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ABSTRACT: Transfer RNA nucleotidyltransferases (CCA-adding enzymes) are responsible for the maturation or repair of the functional 3' end of tRNAs by means of the addition of the essential nucleotides CCA. However, it is unclear how tRNA nucleotidyltransferases polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template. Here we describe the crystal structure of the Archaeoglobus fulgidus tRNA nucleotidyltransferase in complex with tRNA. We also present ternary complexes of this enzyme with both RNA duplex mimics of the tRNA acceptor stem that terminate with the nucleotides C74 or C75, as well as the appropriate incoming nucleoside 5'-triphosphates. A single nucleotide-binding pocket exists whose specificity for both CTP and ATP is determined by the protein side chain of Arg 224 and backbone phosphates of the tRNA, which are non-complementary to and thus exclude UTP and GTP. Discrimination between CTP or ATP at a given addition step and at termination arises from changes in the size and shape of the nucleotide binding site that is progressively altered by the elongating 3' end of the tRNA.
Nature 09/2004; 430(7000):640-5. · 36.28 Impact Factor
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ABSTRACT: We have determined the crystal structure of the HIV type 1 reverse transcriptase complexed with CP-94,707, a new nonnucleoside reverse transcriptase inhibitor (NNRTI), to 2.8-A resolution. In addition to inhibiting the wild-type enzyme, this compound inhibits mutant enzymes that are resistant to inhibition by nevirapine, efavirenz, and delaviridine. In contrast to other NNRTI complexes where tyrosines 181 and 188 are pointing toward the enzyme active site, the binding pocket in this complex has the tyrosines pointing the opposite direction, as in the unliganded protein structure, to accommodate CP-94,707. This conformation of the pocket has not been observed previously in NNRTI complexes and substantially alters the shape and surface features that are available for interactions with the inhibitor. One ring of CP-94,707 makes extensive stacking interactions with tryptophan 229, one of the few residues in the NNRTI-binding pocket that cannot readily mutate to give rise to drug resistance. In this conformation of the pocket, mutations of tyrosines 181 and 188 are less likely to disrupt inhibitor binding. Modeling the asparagine mutation of lysine 103 shows that a hydrogen bond between it and tyrosine 188 could form as readily in the CP-94,707 complex as it does in the apoenzyme structure, providing an explanation for the activity of this inhibitor against this clinically important mutant.
Proceedings of the National Academy of Sciences 08/2004; 101(29):10548-53. · 9.68 Impact Factor
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Thomas A Steitz
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ABSTRACT: The RNA polymerase from phage T7 is a 99kDa single polypeptide that is unrelated to the multisubunit cellular RNA polymerases, but exhibits nearly all of their properties. Six separate crystal structures have enhanced our understanding of promoter DNA recognition, duplex DNA opening, and the transition from the abortive initiation phase to the elongation phase. A major conformational change in the N-terminal domain removes the promoter-binding site, accounting for promoter clearance, and creates a tunnel through which the transcript passes, accounting for the processivity of the elongation phase. Structures of substrate and product complexes show that a rotational conformational change of the fingers domain is associated with translocation and downstream strand separation. The rotation that results in translocation is powered by the release of the pyrophosphate product.
Current Opinion in Structural Biology 03/2004; 14(1):4-9. · 9.42 Impact Factor
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ABSTRACT: RNA polymerase functions like a molecular motor that can convert chemical energy into the work of strand separation and translocation along the DNA during transcription. The structures of phage T7 RNA polymerase in an elongation phase substrate complex that includes the incoming nucleoside triphosphate and a pretranslocation product complex that includes the product pyrophosphate (PPi) are described here. These structures and the previously determined posttranslocation elongation complex demonstrate that two enzyme conformations exist during a cycle of single nucleotide addition. One orientation of a five-helix subdomain is stabilized by the phosphates of either the incoming NTP or by the product PPi. A second orientation of this subdomain is stable in their absence and is associated with translocation of the heteroduplex product as well as strand separation of the downstream DNA. We propose that the dissociation of the product PPi after nucleotide addition produces the protein conformational change resulting in translocation and strand separation.
Cell 03/2004; 116(3):393-404. · 32.40 Impact Factor
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ABSTRACT: The structures of DNA polymerases from different families show common features and significant differences that shed light on the ability of these enzymes to accurately copy DNA and translocate. The structure of a B family DNA polymerase from phage RB69 exhibits an active-site closing conformational change in the fingers domain upon forming a ternary complex with primer template in deoxynucleoside triphosphate. The rotation of the fingers domain alpha-helices by 60 degrees upon dNTP binding is analogous to the changes seen in other families of polymerases. When the 3' terminus is bound to the editing 3' exonuclease active site, the orientation of the DNA helix axis changes by 40 degrees and the thumb domain re-orients with the DNA. Structures of substrate and product complexes of T7 RNA polymerase, a structural homologue of T7 DNA polymerase, show that family polymerases use the rotation conformational change of the fingers domain to translocate down the DNA. The fingers opening rotation that results in translocation is powered by the release of the product pyrophosphate and also enables the Pol I family polymerases to function as a helicase in displacing the downstream non-template strand from the template strand.
Philosophical Transactions of The Royal Society B Biological Sciences 02/2004; 359(1441):17-23. · 6.40 Impact Factor
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ABSTRACT: CCA-adding enzymes catalyze the addition of CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template and have been divided into two classes based on their amino acid sequences. We have determined the crystal structures of a class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA) and its complexes with ATP, CTP, or UTP. Although it and the class II bacterial Bacillus stearothermophilus CCA enzyme (BstCCA) have similar dimensions and domain architectures (head, neck, body, and tail), only the polymerase domain is structurally homologous. Moreover, the relative orientation of the head domain with respect to the body and tail domains, which appear likely to bind tRNA, differs significantly between the two enzyme classes. Unlike the class II BstCCA, this enzyme binds nucleotides nonspecifically in the absence of bound tRNA. The shape and electrostatic charge distribution of the AfCCA enzyme suggests a model for tRNA binding that accounts for the phosphates that are protected from chemical modification by tRNA binding to AfCCA. The structures of the AfCCA enzyme and the eukaryotic poly(A) polymerase are very similar, implying a close evolutionary relationship between them.
Molecular Cell 12/2003; 12(5):1165-72. · 14.18 Impact Factor
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ABSTRACT: During translation, tRNAs cycle through three binding sites on the ribosome: the A, the P, and the E sites. We have determined the structures of complexes between the Haloarcula marismortui large ribosomal subunit and two different E site substrates: a deacylated tRNA acceptor stem minihelix and a CCA-acceptor end. Both of these tRNA mimics contain analogs of adenosine 76, the component responsible for a large proportion of E site binding affinity. They bind in the center of the loop-extension of protein L44e, and make specific contacts with both L44e and 23S rRNA including bases that are conserved in all three kingdoms of life. These contacts are consistent with the footprinting, protection, and cross-linking data that have identified the E site biochemically. These structures explain the specificity of the E site for deacylated tRNAs, as it is too small to accommodate any relevant aminoacyl-tRNA. The orientation of the minihelix suggests that it may mimic the P/E hybrid state. It appears that the E site on the 50S subunit was formed by only RNA in the last common ancestor of the three kingdoms, since the proteins at the E sites of H. marismortui and Deinucoccus radiodurans large subunits are not homologous.
RNA 12/2003; 9(11):1345-52. · 5.09 Impact Factor
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ABSTRACT: Knowledge of the number of macromolecules per crystallographic asymmetric unit is frequently useful in the determination of crystal structures. A method has been developed to establish this number directly from measurements of the volume and macromolecular contents of a crystal. The volume of a crystal is determined by measuring the volume of solvent that it displaces in a fine capillary tube. The macromolecular mass contained in a crystal is measured by dissolving the crystal in a known amount of water or suitable buffer and then measuring the UV absorbance of the solution. The method has been tested successfully on three different crystals of known structures.
Acta Crystallographica Section D Biological Crystallography 11/2003; 59(Pt 10):1793-6. · 12.62 Impact Factor
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ABSTRACT: Recently, the atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with substrates have been determined. These have provided exciting new insights into the principles of RNA structure, the mechanism of the peptidyl-transferase reaction and early events in the evolution of this RNA-protein complex assembly that is essential in all cells. The structures of the large subunit bound to a variety of antibiotics explain the effects of antibiotic resistance mutations and provide promise for the development of new antibiotics.
Trends in Biochemical Sciences 09/2003; 28(8):411-8. · 10.85 Impact Factor
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ABSTRACT: Structures of anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bound to the large ribosomal subunit of Haloarcula marismortui have been determined at 3.0A resolution. Most of these antibiotics bind to sites that overlap those of either peptidyl-tRNA or aminoacyl-tRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H.marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA.
Journal of Molecular Biology 08/2003; 330(5):1061-75. · 4.00 Impact Factor
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ABSTRACT: Until recently, there were no examples of RNAs whose structures had been determined by both NMR and x-ray crystallography, and thus there was no experimental basis for assessing the accuracy of RNA solution structures. A comparison of the solution and the crystal structures of two RNAs is presented, which demonstrates that NMR can produce solution structures that resemble crystal structures and thus validates the application to RNA of a methodology developed initially for the determination of protein conformations. Models for RNA solution structures are appreciably affected by the parameters used for their refinement that describe intramolecular interactions. For the RNAs of interest here, the more realistic those parameters, the greater the similarity between solution structures and crystal structures.
05/2003;
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ABSTRACT: Atomic resolution crystal structures of the large subunit published since the middle of August 2000 prove that the peptidyl transferase center of the ribosome, which is the site of peptide-bond formation, is composed entirely of RNA; the ribosome is a ribozyme. They also demonstrate that alignment of the CCA ends of ribosome-bound peptidyl tRNA and aminoacyl tRNA in the peptidyl transferase center contributes significantly to its catalytic power. Several issues remain unresolved. For example, do any components of the site enhance the rate of peptide-bond formation chemically? Do intact ribosomes make peptide bonds the same way as the isolated large subunits that have been the source of all this atomic resolution structural information?
RNA 03/2003; 9(2):155-9. · 5.09 Impact Factor
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ABSTRACT: Cysteinyl-tRNA synthetase is an essential enzyme required for protein synthesis. Genes encoding this protein have not been identified in Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, or Methanopyrus kandleri. It has previously been proposed that the prolyl-tRNA synthetase (ProRS) enzymes in these organisms recognize either proline or cysteine and can aminoacylate their cognate tRNAs through a dual-specificity mechanism. We report five crystal structures at resolutions between 2.6 and 3.2 A: apo M. jannaschii ProRS, and M. thermautotrophicus ProRS in apo form and in complex with cysteinyl-sulfamoyl-, prolyl-sulfamoyl-, and alanyl-sulfamoyl-adenylates. These aminoacyl-adenylate analogues bind to a single active-site pocket and induce an identical set of conformational changes in loops around the active site when compared with the ligand-free conformation of ProRS. The cysteinyl- and prolyl-adenylate analogues have similar, nanomolar affinities for M. thermautotrophicus ProRS. Homology modeling of tRNA onto these adenylate complexes places the 3'-OH of A76 in an appropriate position for the transfer of any of the three amino acids to tRNA. Thus, these structures explain recent biochemical experiments showing that M. jannaschii ProRS misacylates tRNA(Pro) with cysteine, and argue against the proposal that these archaeal ProRS enzymes possess the dual capacity to aminoacylate both tRNA(Pro) and tRNA(Cys) with their cognate amino acids.
Proceedings of the National Academy of Sciences 03/2003; 100(4):1673-8. · 9.68 Impact Factor
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ABSTRACT: The ribosome crystal structures published in the past two years have revolutionized our understanding of ribonucleoprotein structure, and more specifically, the structural basis of the peptide bonding forming activity of the ribosome. This review concentrates on the crystallographic developments that made it possible to solve these structures. It also discusses the information obtained from these structures about the three-dimensional architecture of the large ribosomal subunit, the mechanism by which it facilitates peptide bond formation, and the way antibiotics inhibit large subunit function. The work reviewed, taken as a whole, proves beyond doubt that the ribosome is an RNA enzyme, as had long been surmised on the basis of less conclusive evidence.
Annual Review of Biochemistry 02/2003; 72:813-50. · 34.32 Impact Factor
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ABSTRACT: CCA-adding enzymes polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template. The 3.0 A resolution crystal structures of the CCA-adding enzyme from Bacillus stearothermophilus and its complexes with ATP or CTP reveal a seahorse-shaped subunit consisting of four domains: head, neck, body, and tail. The head is structurally homologous to the palm domain of DNA polymerase beta but has additional structural features and functions. The neck, body, and tail represent new protein folding motifs. The neck provides a specific template for the incoming ATP or CTP, whereas the body and tail may bind tRNA. Each subunit has one active site capable of switching its base specificity between ATP and CTP, an important component of the CCA-adding mechanism.
Cell 01/2003; 111(6):815-24. · 32.40 Impact Factor
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ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates DNA synthesis from the 3' end of human tRNA(Lys3). We have used cis-acting hammerhead ribozymes to produce homogeneous-length transcribed tRNA(Lys3) and have developed conditions for purifying highly structured RNAs on a modified tube-gel apparatus. Titration experiments show that this RNA can assemble into an initiation complex that contains equimolar amounts of HIV-1 RT, transcribed tRNA(Lys3), and chemically synthesized template RNA. We have purified this complex using gel-filtration chromatography and have found that it is homogeneous with respect to molecular weight, demonstrating that the initiation complex forms a single discrete species at micromolar concentrations. When this initiation complex is supplied with deoxynucleotides, essentially all of the tRNA is used as a primer by HIV-1 RT and is fully extended to the 5' end of the template. Thus, in vitro transcribed tRNA can be used efficiently as a primer by HIV-1 RT. We have also obtained crystals of the HIV-1 initiation complex that require the precisely defined ends of this in vitro transcribed tRNA(Lys3) to grow.
Nucleic Acids Research 12/2002; 30(22):4855-63. · 8.03 Impact Factor
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ABSTRACT: To make messenger RNA transcripts, bacteriophage T7 RNA polymerase (T7 RNAP) undergoes a transition from an initiation phase, which only makes short RNA fragments, to a stable elongation phase. We have determined at 2.1 angstrom resolution the crystal structure of a T7 RNAP elongation complex with 30 base pairs of duplex DNA containing a "transcription bubble" interacting with a 17-nucleotide RNA transcript. The transition from an initiation to an elongation complex is accompanied by a major refolding of the amino-terminal 300 residues. This results in loss of the promoter binding site, facilitating promoter clearance, and creates a tunnel that surrounds the RNA transcript after it peels off a seven-base pair heteroduplex. Formation of the exit tunnel explains the enhanced processivity of the elongation complex. Downstream duplex DNA binds to the fingers domain, and its orientation relative to upstream DNA in the initiation complex implies an unwinding that could facilitate formation of the open promoter complex.
Science 12/2002; 298(5597):1387-95. · 31.20 Impact Factor
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ABSTRACT: The large ribosomal subunit catalyzes peptide bond formation and will do so by using small aminoacyl- and peptidyl-RNA fragments of tRNA. We have refined at 3-A resolution the structures of both A and P site substrate and product analogues, as well as an intermediate analogue, bound to the Haloarcula marismortui 50S ribosomal subunit. A P site substrate, CCA-Phe-caproic acid-biotin, binds equally to both sites, but in the presence of sparsomycin binds only to the P site. The CCA portions of these analogues are bound identically by either the A or P loop of the 23S rRNA. Combining the separate P and A site substrate complexes into one model reveals interactions that may occur when both are present simultaneously. The alpha-NH(2) group of an aminoacylated fragment in the A site forms one hydrogen bond with the N3 of A2486 (2451) and may form a second hydrogen bond either with the 2' OH of the A-76 ribose in the P site or with the 2' OH of A2486 (2451). These interactions position the alpha amino group adjacent to the carbonyl carbon of esterified P site substrate in an orientation suitable for a nucleophilic attack.
Proceedings of the National Academy of Sciences 10/2002; 99(18):11670-5. · 9.68 Impact Factor
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ABSTRACT: The ribosome is a particle made of RNA and protein that is found in abundance in all cells that are actively making protein. It catalyses the messenger RNA-directed synthesis of proteins. Recent structural work has demonstrated a profound involvement of the ribosome's RNA component in all aspects of its function, supporting the hypothesis that proteins were added to the ribosome late in its evolution.
Nature 08/2002; 418(6894):229-35. · 36.28 Impact Factor
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ABSTRACT: Crystal structures of the Haloarcula marismortui large ribosomal subunit complexed with the 16-membered macrolide antibiotics carbomycin A, spiramycin, and tylosin and a 15-membered macrolide, azithromycin, show that they bind in the polypeptide exit tunnel adjacent to the peptidyl transferase center. Their location suggests that they inhibit protein synthesis by blocking the egress of nascent polypeptides. The saccharide branch attached to C5 of the lactone rings extends toward the peptidyl transferase center, and the isobutyrate extension of the carbomycin A disaccharide overlaps the A-site. Unexpectedly, a reversible covalent bond forms between the ethylaldehyde substituent at the C6 position of the 16-membered macrolides and the N6 of A2103 (A2062, E. coli). Mutations in 23S rRNA that result in clinical resistance render the binding site less complementary to macrolides.
Molecular Cell 08/2002; 10(1):117-28. · 14.18 Impact Factor
