Mechanisms for control of nucleoside triphosphate hydrolysis. Effects of DNA and RNA co-factors

Sammanfattning: In energetically unfavourable biological processes "protein machines" utilize chemical energy from the hydrolysis of high-energy phosphate bonds. Thus, the hydrolysis of a nucleoside triphosphate (NTP) to nucleoside diphosphate (NDP) with the release of free orthophosphate results in a liberation of energy. In addition, proteins that work as switches or gates in order to ensure fidelity and directionality to many synthetic and signal transduction processes also use NTP hydrolysis. This thesis describes the studies of the mechanisms involved in controlling the GTP and ATP binding and hydrolysis by the Ffh and FtsY proteins from the bacterial Signal Recognition Particle, SRP, and the origin binding protein, OBP, or UL9 from the Herpes simplex virus type 1 replication machinery.The Ffh and FtsY proteins from Mycoplasma mycoides are unusual GTPases because they act as GAPs for each other. We show that the reciprocal GTPase stimulation occurs when the G-domains of the proteins are combined in vitro. This finding indicates that important elements of the basic GTPase activation mechanism reside in the G-domains as such and that the other domains of Ffh and FtsY only serve to modulate the activation. We also show that binding of GTP to Ffh results in significant conformational changes of the protein. In particular, a region near the C-terminus of the G-domain becomes more ordered as a result of nucleotide binding. This region is close to sites where the G-domain interacts with other domains of the protein. Therefore, the structural changes that we observe may be part of a mechanism where GTP binding induces conformational changes in other domains of Ffh.OBP acts as the initiator for replication of the HSV-1 genome. It acts by converting the double-stranded origin of DNA replication oriS to an activated partially single-stranded conformation referred to as oriS'. The reaction requires ATP hydrolysis. We demonstrate genetically that oriS' most likely is formed in vivo. We also show that oriS' is an efficient activator of ATP hydrolysis and that stimulation of ATP hydrolysis requires binding to a hairpin containing the recognition sequence for OBP and position-specific base-contacts with a 3' single-stranded tail. Gel retardation experiments indicate furthermore that OBP adopts different conformations in the presence of ATP or ADP.Finally, the diverse roles that RNA and DNA have on regulation of hydrolysis of nucleoside triphosphates are discussed.