The BIPA global regulator interacts with Ribosomes

Owens, Roisin Meabh (2002) The BIPA global regulator interacts with Ribosomes. University of Southampton, Doctoral Thesis.

Abstract

Inspection of the BipA sequence shows that it resembles ribosome-binding GTPases such as EF-G and the TetO/TetM family of tetracycline resistance proteins. This raises the possibility that BipA also interacts with ribosomes. A primary aim of these studies was to test this possibility and to characterise the factors that influence the GTPase activity of BipA, using hexahistidine-tagged derivatives. Full-length (His)₆BipA, a C-terminally truncated derivative and a G77V mutant were purified by metal chelate chromatography. Gel filtration experiments were performed to study the interaction of (His)₆BipA with the 70S ribosome. (His)₆BipA eluted rapidly when ribosomes were present but much more slowly when they were not. The profile obtained was similar to that obtained with EF-G under the same conditions. Co-elution of (His)₆BipA occurred in the presence or absence of guanosine nucleotides. In the presence of EF-G, (His)₆BipA did not co-elute with 70S ribosomes, indicating that both factors bind to the same or a closely overlapping site on the ribosome.

Fluorescence titrations revealed that (His)₆BipA has similar affinities for both GDP and GTP (18 μM and 60 μM respectively). When assayed for GTPase activity both the full length and the truncated proteins were found to have basal levels that were stimulated over 10-fold on addition of 70S ribosomes, while the G77V mutant was devoid of such activity. The 70S-stimulated GTPase activities of these proteins were compared with those of EF-G, TetO and EF-Tu. Inhibitor studies showed that (His)₆BipA was sensitive to thiostrepton and α-sarcin. In contrast to EF-G, (His)₆BipA was inhibited by GDP but not by fusidic acid. (His)₆BipA had no discernible ATPase activity.

Taken together with its global regulatory properties, these results suggest that BipA uses a novel mechanism to modulate gene expression.

This record has no associated files available for download.

Contributors

Download statistics