Source: Dissertation Abstracts International, Volume: 74-05(E), Section: B.

Advisers: Thomas A. Steitz; Joan A. Steitz.

In cells, genetic information flows from DNA to RNA to protein according to Crick's Central Dogma, stated in 1957: "Once information has got into a protein it can't get out again" (Crick, 1958). This study explores two independent aspects of this molecular biology paradigm at a structural level using X-ray crystallography: prokaryotic DNA replication and eukaryotic translation.

The first part of this investigation aims to elucidate the mechanism by which the replicative DNA helicase (DnaB hexamer) translocates on single-stranded DNA (ssDNA). The crystal structure of the DnaB hexamer in complex with GDP-AlF 4 and ssDNA reported here reveals that DnaB adopts a spiral staircase lockwasher quaternary structure around an A-form ssDNA with each C-terminal domain coordinating two nucleotides of ssDNA. The structure not only provides the first structural insight into the translocation mechanism of superfamily IV helicases, but also suggests that members of this superfamily employ a novel translocation mechanism that is distinct from those of Rho and E1 helicases. We propose a hand-over-hand mechanism in which sequential hydrolysis of NTP causes a sequential 5'-to-3' movement of the subunits along the helical axis of the lockwasher, resulting in the unwinding of 2 base pairs per subunit.

The goal of the second section of this study is to determine the structure of full-length human cytoplasmic poly(A) binding protein 1 (PABP1) in complex with poly(A)-containing RNAs and/or PABP1-interacting proteins (Paip1). These complexes would provide structural insights into how PABP1 multimerizes on the poly(A) tail of eukaryotic mRNAs as well as how it interacts with its binding partners. While crystallization screens did not yield promising leads, this investigation has established a framework for future structural studies of PABP1 by discovering poly(A)-containing RNA constructs that can prevent multimerization of PABP1 bound to different pieces of RNAs, which is crucial for obtaining homogeneous samples of PABP1:RNA complexes.