We will continue to apply structure/function studies to three aspects of genetic expression and regulation. The goal is to explain function in terms of stereochemical details derived from high resolution crystal structures of relevant macromolecules and their complexes. 1. Initiation of protein synthesis in eukaryotes: Here the aim is to explain the special functions of eukaryotic initiator tRNA in terms of the molecular model of yeast tRNA-i-Met. A stereochemical analysis will be carried out on various misaminoacylated initiator tRNAs to understand the unexpectedly important role of the amino acid side chain. Site-directed mutagenesis will be employed (in collaboration with others) to define which sequence features are responsible for each of the special attributes of eukaryotic tRN-Ai-Met. 2. tRNA/aminoacyl-tRNA synthetase: While trying to prepare X-ray quality crystals of the complex, computer graphic experiments will be carried out to 'dock' the structure of tRNA-i-Met with a model of Met-tRNA synthetase. The principal guides will be direct photocrosslinks and a labeling reaction that simulates the transient reaction of the tRNA's U8 with the synthetase. These """"""""tethers"""""""" indicate the nucleotides of the tRNA and the amino acids of the synthetase that are in contact with one another at the molecular interface of the complex. 3. Bacterial repression: trp repressor/operator system: The recently determined atomic model of the trp repressor will be refined and compared to the high resolution structures of the inactive aporepressor (no bound tryptophan) and the inactive pseudorepressor (trytophan replaced by a competing antagonist) that are currently being determined crystallographically. Efforts will continue to improve the crystalline complexes of repressor and operator. X-ray diffraction analysis will proceed when suitable crystals are prepared. These crystal structures as well as mutational analyses from other labs should help to develop a molecular mechanism for sequence-specific DNA-binding by regulatory proteins and the modulation of genetic regulatory proteins by small ligands. New regulatory systems for future structural studies will be examined, including; (1) The erythromycin resistance protein that binds and modifies a specific residue in ribosomal RNA, (2) the monomeric RNA polymerase/promoter system from the T7 family of bacteriophage and (3) steroid receptor/enhancer system from mammalian cell lines.
| Bell, S D; Kosa, P L; Sigler, P B et al. (1999) Orientation of the transcription preinitiation complex in archaea. Proc Natl Acad Sci U S A 96:13662-7 |
