Some Base Substitutions in the Leader of an Escherichia coli Ribosomal RNA Operon Affect the Structure and Function of Ribosomes: Evidence for a Transient Scaffold Function of the rRNA Leader

Abstract

Recently, we reported on the creation of a systematic series of C to T transition mutations, located between 19 and 45 nucleotides upstream of the mature 16 S RNA 5′ end of a complete, plasmid encoded ribosomal RNA operon. We showed that some of these base transitions have pronounced effects on the growth phenotype of mutant cells, and on the stability of their 16 S RNA as well as on the association capability of their ribosomal subunits. From these observations we concluded that the mutated leader region has post-transcriptional functions in the biogenesis of ribosomes.To further substantiate our conclusions we have now analyzed the growth phenotypes of some leader mutants in more detail, and show here that they are temperature dependent. Furthermore, we have isolated ribosomal RNA, 70 S ribosomes and ribosomal subunits from wild-type and mutant strains and subjected them to a detailed structural and functional analysis. We show that processing and maturation of the 16 S RNA is not altered as a consequence of the base transitions in the leader. In contrast, comparison of the protein composition of wild-type and mutant 30 S particles by two-dimensional gel electrophoresis revealed specific differences. Wild-type 30 S subunits, which are not tightly bound to 50 S, are lacking many ribosomal proteins, while the same fraction of ribosomes from mutant cells has an approximately complete r-protein set, and instead contains some additional non-ribosomal proteins.The translational activity of mutant and wild-type total ribosome preparations was analyzed in vitro. Ribosomes from slowly growing mutants show a significantly reduced in vitro translational activity, which is caused by the 30 S subunits. In contrast, the defects in association reside mainly in the 50 S subunits.Our results demonstrate that some base substitutions in the leader of an Escherichia coli rRNA operon affect the structure and function of ribosomes, although the mutated region is not part of the particles finally formed. This finding implies that at least part of the leader region assists the structure formation of functional 30 S subunits, before it is cut away and discarded.We argue that the rrn leader thus fulfills the functional criteria of a transient molecular scaffold.