MUTATIONS IN THE LEADER REGION OF RIBOSOMAL-RNA OPERONS CAUSE STRUCTURALLY DEFECTIVE 30 S RIBOSOMES AS REVEALED BY IN-VIVO STRUCTURAL PROBING

The biogenesis of functional ribosomes is regulated in a very complex manner, involving different proteins and RNA molecules. RNAs are not o nly essential components of both ribosomal subunits but also transient ly interacting factors during particle formation. In eukaryotes snoRNA s act as molecular chaperones to assist maturation, modification and a ssembly. In a very similar way highly conserved leader sequences of ba cterial rRNA operons are involved in the correct formation of 30 S rib osomal subunits. Certain mutations in the rRNA leader region cause sev ere growth defects due to malfunction of ribosomes which are assembled from such transcription units. To understand how the leader sequences act to facilitate the formation of the correct 30 S subunits we perfo rmed in vivo chemical probing to assess structural differences between ribosomes assembled either from rRNA transcribed from wild-type opero ns or from operons which contain mutations in the rRNA leader region. Cells transformed with plasmids containing the respective rRNA operons were reacted with dimethylsulphate (DMS). Ribosomes were isolated by sucrose gradient centrifugation and modified nucleotides within the 16 S rRNA were identified by primer extension reaction. Structural diffe rences between ribosomes from wild-type and mutant rRNA operons occur in several clusters within the 16 S rRNA secondary structure. The most prominent differences are located in the central domain including the universally conserved pseudoknot structure which connects the 5', the central and the 3' domain of 16 S rRNA. Two other clusters with struc tural differences fall in the 5' domain where the leader had been show n to interact with mature 16 S rRNA and within the ribosomal protein S 4 binding site. The other differences in structure are located in site s which are also known as sites for the action of several antibiotics. The data explain the functional defects of ribosomes from rRNA operon s with leader mutations and help to understand the altered biogenesis pathway from mutations in an rRNA leader region to the formation of fu nctionally defective ribosomes. (C) 1998 Academic Press Limited.