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1.
Protein Sci ; 10(12): 2577-86, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11714926

ABSTRACT

The variable adherence-associated (Vaa) adhesin of the opportunistic human pathogen Mycoplasma hominis is a surface-exposed, membrane-associated protein involved in the attachment of the bacterium to host cells. The molecular masses of recombinant 1 and 2 cassette forms of the protein determined by a light-scattering (LS) method were 23.9 kD and 36.5 kD, respectively, and corresponded to their monomeric forms. Circular dichroism (CD) spectroscopy of the full-length forms indicated that the Vaa protein has an alpha-helical content of approximately 80%. Sequence analysis indicates the presence of coiled-coil domains in both the conserved N-terminal and antigenic variable C-terminal part of the Vaa adhesin. Experimental results obtained with recombinant proteins corresponding to the N- or C-terminal parts of the shortest one-cassette form of the protein were consistent with the hypothesis of two distinct coiled-coil regions. The one-cassette Vaa monomer appears to be an elongated protein with a axial shape ratio of 1:10. Analysis of a two-cassette Vaa type reveals a similar axial shape ratio. The results are interpreted in terms of the topological organization of the Vaa protein indicating the localization of the adherence-mediating structure.


Subject(s)
Adhesins, Bacterial , Bacterial Proteins/chemistry , Membrane Proteins , Mycoplasma hominis/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Chromatography, Affinity , Circular Dichroism , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Light , Models, Molecular , Molecular Sequence Data , Plasmids/metabolism , Polymerase Chain Reaction , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Scattering, Radiation , Software , Water
2.
Mol Cell ; 8(6): 1375-82, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11779511

ABSTRACT

Bacterial release factor RF2 promotes termination of protein synthesis, specifically recognizing stop codons UAA or UGA. The crystal structure of Escherichia coli RF2 has been determined to a resolution of 1.8 A. RF2 is structurally distinct from its eukaryotic counterpart eRF1. The tripeptide SPF motif, thought to confer RF2 stop codon specificity, and the universally conserved GGQ motif, proposed to be involved with the peptidyl transferase center, are exposed in loops only 23 A apart, and the structure suggests that stop signal recognition is more complex than generally believed.


Subject(s)
Escherichia coli Proteins , Peptide Termination Factors/chemistry , Amino Acid Sequence , Conserved Sequence , Crystallization , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Mimicry , Molecular Sequence Data , Nucleic Acid Conformation , Peptide Termination Factors/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Transfer/chemistry , RNA, Transfer/genetics , RNA, Transfer/metabolism , Sequence Alignment
3.
Mol Cell ; 6(5): 1261-6, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11106763

ABSTRACT

The crystal structure of a complex between the protein biosynthesis elongation factor eEF1A (formerly EF-1alpha) and the catalytic C terminus of its exchange factor, eEF1Balpha (formerly EF-1beta), was determined to 1.67 A resolution. One end of the nucleotide exchange factor is buried between the switch 1 and 2 regions of eEF1A and destroys the binding site for the Mg(2+) ion associated with the nucleotide. The second end of eEF1Balpha interacts with domain 2 of eEF1A in the region hypothesized to be involved in the binding of the CCA-aminoacyl end of the tRNA. The competition between eEF1Balpha and aminoacylated tRNA may be a central element in channeling the reactants in eukaryotic protein synthesis. The recognition of eEF1A by eEF1Balpha is very different from that observed in the prokaryotic EF-Tu:EF-Ts complex. Recognition of the switch 2 region in nucleotide exchange is, however, common to the elongation factor complexes and those of Ras:Sos and Arf1:Sec7.


Subject(s)
Nucleotides/metabolism , Peptide Elongation Factor 1/chemistry , Peptide Elongation Factor 1/metabolism , Protein Biosynthesis , RNA, Transfer/metabolism , Binding Sites , Conserved Sequence , Crystallography, X-Ray , Magnesium/metabolism , Models, Molecular , Nucleotides/genetics , Peptide Elongation Factor Tu/chemistry , Peptide Elongation Factor Tu/metabolism , Pliability , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Transfer/chemistry , RNA, Transfer/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity Relationship
4.
Acta Crystallogr D Biol Crystallogr ; 56(Pt 4): 431-41, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10739916

ABSTRACT

Two examples of the application of single-wavelength anomalous dispersion (SAD) in macromolecular structure determination are described, both using the statistical phasing program SHARP. For the holmium-substituted calcium-binding protein psoriasin (22.7 kDa), a set of accurate phases has been obtained to a resolution of 1.05 A without recourse to an atomic model of the molecule. The accuracy of the phases resulted in an electron-density map of a quality comparable to sigma(A)-weighted 2mF(o) - DF(c) maps derived from the final model refined with SHELX97. Comparison of the refined and SAD electron-density maps showed significant discrepancies resulting from the iterative refinement in reciprocal space. Additionally, it is shown that the structure of psoriasin can be determined from native data extending to 2.0 A alone by exploiting the minute anomalous signal from a bound zinc ion.


Subject(s)
Calcium-Binding Proteins/chemistry , Crystallography, X-Ray/methods , Biomarkers, Tumor/chemistry , Computer Simulation , Holmium , Models, Molecular , Protein Conformation , S100 Calcium Binding Protein A7 , S100 Proteins , Sensitivity and Specificity
5.
EMBO J ; 19(4): 489-95, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10675317

ABSTRACT

Some proteins have been shown to mimic the overall shape and structure of nucleic acids. For some of the proteins involved in translating the genetic information into proteins on the ribosome particle, there are indications that such observations of macromolecular mimicry even extend to similarity in interaction with and function on the ribosome. A small number of structural results obtained outside the protein biosynthesis machinery could indicate that the concept of macromolecular mimicry between proteins and nucleic acids is more general. The implications for the function and evolution of protein biosynthesis are discussed.


Subject(s)
Molecular Mimicry , Animals , GTP Phosphohydrolase-Linked Elongation Factors/chemistry , GTP Phosphohydrolase-Linked Elongation Factors/metabolism , Humans , Macromolecular Substances , Models, Molecular , Nucleic Acid Conformation , Nucleic Acids/chemistry , Nucleic Acids/genetics , Nucleotides/chemistry , Protein Biosynthesis , Protein Conformation , Protein Structure, Tertiary , Proteins/chemistry , Proteins/genetics , RNA, Transfer/chemistry , RNA, Transfer/metabolism
6.
J Cataract Refract Surg ; 26(1): 83-7, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10646152

ABSTRACT

PURPOSE: To compare the effect of 2 contemporary sutureless cataract surgery incisions on corneal astigmatism 1 year after surgery. SETTING: Outpatient Clinic, Department of Ophthalmology, Vejle Hospital, Denmark. METHODS: Sixty-nine patients who had cataract surgery in 1997 with a 4.0 mm temporal clear corneal (n = 32) or superior scleral (n = 37) incision were examined 1 year postoperatively. Surgically induced astigmatism was analyzed by vector analysis, vector decomposition, and polar values using preoperative and postoperative keratometric readings. RESULTS: Vector analysis revealed a slightly lower median induced cylinder in the clear corneal patients; 0.41 diopter (D) (95% confidence limits [CL] 0.24 to 0.67 D) versus 0.61 D (95% CL 0.49 to 0.73 D) (P < .05). Decomposition of the induced cylinder (against the rule/total) showed statistically significant differences in the direction of the cylinder; 0.21 (95% CL 0.03 to 0.41) versus 0.90 (95% CL 0.82 to 0.95) (P < .00001). The directional difference was confirmed by polar values. CONCLUSION: Both incisions induced low and comparable amounts of astigmatism. The directional differences were modest. Findings in previous studies of a poor outcome after clear corneal incisions were not confirmed in this long-term follow-up.


Subject(s)
Astigmatism/physiopathology , Cataract Extraction/adverse effects , Cornea/physiopathology , Cornea/surgery , Sclera/surgery , Aged , Aged, 80 and over , Astigmatism/etiology , Cornea/pathology , Follow-Up Studies , Humans , Lens Implantation, Intraocular , Middle Aged , Minimally Invasive Surgical Procedures , Refraction, Ocular , Retrospective Studies , Treatment Outcome
7.
FEBS Lett ; 452(1-2): 41-6, 1999 Jun 04.
Article in English | MEDLINE | ID: mdl-10376675

ABSTRACT

Protein biosynthesis is controlled by a number of proteins external to the ribosome. Of these, extensive structural investigations have been performed on elongation factor-Tu and elongation factor-G. This now gives a rather complete structural picture of the functional cycle of elongation factor-Tu and especially of the elongation phase of protein biosynthesis. The discovery that three domains of elongation factor-G are structurally mimicking the amino-acylated tRNA in the ternary complex of elongation factor-Tu has been the basis of much discussion of the functional similarities and functional differences of elongation factor-Tu and elongation factor-G in their interactions with the ribosome. Elongation factor-G:GDP is now thought to leave the ribosome in a state ready for checking the codon-anticodon interaction of the aminoacyl-tRNA contained in the ternary complex of elongation factor-Tu. Elongation factor-G does this by mimicking the shape of the ternary complex. Other translation factors such as the initiation factor-2 and the release factor 1 or 2 are also thought to mimic tRNA. These observations raise questions concerning the possible evolution of G-proteins involved in protein biosynthesis.


Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Models, Molecular , Molecular Mimicry , Protein Conformation , Structure-Activity Relationship
8.
Structure ; 7(2): 143-56, 1999 Feb 15.
Article in English | MEDLINE | ID: mdl-10368282

ABSTRACT

BACKGROUND: . The translation elongation factor EF-Tu in its GTP-bound state forms a ternary complex with any aminoacylated tRNA (aa-tRNA), except initiator tRNA and selenocysteinyl-tRNA. This complex delivers aa-tRNA to the ribosomal A site during the elongation cycle of translation. The crystal structure of the yeast Phe-tRNAPhe ternary complex with Thermus aquaticus EF-Tu-GDPNP (Phe-TC) has previously been determined as one representative of this general yet highly discriminating complex formation. RESULTS: The ternary complex of Escherichia coli Cys-tRNACys and T. aquaticus EF-Tu-GDPNP (Cys-TC) has been solved and refined at 2.6 degrees resolution. Conserved and variable features of the aa-tRNA recognition and binding by EF-Tu-GTP have been revealed by comparison with the Phe-TC structure. New tertiary interactions are observed in the tRNACys structure. A 'kissing complex' is observed in the very close crystal packing arrangement. CONCLUSIONS: The recognition of Cys-tRNACys by EF-Tu-GDPNP is restricted to the aa-tRNA motif previously identified in Phe-TC and consists of the aminoacylated 3' end, the phosphorylated 5' end and one side of the acceptor stem and T stem. The aminoacyl bond is recognized somewhat differently, yet by the same primary motif in EF-Tu, which suggests that EF-Tu adapts to subtle variations in this moiety among all aa-tRNAs. New tertiary interactions revealed by the Cys-tRNACys structure, such as a protonated C16:C59 pyrimidine pair, a G15:G48 'Levitt pair' and an s4U8:A14:A46 base triple add to the generic understanding of tRNA structure from sequence. The structure of the 'kissing complex' shows a quasicontinuous helix with a distinct shape determined by the number of base pairs.


Subject(s)
Guanosine Triphosphate/chemistry , Peptide Elongation Factor Tu/chemistry , RNA, Transfer, Cys/chemistry , Thermus/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Crystallography, X-Ray , Escherichia coli/chemistry , GTP Phosphohydrolase-Linked Elongation Factors/chemistry , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , RNA, Bacterial/chemistry , RNA, Transfer, Phe/chemistry , RNA-Binding Proteins/chemistry , Sequence Alignment
9.
Biochemistry ; 38(6): 1695-704, 1999 Feb 09.
Article in English | MEDLINE | ID: mdl-10026247

ABSTRACT

The crystal structure of human psoriasin (S100A7) in the native, calcium-bound form has been determined from two crystal forms of the protein crystallized with and without divalent zinc. The overall structures of the dimeric protein closely resemble the previously determined holmium-substituted structure. The structures also reveal a zinc-binding site of the protein, which is formed by three histidines and an aspartate residue. Together, these residues coordinate the zinc ion in a way similar to the pattern seen in certain metalloproteases and in particular the collagenase family of proteins. Sequence comparison suggests that this zinc site is present in a number of the remaining members of the S100 family. The structure of S100A7 crystallized in the absence of zinc further shows that loss of zinc results in a reorganization of the adjacent empty and distorted EF-hand loop, causing it to resemble a calcium-loaded EF-hand.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium/metabolism , Zinc/metabolism , Amino Acid Sequence , Binding Sites , Calcium/chemistry , Calcium-Binding Proteins/metabolism , Computer Simulation , Crystallization , Crystallography, X-Ray , Humans , Metalloendopeptidases/chemistry , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , S100 Calcium Binding Protein A7 , S100 Proteins/chemistry , S100 Proteins/metabolism , Sequence Alignment , Zinc/chemistry
10.
Sci Prog ; 82 ( Pt 4): 295-312, 1999.
Article in English | MEDLINE | ID: mdl-10701337

ABSTRACT

Metal ions are used in a variety of ways in our cells to regulate, activate, and stabilise specific protein molecules. In this review, we describe some of the regulatory functions of calcium and zinc that have been examined using X-ray crystallographic structural studies of specific proteins. These studies indicate that very precise control of cellular activity can be achieved by exploiting the specific physio-chemical properties of different metal ions.


Subject(s)
Calcium-Binding Proteins/chemistry , Protein Binding/physiology , Zinc/metabolism , Amino Acid Sequence , Binding Sites , Calcium Signaling , Calcium-Binding Proteins/physiology , Humans , Molecular Sequence Data , Protein Conformation
11.
Structure ; 6(4): 477-89, 1998 Apr 15.
Article in English | MEDLINE | ID: mdl-9562557

ABSTRACT

BACKGROUND: The S100 family consists of small acidic proteins, belonging to the EF-hand class of calcium-binding proteins. They are primarily regulatory proteins, involved in cell growth, cell structure regulation and signal transduction. Psoriasin (S100A7) is an 11.7 kDa protein that is highly upregulated in the epidermis of patients suffering from the chronic skin disease psoriasis. Although its exact function is not known, psoriasin is believed to participate in the biochemical response which follows transient changes in the cellular Ca2+ concentration. RESULTS: The three-dimensional structure of holmium-substituted psoriasin has been determined by multiple anomalous wavelength dispersion (MAD) phasing and refined to atomic resolution (1.05 A). The structure represents the most accurately determined structure of a calcium-binding protein. Although the overall structure of psoriasin is similar to those of other S100 proteins, several important differences exist, mainly in the N-terminal EF-hand motif that contains a distorted loop and lacks a crucial calcium-binding residue. It is these minor differences that may account for the different specificities among members of this family. CONCLUSIONS: The structure of human psoriasin reveals that this protein, in contrast to other S100 proteins with known structure, is not likely to strongly bind more than one calcium ion per monomer. The present study contradicts the idea that calcium binding induces large changes in conformation, as suggested by previously determined structures of apo forms of S100 proteins. The substitution of Ca2+ ions in EF-hands by lanthanide ions may provide a general vehicle for structure determination of S100 proteins by means of MAD phasing.


Subject(s)
Calcium-Binding Proteins/chemistry , S100 Proteins , Calcium/metabolism , Dimerization , Epidermis/chemistry , Holmium/chemistry , Humans , Lanthanum/chemistry , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Psoriasis/physiopathology , S100 Calcium Binding Protein A7 , Scattering, Radiation , Sequence Alignment
12.
Fold Des ; 2(3): S7-11, 1997.
Article in English | MEDLINE | ID: mdl-9218959

ABSTRACT

Elongation factor Tu (EF-Tu) is a G-protein which, in its active GTP conformation, protects and carries aminoacylated tRNAs (aa-tRNAs) to the ribosome during protein biosynthesis. EF-Tu consists of three structural domains of which the N-terminal domain consists of two special regions (switch I and switch II) which are structurally dependent on the type of the bound nucleotide. Structural studies of the complete functional cycle of EF-Tu reveal that it undergoes rather spectacular conformational changes when activated from the EF-Tu.GDP form to the EF-Tu.GTP form. In its active form, EF-Tu.GTP without much further structural change interacts with aa-tRNAs in the so-called ternary complex. The conformational changes of EF-Tu involve rearrangements of the secondary structures of both the switch I and switch II regions. As the switch II region forms part of the interface between domains 1 and 3, its structural rearrangement results in a very large change of the position of domain 1 relative to domains 2 and 3. The overall shape of the ternary complex is surprisingly similar to the overall shape of elongation factor G (EF-G). Thus, three domains of the protein EF-G seem to mimic the tRNA part of the ternary complex. This macromolecular mimicry has profound implications for the function of the elongation factors on the ribosome.


Subject(s)
Molecular Mimicry , Protein Biosynthesis , Amino Acid Sequence , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Peptide Chain Elongation, Translational , Peptide Elongation Factor Tu/chemistry , Peptide Elongation Factor Tu/genetics , Peptide Elongation Factor Tu/metabolism , Protein Folding , Protein Structure, Secondary , Sequence Homology, Amino Acid
13.
Methods Enzymol ; 277: 173-208, 1997.
Article in English | MEDLINE | ID: mdl-18488310
14.
Structure ; 4(10): 1141-51, 1996 Oct 15.
Article in English | MEDLINE | ID: mdl-8939739

ABSTRACT

BACKGROUND: Elongation factor Tu (EF-Tu) in its GTP conformation is a carrier of aminoacylated tRNAs (aa-tRNAs) to the ribosomal A site during protein biosynthesis. The ribosome triggers GTP hydrolysis, resulting in the dissociation of EF-Tu-GDP from the ribosome. The affinity of EF-Tu for other molecules involved in this process, some of which are unknown, is regulated by two regions (Switch I and Switch II) that have different conformations in the GTP and GDP forms. The structure of the GDP form of EF-Tu is known only as a trypsin-modified fragment, which lacks the Switch I, or effector, domain. The aim of this work was to establish the overall structure of intact EF-Tu-GDP, in particular the structure of the effector domain. RESULTS: The crystal structures of intact EF-Tu-GDP from Thermus aquaticus and Escherichia coli have been determined at resolutions of 2.7 A and 3.8 A, respectively. The structures confirm the domain orientation previously found in the structure of partially trypsin-digested EF-Tu-GDP. The structures of the effector region in T. aquaticus and E. coli EF-Tu-GDP are very similar. The C-terminal part of the effector region of EF-Tu-GDP is a beta hairpin; in EF-Tu-GTP, this region forms an alpha helix. This conformational change is not a consequence of crystal packing. CONCLUSIONS: EF-Tu undergoes major conformational changes upon GTP hydrolysis. Unlike other GTP-binding proteins, EF-Tu exhibits a dramatic conformational change in the effector region, involving an unwinding of a small helix and the formation of a beta hairpin structure. This change is presumably involved in triggering the release of tRNA, and EF-Tu, from the ribosome.


Subject(s)
Bacterial Proteins/chemistry , Guanosine Diphosphate/chemistry , Peptide Elongation Factor Tu/chemistry , Protein Structure, Secondary , Binding Sites , Computer Simulation , Crystallography , Escherichia coli , Models, Molecular , Molecular Sequence Data , Species Specificity , Thermus
15.
FASEB J ; 10(12): 1347-68, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8903506

ABSTRACT

GTP binding proteins (G-proteins) have wide-ranging functions in biology, being involved in cell proliferation, signal transduction, protein synthesis, and protein targeting. Common to their functioning is that they are active in the GTP-bound form and inactive in the GDP-bound form. The protein synthesis elongation factor EF-Tu was the first G-protein whose nucleotide binding domain was solved structurally by X-ray crystallography to yield a structural definition of the GDP-bound form, but a still increasing number of new structures of G-proteins are appearing in the literature, in both GDP and GTP bound forms. A common structural core for nucleotide binding is present in all these structures, and this core has long been known to include common consensus sequence elements involved in binding of the nucleotide. Nevertheless, subtle changes in the common sequences reflect functional differences. Therefore, it becomes increasingly important to focus on how these differences are reflected in the structures, and how these structural differences are related to function. The aim of this review is to describe to what extent this structural motif for GDP/GTP binding is common to other known structures of this class of proteins. We first describe the common structural core of the G-proteins. Next, examples are based on information available on the Ras protein superfamily, the targeting protein ARF, elongation factors EF-Tu and EF-G, and the heterotrimeric G-proteins. Finally, we discuss the important structures of complexes between GTP binding proteins and their substrates that have appeared in the literature recently.


Subject(s)
Bacterial Proteins , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Guanosine Triphosphate/metabolism , Adenylosuccinate Synthase/chemistry , Animals , Binding Sites , Conserved Sequence , Humans , Membrane Proteins/chemistry , Methyl-Accepting Chemotaxis Proteins , Models, Molecular
16.
Curr Opin Biotechnol ; 7(4): 369-75, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8768893

ABSTRACT

The past year has brought some notable advances in our understanding of the structure and function of elongation factors (EFs) involved in protein biosynthesis. The structures of the ternary complex of aminoacylated tRNA with EF-Tu.GTP and of the complex EF-Tu.EF-Ts have been determined. Within the same period, new cryo-electron microscopy reconstructions of ribosome particles have been obtained.


Subject(s)
Bacterial Proteins/biosynthesis , Peptide Chain Elongation, Translational , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/metabolism , Guanosine Triphosphate/metabolism , Hydrolysis , Models, Biological , Models, Molecular , Peptide Elongation Factor Tu/chemistry , Peptide Elongation Factor Tu/metabolism , Protein Conformation , Ribosomes/chemistry , Ribosomes/ultrastructure
18.
Biochimie ; 78(11-12): 921-33, 1996.
Article in English | MEDLINE | ID: mdl-9150869

ABSTRACT

The refined crystal structure of the ternary complex of yeast Phe-tRNAPhe, Thermus aquaticus elongation factor EF-Tu and the non-hydrolyzable GTP analog, GDPNP, reveals many details of the EF-Tu recognition of aminoacylated tRNA (aa-tRNA). EF-Tu-GTP recognizes the aminoacyl bond and one side of the backbone fold of the acceptor helix and has a high affinity for all ordinary elongator aa-tRNAs by binding to this aa-tRNA motif. Yet, the binding of deacylated tRNA, initiator tRNA, and selenocysteine-specific tRNA (tRNASec) is effectively discriminated against. Subtle rearrangements of the binding pocket may occur to optimize the fit to any side chain of the aminoacyl group and interactions with EF-Tu stabilize the 3'-aminoacyl isomer of aa-tRNA. A general complementarity is observed in the location of the binding sites in tRNA for synthetases and for EF-Tu. The complex formation is highly specific for the GTP-bound conformation of EF-Tu, which can explain the effects of various mutants.


Subject(s)
Guanosine Triphosphate/chemistry , Peptide Elongation Factor Tu/chemistry , Protein Folding , RNA, Transfer, Phe/chemistry , Aspartic Acid , Binding Sites , Crystallography, X-Ray , Guanosine Triphosphate/metabolism , Guanylyl Imidodiphosphate/chemistry , Guanylyl Imidodiphosphate/metabolism , Models, Structural , Peptide Elongation Factor Tu/metabolism , Protein Binding , RNA, Transfer, Phe/metabolism , Saccharomyces cerevisiae/metabolism , Thermus/metabolism
19.
Science ; 270(5241): 1464-72, 1995 Dec 01.
Article in English | MEDLINE | ID: mdl-7491491

ABSTRACT

The structure of the ternary complex consisting of yeast phenylalanyl-transfer RNA (Phe-tRNAPhe), Thermus aquaticus elongation factor Tu (EF-Tu), and the guanosine triphosphate (GTP) analog GDPNP was determined by x-ray crystallography at 2.7 angstrom resolution. The ternary complex participates in placing the amino acids in their correct order when messenger RNA is translated into a protein sequence on the ribosome. The EF-Tu-GDPNP component binds to one side of the acceptor helix of Phe-tRNAPhe involving all three domains of EF-Tu. Binding sites for the phenylalanylated CCA end and the phosphorylated 5' end are located at domain interfaces, whereas the T stem interacts with the surface of the beta-barrel domain 3. The binding involves many conserved residues in EF-Tu. The overall shape of the ternary complex is similar to that of the translocation factor, EF-G-GDP, and this suggests a novel mechanism involving "molecular mimicry" in the translational apparatus.


Subject(s)
Guanosine Triphosphate/analogs & derivatives , Peptide Elongation Factor Tu/chemistry , RNA, Transfer, Amino Acyl/chemistry , Amino Acid Sequence , Anticodon , Base Sequence , Binding Sites , Crystallography, X-Ray , Guanosine Diphosphate/chemistry , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , Histidine/metabolism , Lysine/metabolism , Models, Molecular , Molecular Mimicry , Molecular Sequence Data , Nucleic Acid Conformation , Peptide Elongation Factor G , Peptide Elongation Factor Tu/metabolism , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/metabolism , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , Peptide Termination Factors/chemistry , Peptide Termination Factors/metabolism , Prokaryotic Initiation Factor-2 , Protein Biosynthesis , Protein Conformation , Protein Structure, Secondary , RNA, Transfer, Amino Acyl/metabolism , Ribosomes/metabolism , Thermus
20.
FEBS Lett ; 356(2-3): 165-8, 1994 Dec 19.
Article in English | MEDLINE | ID: mdl-7805830

ABSTRACT

Elongation factor Tu (EF-Tu) is the most abundant protein in prokaryotic cells. Its general function in protein biosynthesis is well established. It is a member of the large family of G-proteins, all of which bind guanosine phosphates (GDP or GTP) as cofactors. In its active GTP bound state EF-Tu binds aminoacylated tRNA (aa-tRNA) forming the ternary complex EF-Tu:GTP:aa-tRNA. The ternary complex interacts with the ribosome where the anticodon on tRNA recognises a codon on mRNA, GTPase activity is induced and inactive EF-Tu:GDP is released. Here we report the successful crystallization of a ternary complex of Thermus aquaticus EF-Tu:GDPNP and yeast Phe-tRNA(Phe) after its purification by HPLC.


Subject(s)
Guanosine Triphosphate/chemistry , Peptide Elongation Factor Tu/chemistry , RNA, Transfer, Phe/chemistry , Chromatography, Gel , Chromatography, High Pressure Liquid , Crystallization , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Guanosine Triphosphate/isolation & purification , Guanosine Triphosphate/metabolism , Guanylyl Imidodiphosphate/metabolism , Peptide Elongation Factor Tu/isolation & purification , Peptide Elongation Factor Tu/metabolism , RNA, Transfer, Phe/isolation & purification , RNA, Transfer, Phe/metabolism , Saccharomyces cerevisiae/metabolism , Thermus/metabolism
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