF-Tu.EF-Ts complex from Thermus thermophilus.

In order to study nucleotide exchange mechanisms in GTP-binding proteins, we have determined the crystal structure of the complex formed by the elongation factor Tu (EF-Tu) and its exchange factor Ts (EF-Ts) from Thermus thermophilus. The complex is a dyad symmetrical heterotetramer in which each EF-Tu, through a bipartite interface, interacts with two subunits of EF-Ts, explaining the need for a dimeric exchange factor. The architecture of the assembly is distinctly different from that of the corresponding heterodimeric E. coli complex, in which the monomeric E. coli EF-Ts remarkably forms essentially the same bipartite interface with EF-Tu through a sequence/structural repeat. GDP is released primarily by a Ts-induced peptide flip in the nucleotide binding pocket that disrupts hydrogen bonds to the phosphates and repositions the peptide carbonyl so as to sterically and electrostatically eject the GDP. The exchange mechanism may have useful implications for receptor-induced exchange in heterotrimeric G proteins.

Amino Acid Sequence↗
PubMed1997

Ni2+-binding RNA motifs with an asymmetric purine-rich internal loop and a G-A base pair.

RNA molecules with high affinity for immobilized Ni2+ were isolated from an RNA pool with 50 randomized positions by in vitro selection-amplification. The selected RNAs preferentially bind Ni2+ and Co2+ over other cations from first series transition metals. Conserved structure motifs, comprising about 15 nt, were identified that are likely to represent the Ni2+ binding sites. Two conserved motifs contain an asymmetric purine-rich internal loop and probably a mismatch G-A base pair. The structure of one of these motifs was studied with proton NMR spectroscopy and formation of the G-A pair at the junction of helix and internal loop was demonstrated. Using Ni2+ as a paramagnetic probe, a divalent metal ion binding site near this G-A base pair was identified. Ni2+ ions bound to this motif exert a specific stabilization effect. We propose that small asymmetric purine-rich loops that contain a G-A interaction may represent a divalent metal ion binding site in RNA.

Adenine↗
PubMed1996

Structure and importance of the dimerization domain in elongation factor Ts from Thermus thermophilus.

Elongation factor Ts (EF-Ts) functions as a nucleotide-exchange factor by binding elongation factor Tu (EF-Tu) and accelerating the GDP dissociation from EF-Tu; thus EF-Ts promotes the transition of EF-Tu from the inactive GDP form to the active GTP form. Thermus thermophilus EF-Ts exists as a stable dimer in solution which binds two molecules of EF-Tu to form a (EF-Tu.EF-Ts)2 heterotetramer. Here we report the crystal structure of the dimerization domain of EF-Ts from T. thermophilus refined to 1.7 A resolution. A three-stranded antiparallel beta-sheet from each subunit interacts to form a beta-sandwich that serves as an extensive dimer interface tethered by a disulfide bond. This interface is distinctly different from the predominantly alpha-helical one that stabilizes the EF-Ts dimer from Escherichia coli [Kawashima, T., et al. (1996) Nature 379, 511-518]. To test whether the homodimeric form of T. thermophilus EF-Ts is necessary for catalyzing nucleotide exchange, the present structure was used to design mutational changes within the dimer interface that disrupt the T. thermophilus EF-Ts dimer but not the tertiary structure of the subunits. Surprisingly, EF-Ts monomers created in this manner failed to catalyze nucleotide exchange in EF-Tu, indicating that, in vitro. T. thermophilus EF-Ts functions only as a homodimer.

Amino Acid Sequence↗
PubMed1996

Limited proteolysis and amino acid replacements in the effector region of Thermus thermophilus elongation factor Tu.

The effector region of the elongation factor Tu (EF-Tu) from Thermus thermophilus was modified by limited proteolysis or via site-directed mutagenesis. The biochemical properties of the obtained EF-Tu variants were investigated with respect to partial reactions of the functional cycle of EF-Tu. EF-Tu that was cleaved at the Arg59-Gly60 peptide bond [EF-Tu-(1-59)/EF-Tu-(60-405)] bound GDP, EF-Ts and aminoacyl-tRNA, had normal intrinsic GTPase activity and was active in poly(U)-dependent poly(Phe) synthesis. However, the GTPase activity of EF-Tu-(1-59)/EF-Tu-(60-405) was not stimulated by T. thermophilus 70S ribosomes, and its GTP-dissociation rate was increased compared with that of intact EF-Tu. EF-Tu cleaved at the Lys52-Ala53 peptide bond has properties similar to EF-Tu-(1-59)/EF-Tu-(60-405). By means of site-directed mutagenesis, Glu55 was replaced by Leu, Glu56 by Ala and Arg59 by Thr in T. thermophilus EF-Tu. These amino acid substitutions did not substantially affect either the affinity of EF-Tu. GTP for aminoacyl-tRNA or the interactions with GDP, GTP or EF-Ts. Similarly the intrinsic GTPase activity is not influenced. Replacement of Glu56 by Ala led to strong reduction in the ribosome-induced GTPase activity. This effect is specific since replacement of the neighbouring Glu55 by Leu did not affect the ribosome-induced GTPase activity. The results demonstrate that the structure of the effector region of EF-Tu in the vicinity of Arg59 is important for the control of the GTPase activity by ribosomes.

Amino Acid Sequence↗
PubMed1996

NMR evidence for helix geometry modifications by a G-U wobble base pair in the acceptor arm of E. coli tRNA(Ala).

A ribooligonucleotide duplex representing the acceptor stem of E. coli RNA(Ala) with a G3-U70 wobble base pair, which is the main identity element for the recognition by the alanine-tRNA synthetase, has been characterized by 2D-NMR, as having two sequence variants with a regular Watson-Crick G3-C70 and an I3-U70 wobble pair, respectively. As compared to a regular A-RNA, the G-U base pair gives rise to variations of the local helix geometry which are reflected in distinct local chemical shift changes. Structural differences between the duplex possessing an I3-U70 base pair and the wild-type G3-U70 sequence have also been found. The nucleotides in the ubiquitous single-stranded NCCA terminus display a surprisingly high degree of stacking order, especially between A73, C74, and C75.

Base Composition↗
PubMed1996

Elongation factor Ts from Thermus thermophilus-- overproduction in Escherichia coli, quaternary structure and interaction with elongation factor Tu.

The gene encoding the elongation factor Ts from Thermus thermophilus was sequenced, cloned and the protein overproduced in Escherichia coli. In comparison to the EF-Ts from E. coli with 282 amino acid residues, EF-Ts from T. thermophilus is considerably shorter, differing by 86 amino acids. EF-Ts from the thermophile is stable at high temperatures, which facilitates its separation from E. coli proteins. Purified T. thermophilus EF-Ts forms a homodimer with a disulfide bridge between the two cysteine residues at position 190. The modification of Cys19O by iodoacetamide affects neither the dimerization nor the ability of EF-Ts to facilitate the nucleotide exchange of elongation factor Tu. The disulfide bridge was detected only in purified EF-TS, but not in protein extracts immediately after cell disruption. The physiological role of this disulfide bridge remains, therefore, unclear. Besides the quaternary (EF-TU . EF-Ts)2 complex, a ternary EF-TU . EF-Ts2 complex was detected by gel permeation chromatography and polyacrylamide gel electrophoresis. Trypsin cleavage after Lys48 or modification of Cys78 yield inactive EF-Ts, that does not bind to EF-Tu but is still capable of forming homodimers.

Amino Acid Sequence↗
PubMed1996

Antideterminants present in minihelix(Sec) hinder its recognition by prokaryotic elongation factor Tu.

During protein biosynthesis, all aminoacylated elongator tRNAs except selenocysteine-inserting tRNA Sec form ternary complexes with activated elongation factor. tRNA Sec is bound by its own translation factor, an elongation factor analogue, e.g. the SELB factor in prokaryotes. An apparent reason for this discrimination could be related to the unusual length of tRNA Sec amino acid-acceptor branch formed by 13 bp. However, it has been recently shown that an aspartylated minihelix of 13 bp derived from yeast tRNA Asp is an efficient substrate for Thermus thermophilus EF-Tu-GTP, suggesting that features other than the length of tRNA Sec prevent its recognition by EF-Tu-GTP. A stepwise mutational analysis of a minihelix derived from tRNA Sec in which sequence elements of tRNA Asp were introduced showed that the sequence of the amino acid- acceptor branch of Escherichia coli tRNA Sec contains a specific structural element that hinders its binding to T.thermophilus EF-Tu-GTP. This antideterminant is located in the 8th, 9th and 10th bp in the acceptor branch of tRNA Sec, corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem. The function of this C7.G66/G49.U65/C50.G64 box was tested by its transplantation into a minihelix derived from tRNA Asp, abolishing its recognition by EF-Tu-GTP. The specific role of this nucleotide combination is further supported by its absence in all known prokaryotic elongator tRNAs.

Base Sequence↗
PubMed1996

Conservation in evolution for a small monomeric phenylalanyl-tRNA synthetase of the tRNA(Phe) recognition nucleotides and initial aminoacylation site.

We previously showed that yeast mitochondrial phenylalanyl-tRNA synthetase (MSF protein) is evolutionarily distant to the cytoplasmic counterpart based on a high degree of divergence in protein sequence, molecular mass, and quaternary structure. Using yeast cytoplasmic tRNA(Phe) which is efficiently aminoacylated by MSF protein, we report here the tRNA(Phe) primary site of aminoacylation and the identity determinants for MSF protein. As for the cytoplasmic phenylalanyl-tRNA synthetase (Sampson, J. R., Di Renzo, A. B., Behlen, L. S., & Uhlenbeck, O. C. (1989) Science 243, 1363-1366), MSF protein recognizes nucleotides from the anticodon and the acceptor end including base A73 and, as shown here, adjacent G1-C72 base pair or at least C72 base. This indicates that the way of tRNA(Phe) binding for the two phenylalanine enzymes is conserved in evolution. However, tRNA(Phe) tertiary structure seems more critical for the interaction with the cytoplasmic enzyme than with MSF protein, and unlike cytoplasmic phenylalanyl-tRNA synthetase, the small size of the monomeric MSF protein probably does not allow contacts with residue 20 at the top corner of the L molecule. We also show that MSF protein preferentially aminoacylates the terminal 2'-OH group of tRNA(Phe) but with a catalytic efficiency for tRNA(Phe)-CC-3'-deoxyadenosine reduced 100-fold from that of native tRNA(Phe), suggesting a role of the terminal 3'-OH in catalysis. The loss is only 1.5-fold when tRNA(Phe)-CC-3'-deoxyadenosine is aminoacylated by yeast cytoplasmic PheRS (Sprinzl, M., & Cramer, F. (1973) Nature 245, 3-5), indicating mechanistic differences between the two PheRS's active sites for the amino acid transfer step.

Amino Acid Sequence↗
PubMed1995

Site-directed mutagenesis of Thermus thermophilus EF-Tu: the substitution of threonine-62 by serine or alanine.

The invariant threonine-62, which occurs in the effector region of all GTP/GDP-binding regulatory proteins, was substituted via site-directed mutagenesis by alanine and serine in the elongation factor Tu from Thermus thermophilus. The altered proteins were overproduced in Escherichia coli, purified and characterized. The EF-Tu T62S variant had similar properties with respect to thermostability, aminoacyl-tRNA binding, GTPase activity and in vitro translation as the wild-type EF-Tu. In contrast, EF-Tu T62A is severely impaired in its ability to sustain polypeptide synthesis and has only very low intrinsic and ribosome-induced GTPase activity. The affinity of aminoacyl-tRNA to the EF-Tu T62A.GTP complex is almost 40 times lower as compared to the native EF-Tu.GTP. These observations are in agreement with the tertiary structure of EF-Tu.GTP, in which threonine-62 is interacting with the Mg2+ ion, gamma-phosphate of GTP and a water molecule, which is presumably involved in the GTP hydrolysis.

Alanine↗
PubMed1995

Properties of isolated domains of the elongation factor Tu from Thermus thermophilus HB8.

The relative contributions of the three domains of elongation factor Tu (EF-Tu) to the factor's function and thermal stability were established by dissecting the domains apart with recombination techniques. Domain I (EF-TuI), domains I/II (EF-TuI/II) and domain III (EF-TuIII) of the EF-Tu from Thermus thermophilus HB8 comprising the amino acids 1-211, 1-312 and 317-405, respectively, were overproduced in Escherichia coli and purified. A polypeptide consisting of domain II and III (EF-TuII/III) was prepared by limited proteolysis of native EF-Tu with V8 protease from Staphylococcus aureus [Peter, M. E., Reiser, C. O. A., Schirmer, N. K., Kiefhaber, T., Ott, G., Grillenbeck, N. W. & Sprinzl, M. (1990) Nucleic Acids Res. 18, 6889-6893]. As determined by circular dichroism spectrometry, the isolated domains have the secondary structure elements found in the native EF-Tu. GTP and GDP binding as well as GTPase activity are maintained by the EF-TuI and EF-TuI/II; however, the rate of GDP dissociation from EF-TuI . GDP and EF-TuI/II . GDP complex is increased as compared to native EF-Tu . GDP, reflecting a constraint imposed by domain III on the ability to release the nucleotide from its binding pocket located in domain I. A weak interaction of Tyr-tRNATyr with the EF-TuI . GTP suggests that domain I provides a part of the structure interacting with aminoacyl-tRNA. The domain III is capable of regulating the rate of GTPase in EF-Tu, since the polypeptide consisting only of domains I/II has a 39-fold higher intrinsic GTPase compared to the native EF-Tu. No in vitro poly(U)-dependent poly(Phe) synthesis was detectable with a mixture of equimolar amounts of domains I/II and domain III, demonstrating the necessity of covalent linkage between the domains of EF-Tu for polypeptide synthesis. In contrast to native EF-Tu and EF-TuII/III, EF-TuI and, to a lesser extent the polypeptide consisting of domains I/II, are unstable at elevated temperatures. This indicates that domains II/III strongly contribute to the thermal stability of this T. thermophilus EF-Tu. Deletion of amino acid residues 181-190 from domain I of T. thermophilus EF-Tu decreases the thermostability to that of EF-Tu from E. coli, which does not have these residues. Interdomain interactions must be important for the stabilisation of the structure of domain I, since isolated T. thermophilus EF-TuI is thermolabile despite the presence of the 181-190 loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Interaction of guanosine nucleotides and their analogs with elongation factor Tu from Thermus thermophilus.

Transient kinetic experiments on the interaction of nucleotide-free EF-Tu from Thermus thermophilus with nucleotides using intrinsic protein fluorescence, extrinsic nucleotide fluorescence and fluorescence resonance energy transfer show that nucleotide binding is in general at least a two-step process. The first step is a weak initial binding, which is followed by a relatively slow isomerization of the protein-nucleotide complex in which changes of both intrinsic and extrinsic fluorescence, as well as energy transfer, occur. The values obtained for the equilibrium and kinetic constants confirm the earlier observation that EF-Tu has a higher affinity for GDP than GTP. This is mainly due to a lower dissociation rate constant for GDP, in combination with a somewhat higher effective association rate constant. Modifications of the triphosphate moiety of GTP are quite well tolerated by EF-Tu, with GTP gamma S displaying the same affinity as GTP and with GppNHp and GppCH2p being only ca. 2-3-fold less strongly bound. Caged GTP is bound about 6-fold more weakly than GTP. These results suggest that the binding of GppNHp and GppCH2p is likely to be similar to that of GTP. The photolytic protecting group of caged GTP (or the loss of one of the negative charges on the gamma-phosphate group) appears to interfere to a certain extent with the interaction with the protein, but the affinity is high enough to permit generation of 1:1 complexes for dynamic structural studies. Discrimination between GDP and ADP is dramatic, with a difference of 6 orders of magnitude in affinity.

Adenosine Diphosphate↗