Book of Abstracts: Albany 2009

category image Albany 2009
Conversation 16
June 16-20 2009
© Adenine Press (2008)

The Ribosomal Stalk Plays a Key-role in the Translation Initiation in Bacteria

Fast association of the ribosomal subunits during translation initiation requires the presence of initiation factor 2 (IF2) on the 30S-preinitiation complex (30S-preI) containing mRNA and the initiator tRNA. But how the 50S recognizes the IF2 bound 30S-preI is not known. Our results from the parallel fast kinetic measurements of the different steps of initiation show that the ribosomal ?stalk?, composed of the L12 proteins, constitutes the key component on the 50S subunit for IF2 recognition.

Depletion of the L12 proteins from 50S has no effect on the association of the naked subunits, suggesting that the L12 protein is not a structural element on the 50S essential for the subunit association. Also, L12 depletion does not alter the rates of the subunit association when all other components of the pre-initiation complex except IF2 are present on the 30S. When IF2 is added on the 30S-preI a very fast rate of subunit association is obtained with normal 50S (ka=130 μM-1s-1), which decreases significantly (30 ? 40 fold) upon removal of L12 from it. These data clearly suggest that IF2 and L12 are two recognition markers on the 30S-preI and the 50S subunits respectively; the absence of any of the two results in a rather inefficient association of the subunits. In parallel, we have studied the role of the L12 protein in the stimulation of the GTPase activity of IF2. L12 depleted 50S when associated with the 30S-PreI showed essentially same rates of GTP hydrolysis and Pi release as with normal 50S. Also, there is no direct effect of L12 depletion on the rate of IF2 release. These results, in contrast to the earlier studied cases of EF-G and EF-Tu, suggest that the L12 protein is not involved in the GTPase activation on IF2. We also confirm that the GTP hydrolysis and Pi release are not essential for the association step, but crucial for the release of IF2-GDP from the 70S initiation complex.

In summary, it is evident from our data that the main role of the ribosomal stalk in translation initiation is the recognition and recruitment of IF2 which in turn brings the 30S-PreI to the 50S and results in a fast subunit association. When L12 is removed from the 50S the subunit association becomes slower. As a consequence of the slow subunit association the subsequent steps such as GTP hydrolysis and Pi release by IF2 and IF2 release from the 70S-initiation complex become apparently slower but their individual rates remain unaffected. So, for IF2-GTPase L12 does not work as a GTPase activator protein (GAP).

Chenhui Huang
Chandra Sekhar Mandava
Suparna Sanyal

Dept. of Cell and Molecular Biology
Uppsala University, Box-596
BMC, 75124, Uppsala