Source: Dissertation Abstracts International, Volume: 75-05(E), Section: B.

Adviser: Thomas A. Steitz.

All cells synthesize proteins for use as enzymes, structural supports, transportation, signaling. These proteins are synthesized in a message directed fashion using a macromolecular complex of RNA and protein called the ribosome. This process is tightly regulated and uses different protein and RNA factors at each step in the process. The synthesis of proteins in prokaryotes is divided into four distinct processes: initiation, elongation, termination, and recycling. Initiation of translation in prokaryotes is guided by three initiation factors (IF1, IF2, and IF3). These factors direct the process in which mRNA, initiator tRNA, and the ribosomal subunits join to form a 70S ribosomal initiation complex ready for translation of the message into a protein.

IF2 is a GTPase that functions to position the initiator tRNA within the 30S ribosomal initiation complex and promote the complex to join with the 50S ribosomal subunit to form a 70S ribosome. A 3.1A-resolution X-ray crystal structure was determined from full-length IF2 from Thermus thermophilus in the apo form and GDP bound form. Each structure had a distinct conformation that together demonstrates the flexibility of IF2.

Unlike all other translational GTPases, the effector domain of IF2 does not form a stable contact with the switch II region of the GTPase domain. The domain organization of IF2 is incompatible with the "articulated lever'' mechanism of communication between the GTPase and initiator tRNA binding domains that has been proposed for eIF5B, a related protein in eukaryotes. Previous NMR experiments, cryo-EM reconstructions, and these structures show that IF2 is a flexible protein with dynamic conformations that largely decouple the G-nucleotide bound state of IF2 with its biological function of interacting with ribosomal complexes and initiator tRNA.

Ribozymes are noncoding RNA enzymes that perform chemistry with rates of catalysis that approach those of protein enzymes. Ribozymes function in protein synthesis, RNA processing, self-splicing and self-cleavage in a constitutive or metabolite-dependent manner. Nine classes of ribozymes have been previously discovered in nature. These classes include ribosomes, introns, hammerhead, HDV, and glmS ribozyme classes found in prokaryotes and eukaryotes. Recently, the Breaker lab has discovered and characterized a new ribozyme class as a self-cleaving ribozyme that is present in all kingdoms of life. So far, the biological role of this ribozyme is unknown.

Crystals of a Twister construct derived from an Oryza sativa (Rice) sequence have been the most promising for successful structure determination, although they have been difficult to reproduce. A crystal structure of Twister may help elucidate how it is able to achieve such a high rate of catalysis.