Our general goal is to understand the biological functions of macromolecules in terms of their detailed molecular structure. Of particular interest are the molecular mechanisms by which those proteins and nucleic acids involved in the central dogma of molecular biology (DNA replication, transcription, translation and genetic recombination) achieve their biological function. Virtually all aspects of the maintenance, rearrangement and expression of information stored in the genome involve interactions between proteins and nucleic acids.
Our recent accomplishments have included the determination of the atomic structure of the 50S
ribosomal subunit and its complexes with substrate, intermediate and product analogues as well
as complexes with more than a dozen antibiotics. These structures establish that the ribosome
is a ribozyme, provide insights into the mechanism of peptide bond formation and show how several
classes of antibiotics function. In the area of transcription, six structures of T7 RNA polymerase
captured in various functional states show the structural basis of the transition from the initiation
to elongation phase, which involves a large protein structural rearrangement. They explain the basis
of promoter clearance, processivity of the elongation phase, translocation and strand separation.
The structures of the CCA-adding enzyme captured in each state of CCA incorporation onto tRNA explain
the enzyme's specificity for nucleotide incorporation in the absence of a nucleic acid template.
The structure of a recombination intermediate of γδ resolvase suggests that site specific recombination
by this enzyme is achieved by subunit rotation.
A space filling representation of the 50S subunit (RNA in white and protein in yellow) with the 3 tRNA molecules from the 70S complex superimposed. The structure has been split through the tunnel.
Future directions will focus on the complex macromolecular assemblies
that are the functional machines in these processes, including the ribosome and the replisome.
For example, we wish to establish the atomic structures of the ribosome captured in the act of protein
synthesis in each of its conformational states with elongation factors as well as interacting with the
proteins involved in protein secretion. Likewise, a mechanistic understanding of replication and
recombination will require structures of the assemblies in each step of their functioning. Hypotheses
arising from these structures will be tested using site directed mutagenesis and biochemical studies to
relate structure to function.
50S Ribosomal Subunit of Haloarcula Marismortui
Ban, N. Nissen, P., Hansen, J., Moore, P.B. and Steitz, T.A. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289: 905-920 (2000).
Yin, Y.W. and Steitz, T.A. Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase. Science 298: 1387-1395 (2002).
Xiong, Y. and Steitz, T.A. Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template. Nature 430: 640-645 (2004).
Li, W., Kamtekar, S., Xiong, Y., Sarkis, G., Grindley, N.D.F. and Steitz, T.A. Structure of a synaptic γδ resolvase tetramer covalently linked to two cleaved DNAs. Science 309: 1210-1215 (2005).
Tu, D., Blaha, G., Moore, P.B. and Steitz, T.A. Structures of MLSBK antibiotics bound to mutated large ribosomal subunits provide a structural explanation for resistance. Cell 121: 257-270 (2005).
Schmeing, T.M., Huang, K.S., Strobel, S.A. and Steitz, T.A. Ribosomal protection of peptidyl-tRNA from hydrolysis using an induced fit mechanism. Nature 438: 520-524 (2005).