Poster abstracts
Poster number 55 submitted by Benjamin Warner
Contribution of an alternative 16S rRNA helix to biogenesis of the 30S subunit of the ribosome
Benjamin Warner (Department of Microbiology, Center for RNA Biology)
Abstract:
30S subunits become inactive upon exposure to low Mg2+ concentration, due to a reversible conformational change that entails nucleotides (nt) in the neck helix (h28) and 3’ tail of 16S rRNA. This active-to-inactive transition involves partial unwinding of h28 and re-pairing of nt 921-923 with nt 1532-1534, which requires flipping of the 3’ tail by ~180 degrees. A growing body of evidence suggests that immature 30S particles adopt the inactive conformation in the cell, and transition to the active state occurs at a late stage of maturation. Here, we target nucleotides that form the alternative helix (hALT) of the inactive state. Using an orthogonal ribosome system, we find that disruption of hALT decreases translation activity in the cell modestly, by ~2-fold, without compromising ribosome fidelity. Ribosomes carrying substitutions at positions 1532-1533 support growth of E. coli strain Δ7 prrn (which carries a single rRNA operon), albeit at rates 10-20% slower than wild-type ribosomes. These mutant Δ7 prrn strains accumulate free 30S particles and precursor 17S rRNA, indicative of 30S biogenesis defects. Biochemical analysis of purified 30S subunits harboring the tandem mutation U1532A / C1533G suggests that hALT stabilizes the inactive state by 1.2 kcal/mol with little-to-no impact on the active state or the transition state of conversion.
Keywords: Translation, 70S ribosome, tRNA