Thomas A. Steitz

Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees.

The 3 angstrom resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with a 30-base pair DNA sequence shows that the DNA is bent by 90 degrees. This bend results almost entirely from two 40 degrees kinks that occur between TG/CA base pairs at positions 5 and 6 on each side of the dyad axis of the complex. DNA sequence discrimination by CAP derives both from sequence-dependent distortion of the DNA helix and from direct hydrogen-bonding interactions between three protein side chains and the exposed edges of three base pairs in the major groove of the DNA. The structure of this transcription factor--DNA complex provides insights into possible mechanisms of transcription activation.

Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism.

The refined crystal structures of the large proteolytic fragment (Klenow fragment) of Escherichia coli DNA polymerase I and its complexes with a deoxynucleoside monophosphate product and a single‐stranded DNA substrate offer a detailed picture of an editing 3′‐5′ exonuclease active site. The structures of these complexes have been refined to R‐factors of 0.18 and 0.19 at 2.6 and 3.1 A resolution respectively. The complex with a thymidine tetranucleotide complex shows numerous hydrophobic and hydrogen‐bonding interactions between the protein and an extended tetranucleotide that account for the ability of this enzyme to denature four nucleotides at the 3′ end of duplex DNA. The structures of these complexes provide details that support and extend a proposed two metal ion mechanism for the 3′‐5′ editing exonuclease reaction that may be general for a large family of phosphoryltransfer enzymes. A nucleophilic attack on the phosphorous atom of the terminal nucleotide is postulated to be carried out by a hydroxide ion that is activated by one divalent metal, while the expected pentacoordinate transition state and the leaving oxyanion are stabilized by a second divalent metal ion that is 3.9 A from the first. Virtually all aspects of the pretransition state substrate complex are directly seen in the structures, and only very small changes in the positions of phosphate atoms are required to form the transition state.

DNA Polymerases: Structural Diversity and Common Mechanisms

Possibly the earliest enzymatic activity to appear in evolution was that of the polynucleotide polymerases, the ability to replicate the genome accurately being a prerequisite for evolution itself. Thus, one might anticipate that the mechanism by which all polymerases work would be both simple and universal. Further, these enzymatic scribes must faithfully copy the sequences of the genome into daughter nucleic acid or the information contained within would be lost; thus some mechanism of assuring fidelity is required. Finally, all classes of polynucleotide polymerases must be able to translocate along the template being copied as synthesis proceeds. The crystal structures of numerous DNA polymerases from different families suggest that they all utilize an identical two-metalioncatalyzed polymerase mechanism but differ extensively in many of their structural features. From amino acid sequence comparisons (1) as well as crystal structure analyses (2), the DNA polymerases can be divided into at least five different families, and representative crystal structures are known for enzymes in four of these families. Perhaps the best studied of these families is the DNA polymerase I (pol I) or A polymerase family, which includes the Klenow fragments of Escherichia coli and a Bacillus DNA polymerase I, Thermus aquaticus DNA polymerase, and the T7 RNA and DNA polymerases, all of whose crystal structures are known (3–11). The second family of DNA-dependent DNA polymerases is DNA polymerase a (pol a) or B family DNA polymerase. All eukaryotic replicating DNA polymerases and the polymerases from phages T4 and RB69 belong to this family, and a crystal structure of the RB69 polymerase shows some similarities to the pol I family enzymes and numerous differences (12). Reverse transcriptases (RT), RNA-dependent RNA polymerases, and telomerase appear to show some common structural similarities, whereas the structure of DNA polymerase b shows no structural relatedness to any of these previous families (13, 14). On the basis of amino acid sequence comparisons but no crystal structures, it appears that the bacterial DNA polymerase III enzymes also form a family that is unrelated to the polymerases of known structure (1). Independent of their detailed domain structures, all polymerases whose structures are known presently appear to share a common overall architectural feature. They have a shape that can be compared with that of a right hand and have been described as consisting of “thumb,” “palm,” and “fingers” domains (15). The function of the palm domain appears to be catalysis of the phosphoryl transfer reaction whereas that of the fingers domain includes important interactions with the incoming nucleoside triphosphate as well as the template base to which it is paired. The thumb on the other hand may play a role in positioning the duplex DNA and in processivity and translocation. Although the palm domain appears to be homologous among the pol I, pol a, and RT families, the fingers and thumb domains are different in all four of these families for which structures are known to date (16). Here the functional and structural similarities and differences among the polymerases of known structure are explored. Of particular interest are the role of editing in the fidelity of copying, the common enzymatic mechanism of polymerases, and the manners in which different domain structures function in the polymerase reaction in analogous ways.

THE KINK-TURN: A NEW RNA SECONDARY STRUCTURE MOTIF

Analysis of the Haloarcula marismortui large ribosomal subunit has revealed a common RNA structure that we call the kink‐turn, or K‐turn. The six K‐turns in H.marismortui 23S rRNA superimpose with an r.m.s.d. of 1.7 Å. There are two K‐turns in the structure of Thermus thermophilus 16S rRNA, and the structures of U4 snRNA and L30e mRNA fragments form K‐turns. The structure has a kink in the phosphodiester backbone that causes a sharp turn in the RNA helix. Its asymmetric internal loop is flanked by C–G base pairs on one side and sheared G–A base pairs on the other, with an A‐minor interaction between these two helical stems. A derived consensus secondary structure for the K‐turn includes 10 consensus nucleotides out of 15, and predicts its presence in the 5′‐UTR of L10 mRNA, helix 78 in Escherichia coli 23S rRNA and human RNase MRP. Five K‐turns in 23S rRNA interact with nine proteins. While the observed K‐turns interact with proteins of unrelated structures in different ways, they interact with L7Ae and two homologous proteins in the same way.

RNA tertiary interactions in the large ribosomal subunit: The A-minor motif

Analysis of the 2.4-Å resolution crystal structure of the large ribosomal subunit from Haloarcula marismortui reveals the existence of an abundant and ubiquitous structural motif that stabilizes RNA tertiary and quaternary structures. This motif is termed the A-minor motif, because it involves the insertion of the smooth, minor groove edges of adenines into the minor groove of neighboring helices, preferentially at C-G base pairs, where they form hydrogen bonds with one or both of the 2′ OHs of those pairs. A-minor motifs stabilize contacts between RNA helices, interactions between loops and helices, and the conformations of junctions and tight turns. The interactions between the 3′ terminal adenine of tRNAs bound in either the A site or the P site with 23S rRNA are examples of functionally significant A-minor interactions. The A-minor motif is by far the most abundant tertiary structure interaction in the large ribosomal subunit; 186 adenines in 23S and 5S rRNA participate, 68 of which are conserved. It may prove to be the universally most important long-range interaction in large RNA structures.

Structure of the Replicating Complex of a Pol α Family DNA Polymerase

Abstract We describe the 2.6 A resolution crystal structure of RB69 DNA polymerase with primer-template DNA and dTTP, capturing the step just before primer extension. This ternary complex structure in the human DNA polymerase α family shows a 60° rotation of the fingers domain relative to the apo-protein structure, similar to the fingers movement in pol I family polymerases. Minor groove interactions near the primer 3′ terminus suggest a common fidelity mechanism for pol I and pol α family polymerases. The duplex product DNA orientation differs by 40° between the polymerizing mode and editing mode structures. The role of the thumb in this DNA motion provides a model for editing in the pol α family.

The Structures of Four Macrolide Antibiotics Bound to the Large Ribosomal Subunit

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.

Crystal Structure of a pol α Family Replication DNA Polymerase from Bacteriophage RB69

Abstract The 2.8 A resolution crystal structure of the bacteriophage RB69 gp43, a member of the eukaryotic pol α family of replicative DNA polymerases, shares some similarities with other polymerases but shows many differences. Although its palm domain has the same topology as other polymerases, except rat DNA polymerase β, one of the three carboxylates required for nucleotidyl transfer is located on a different β strand. The structures of the fingers and thumb domains are unrelated to all other known polymerase structures. The editing 3′–5′ exonuclease domain of gp43 is homologous to that of E. coli DNA polymerase I but lies on the opposite side of the polymerase active site. An extended structure-based alignment of eukaryotic DNA polymerase sequences provides structural insights that should be applicable to most eukaryotic DNA polymerases.

Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2•5 Å resolution

The structure of a dimer of the Escherichia coli catabolite gene activator protein has been refined at 2.5 A resolution to a crystallographic R-factor of 20.7% starting with coordinates fitted to the map at 2.9 A resolution. The two subunits are in different conformations and each contains one bound molecule of the allosteric activator, cyclic AMP. The amino-terminal domain is linked to the smaller carboxy-terminal domain by a nine-residue hinge region that exists in different conformations in the two subunits, giving rise to approximately a 30 degree rotation between the positions of the small domains relative to the larger domains. The amino-terminal domain contains an antiparallel beta-roll structure in which the interstrand hydrogen bonding is well-determined. The beta-roll can be described as a long antiparallel beta-ribbon that folds into a right-handed supercoil and forms part of the cyclic AMP binding site. Each cyclic AMP molecule is in an anti conformation and has ionic and hydrogen bond interactions with both subunits.

Placement of protein and RNA structures into a 5 A-resolution map of the 50S ribosomal subunit.

We have calculated at 5.0 Å resolution an electron-density map of the large 50S ribosomal subunit from the bacterium Haloarcula marismortui by using phases derived from four heavy-atom derivatives, intercrystal density averaging and density-modification procedures. More than 300 base pairs of A-form RNA duplex have been fitted into this map, as have regions of non-A-form duplex, single-stranded segments and tetraloops. The long rods of RNA crisscrossing the subunit arise from the stacking of short, separate double helices, not all of which are A-form, and in many places proteins crosslink two or more of these rods. The polypeptide exit channel was marked by tungsten cluster compounds bound in one heavy-atom-derivatized crystal. We have determined the structure of the translation-factor-binding centre by fitting the crystal structures of the ribosomal proteins L6, L11 and L14, the sarcin–ricin loop RNA, and the RNA sequence that binds L11 into the electron density. We can position either elongation factor G or elongation factor Tu complexed with an aminoacylated transfer RNA and GTP onto the factor-binding centre in a manner that is consistent with results from biochemical and electron microscopy studies.