|
Structural Basis of Replication and Gene Expression
Summary: Thomas Steitz uses the methods of x-ray crystallography and molecular biology to establish the structures and mechanisms of the proteins and nucleic acids involved in gene expression, replication, and recombination.
Our general long-term goal is to determine the detailed molecular mechanisms by which the proteins and nucleic acids involved in the central dogma of molecular biology (DNA replication, transcription, and translation) achieve their biological functions. Virtually all aspects of the maintenance and expression of information stored in the genome involve interactions between proteins and nucleic acids. Over the past two and a half decades we have obtained detailed structural insights into the mechanisms by which specific proteins and nucleic acids catalyze and control the fundamental processes of DNA replication, mRNA synthesis, and protein synthesis, as well as DNA recombination.
DNA Replication
To establish the structural basis of DNA replication, we have been studying DNA polymerases and associated proteins involved in replication. Following on our earlier structures of Escherichia coli DNA polymerase I Klenow fragment and Thermus aquaticus DNA polymerase and their DNA substrate complexes, we established the crystal structure of a replicative DNA polymerase (from phage RB69) that is homologous to the eukaryotic B-family polymerases. The structures of the RB69 polymerase complexed with duplex DNA substrate, bound both at the editing site and the polymerase site, and of the sliding clamp complexed with a polymerase carboxyl-terminal peptide allowed construction of a replisome core structure. These structures showed that this macromolecular machine, charged with the responsibility of faithfully copying the DNA genome, undergoes large conformational changes throughout its catalytic cycle. These changes are associated with the enzyme's fidelity-enhancing mechanisms and translocation.
We have most recently determined the structures of another B-family DNA polymerase, φ29 DNA polymerase, which initiates replication by attaching the first nucleotide of the phage genome to a serine side chain of protein primer called terminal protein, as well as its binary and ternary substrate complexes. The basis of DNA strand displacement activity exhibited by this enzyme is explained by the template strand passing through a tunnel that is too small to accommodate the nontemplate strand that is displaced. The extreme processivity of this polymerase is explained by its topological encirclement of the substrate and product DNA at the active site. The structure of the φ29 DNA polymerase bound to terminal protein provides the first structural insights into the mechanism of protein-primed DNA replication, suggesting that a four-helix domain containing the priming serine must back out of the duplex DNA product-binding site as DNA synthesis proceeds.
We have recently made significant progress in our structural studies of eubacterial replication. The structure of the T. aquaticus DNA polymerase III exhibits no similarity to that of the archaeal or eukaryotic replicating polymerases, but rather possesses a catalytic domain that is homologous to that of DNA polymerase β. The possibility that the last common ancestor had a ribozoyme-replicating polymerase is raised. The structures of the hexomeric DnaB helicase, its complex with the helicase binding domain of primase, and its complex with primase are beginning to illuminate the structural bases of primosome function. (This work was supported in part by a grant from the National Institutes of Health.)
Site-Specific Recombination
Transposable elements encode recombination proteins that catalyze recombination of DNA at specific sequences. We have recently determined the structure of γδ resolvase synaptic tetramer bound to two DNA duplexes captured in an intermediate state of the recombination process. The DNA substrate is cleaved and covalently linked to the protein, but the ends to be recombined are separated by 50 Å. The very flat interface between the protein dimers linked to the DNAs to be recombined suggests that recombination is achieved by an unprecedented 180° rotation of one dimer relative to the other. (This work was supported in part by a grant from the National Institutes of Health.)
Transcription
Genes encoded in the DNA are transcribed into messenger RNA by DNA-dependent RNA polymerases that can initiate RNA synthesis at a specific DNA sequence, the promoter. To understand this process and its regulation and to explain how RNA polymerases differ from DNA polymerases, we have determined the crystal structures of T7 RNA polymerase complexed with a transcriptional inhibitor, T7 lysozyme, and several complexes with promoter DNAs, messenger RNAs, and incoming nucleoside triphosphate. These structures show how portions of the RNA polymerase recognize the bases in the duplex DNA promoter and how the enzyme denatures part of the promoter to form a transcription initiation bubble. In an initiation complex, three nucleotides of messenger RNA are seen base-paired to the template strand. We have also captured this polymerase in a transcription elongation phase as a complex with 30 base pairs of DNA and a 17-nucleotide RNA transcript. The transition from the initiation to the elongation phases of transcription is accompanied by a massive structural rearrangement of the amino-terminal domain; this structural change eliminates the promoter DNA-binding site on the enzyme and creates a tunnel through which the transcript exits the enzyme, thus explaining the observed high processivity of the elongation phase.
We have now shown how the free energy available from nucleotide incorporation is structurally coupled to translocation and DNA strand displacement. The structure of T7 RNA polymerase elongation complexes captured at each step of nucleotide incorporation shows a 22° rotation of a five-helix subdomain upon nucleoside triphosphate binding and upon pyrophosphate release. The conformational change that results from pyrophosphate release produces both the translocation of the product heteroduplex and the strand separation of downstream duplex DNA. (This work was supported in part by a grant from the National Institutes of Health.)
Translation
Our structural studies of the proteins and nucleic acids involved in translating the gene sequence carried in the messenger RNA into the protein products are providing insights into the translation of the genetic code. This includes our earlier structural studies of aminoacyl-tRNA synthetases, as well as more recent structural studies explaining how the CCA-adding enzyme is able to mature or repair the 3' CCA end of tRNA without using a nucleic acid template. We have established the structures of the CCA-adding enzyme captured in the steps of adding penultimate C and final A as well as the product tRNA. The enzyme active site changes shape in response to the growing terminus and uses a protein Arg and tRNA backbone phosphates as a template for the incoming nucleotide.
We have been pursuing high-resolution structural studies of the machine that synthesizes proteins, the ribosome. We have determined the atomic structure of the large (molecular weight 1.6 million) ribosomal subunit that catalyzes the formation of the peptide bond. The structures of the large subunit with either substrate or product analogs bound to the active site of peptide synthesis show the peptidyltransferase center that is made entirely of RNA. The ribosome is in fact an RNA enzyme, a ribozyme. Ribosomal RNA positions the substrate α-amino group appropriately for attack of the peptidyl-tRNA, and it also interacts with the latter's A76 2'-OH group, which may function as a proton shuttle between the α-amino group and the A76 3'-OH. These observations prove that RNA enzymes preceded protein enzymes in evolution, since the machine that makes proteins must have predated proteins.
We have also established the structures of more than a dozen different antibiotics that target the large ribosomal subunit in complex with the large subunit. These structures not only establish how these antibiotics stop peptide synthesis but also provide the basis for structure-based design of new antibiotics that will be effective against ribosomes containing antibiotic-resistance mutations. (This work was supported in part by grants from the National Institutes of Health and the Agouron Institute.)
Last updated: December 3, 2007
|
