Use of Lead(II) to Probe the Structure of Large RNA's. Conformation of the 3′ Terminal Domain of E. coli 16S rRNA and its Involvement in Building the tRNA Binding Sites

Abstract

The present work shows that lead(II) can be used as a convenient structure probe to map the conformation of large RNA's and to follow discrete conformational changes at different functional states. We have investigated the conformation of the 3′ domain of the E. coli 16S rRNA (nucleotides 1295–1542) in its naked form, in the 30S subunit and in the 70S ribosome. Our study clearly shows a preferential affinity of Pb(II) for interhelical and loop regions and suggests a high sensitivity for dynamic and flexible regions. Within 30S subunits, some cleavages are strongly decreased as the result of protein-induced protection, while others are enhanced suggesting local conformational ajustments. These rearrangements occur at functionally strategic regions of the RNA centered around nucleotides 1337,1400,1500 and near the 3′ end of the RNA The association of 30S and 50S subunits causes further protections at several nucleotides and some enhanced reactivities that can be interpreted in terms of subunits interface and allosteric transitions. The binding of E. coli tRNA-Phe to the 70S ribosome results in message-independent (positions 1337 and 1397) and message-dependent (1399–1400, 1491–1492 and 1505) protections. Athird class ofprotection(1344–1345,1393–1395,1403–1409,1412–1414, 1504, 1506–1507 and 1517–1519) is observed in message-directed 30S subunits, which are induced by both tRNA binding and 50S subunit association. This extensive reduction of reactivity most probably reflects an allosteric transition rather than a direct shielding.