RNA



Metal ion probing of rRNAs: Evidence for evolutionarily conserved divalent cation binding pockets


NORBERT  POLACEK a1 and ANDREA  BARTA a1c1
a1 Institute of Biochemistry, University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria

Abstract

Ribosomes are multifunctional RNP complexes whose catalytic activities absolutely depend on divalent metal ions. It is assumed that structurally and functionally important metal ions are coordinated to highly ordered RNA structures that form metal ion binding pockets. One potent tool to identify the structural surroundings of high-affinity metal ion binding pockets is metal ion-induced cleavage of RNA. Exposure of ribosomes to divalent metal ions, such as Pb2+, Mg2+, Mn2+, and Ca2+, resulted in site-specific cleavage of rRNAs. Sites of strand scission catalyzed by different cations accumulate at distinct positions, indicating the existence of general metal ion binding centers in the highly folded rRNAs in close proximity to the cleavage sites. Two of the most efficient cleavage sites are located in the 5′ domain of both 23S and 16S rRNA, regions that are known to self-fold even in the absence of ribosomal proteins. Some of the efficient cleavage sites were mapped to the peptidyl transferase center located in the large ribosomal subunit. Furthermore, one of these cleavages was clearly diminished upon AcPhe-tRNA binding to the P site, but was not affected by uncharged tRNA. This provides evidence for a close physical proximity of a metal ion to the amino acid moiety of charged tRNAs. Interestingly, comparison of the metal ion cleavage pattern of eubacterial 70S with that of human 80S ribosomes showed that certain cleavage sites are evolutionarily highly conserved, thus demonstrating an identical location of a nearby metal ion. This suggests that cations, bound to evolutionarily constrained binding sites, are reasonable candidates for being of structural or functional importance.

(Received February 27 1998)
(Revised March 26 1998)
(Accepted June 23 1998)


Key Words: metal ion catalysis; metal ion cleavage; peptidyl transferase; rRNA structure.

Correspondence:
c1 Reprint requests to: Andrea Barta, Institute of Biochemistry, University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria; e-mail: andrea@bch.univie.ac.at.