Ya-ming Hou

RNAs tRNAs interact with aminoacyl tRNA synthetases, elongationfactor Tu EF-Tu, and ribosomes to participate in the decoding of geneticinformation. Studies of the molecular interactions between tRNAs andsynthetases have led to insights into how synthetases distinguish tRNAs onthe basis of different sets of nucleotides that are unique to each tRNA forspecific aminoacylation. Little is known about the interactions betweentRNAs and EF-Tu, and between tRNAs and ribosomes, although the generalbelief is that these interactions depend on nucleotides that are common tomost tRNAs. The sequences of tRNAs contain 15 conserved and 17 semi-conservednucleotides that are common to most tRNAs. These nucleotides establish anetwork of hydrogen bonding interactions the tertiary interactions thatcollectively contribute to the folded, L-shaped three-dimensional tRNAstructure. Preliminary studies show that substitutions at some of theconserved and semi-conserved nucleotides result in non-functional tRNAs inE coli. The molecular basis for such effects is not understood, becausesubstitutions may disrupt the tertiary interactions, or they may eliminatedeterminants that are recognized by EF-Tu or ribosomes. Among the latterpossibilities, the goal is to determine those substitutions that affect theinteraction with EF-Tu. The emphasis on EF-Tu is because this protein hasa well defined crystal structure that can provide the framework forunderstanding its interaction with tRNAs. This proposal has two principle objectives. First, we will establishfunctional and non-functional nucleotides at each of the tertiaryinteractions that define the tRNA structure. Nucleotide substitutions willbe systematically introduced and tested for their effects on the structureand function of a specific tRNA. Based on the crystal structure of yeasttRNAPhe, we will determine whether we can predict the types of nucleotidesubstitutions that can maintain the structure. Second, for thosesubstitutions that maintain the structure but not the function, we willdetermine whether they eliminate the ability of tRNA to interact withEF-Tu. When substitutions that result in defective interaction with EF-Tuare identified, we will take a genetic approach to look for mutations inEF-Tu that compensate for the defect. These EF-Tu mutants, referred to assecond-site revertants, will harbor mutations at sites that are criticalfor tRNA interaction. The proposed studies will not only advance our functional understanding ofthe structure of tRNA, but will also shed light on the molecular contactsbetween EF-Tu and tRNA. This insight will form the basis upon whichmodeling of the crystal structures of EF-Tu and tRNA can be established tounderstand the mechanism of EF-Tu and tRNA interaction that underlies thefidelity of genetic information transfer.