Conformational changes induced in the Saccharomyces cerevisiae GTPase-associated rRNA by ribosomal stalk components and a translocation inhibitor

The yeast ribosomal GTPase associated center is made of parts of the 26S rR NA domains II and VI, and a number of proteins including P0, P1 alpha, P1 b eta, P2 alpha, P2 beta and L12, Mapping of the rRNA neighborhood of the pro teins was performed by footprinting in ribosomes from yeast strains lacking different GTPase components, The absence of protein PO dramatically increa ses the sensitivity of the defective ribosome to degradation hampering the RNA footprinting, In ribosomes lacking the P1/P2 complex, protection of a n umber of nucleotides is detected around positions 840, 880, 1100, 1220-1280 and 1350 in domain II as well as in several positions in the domain VI alp ha -sarcin region, The protection pattern resembles the one reported for th e interaction of elongation factors in bacterial systems. The results exclu de a direct interaction of these proteins with the rRNA and are compatible with an increase in the ribosome affinity for EF-2 in the absence of the ac idic P proteins, Interestingly, a sordarin derivative inhibitor of EF-2 cau ses an opposite effect, increasing the reactivity in positions protected by the absence of P1/P2, Similarly, a deficiency in protein L12 exposes nucle otides G1235, G1242, A1262, A1269, A1270 and A1272 to chemical modification , thus situating the protein binding site in the most conserved part of the 26S rRNA, equivalent to the bacterial protein L11 binding site.