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 three 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. 11
Site-Specific Recombination
Transposable elements encode
recombination proteins that catalyze recombination of DNA at specific
sequences. Our structure of a γδ resolvase synaptic tetramer bound to two DNA
duplexes captured in an intermediate state of the recombination process shows a
cleaved DNA substrate covalently linked to the protein, with the ends to be
recombined 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.
Our recent structure of a synaptic tetramer of the homologous Gin recombinase
exhibits a state in which one dimer is in a rotated position relative to the other
dimer when compared with their orientation in γδ resolvase, consistent with the
rotation hypothesis. 12
Transcription Genes encoded in DNA are transcribed into mRNA by
DNA-dependent RNA polymerases that can initiate RNA synthesis at a specific
promoter sequence. To understand this process and its regulation and to explain
how RNA polymerases differ from DNA polymerases, we have determined the crystal
structures of several T7 RNA polymerase complexes with promoter DNAs, mRNAs,
and incoming NTP. These structures show how portions of the RNA polymerase
recognize the bases in the duplex DNA promoter and denature part of the
promoter to form a transcription initiation bubble. In an initiation complex,
three nucleotides of transcript 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,
which eliminates the promoter DNA-binding site on the enzyme and creates a
tunnel through which the transcript exits the enzyme, thus explaining the high
processivity of the elongation phase. Our recent structures of initiation
complexes with either a 7- or 8-nucleotide transcript show intermediates in
this structural transition in which the promoter binding domain rotates by 45°
to accommodate the growing transcript.
The structures
of T7 RNA polymerase elongation complexes captured at each step of nucleotide
incorporation show a 22° rotation of a five-helix subdomain upon NTP binding
and upon pyrophosphate release. The conformational change that accompanies
pyrophosphate release produces both the translocation of the product
heteroduplex and the strand separation of downstream duplex DNA.
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.
We have been
pursuing high-resolution structural studies of the machine that synthesizes
proteins, the ribosome, and have determined the atomic structure of the 1.6-mDa
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 a 13
peptidyltransferase center that is made entirely of RNA.
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. More recently, we have obtained the structure of the 70S ribosome
complexed with fMet-tRNA in the P site and an essential protein factor EF-P
that is seen to be interacting with the tRNA and a rearranged L1 stalk,
suggesting that it may be stimulating the first step of protein synthesis by
correctly positioning the fMet-tRNA in the P site.
We have also
established the structures of nearly two dozen different antibiotics that
target the large ribosomal subunit in complex with the large subunit as well as
complexes with a T. thermophilus 70S ribosome, including two members of
the tuberactinomycin family of antibiotics that are used to treat tuberculosis.
These structures not only establish how these antibiotics stop peptide
synthesis but also are providing the basis for structure-based design of new
antibiotics (by Rib-X Pharmaceuticals, Inc.) that are effective against
ribosomes containing antibiotic-resistance mutations.
Portions of
this work were supported in part by grants from the National Institutes of
Health and the Agouron Institute.
As of February
09, 2011
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