The Escherichia coli ribosomal RNA leader: a structural and functional investigation.

The structure of the E. coli ribosomal RNA leader was analyzed by treatment with single and double strand specific ribonucleases and by chemical modification. The experimentally derived data together with secondary structure calculations according to minimum free energy was used to construct a secondary structure model. The binding of purified Nus proteins to the ribosomal leader RNA was further tested. Contrary to the recently reported interactions of NusB and NusE with a nut RNA sequence we obtained evidence that the presence of NusA and NusE resulted in protection against hydroxyl radical reaction of the leader nut elements boxA, boxB and boxC. The possible significance of this interaction is discussed. In the second part of the study we analyzed effects of leader mutations, which are known to affect cell growth, on the activity of ribosomes in vivo. A system was used able to distinguish the proportion of ribosomes assembled from rRNA of chromosomal origin (wild type) and plasmid origin (mutant). It turned out that the amount of 16S RNA transcribed from genes with point mutations in the leader region decreased if ribosomal pools with different translational activities were compared. High amounts of transcripts from mutant operons were present in the free ribosomal RNA and the free 30S fractions. Significantly less 16S RNA transcripts from the mutated genes were detected in the functionally active and homogeneous 70S tight couple preparations, and even less in the polysome fraction involved in active translation. The results allow a better understanding of the function of rRNA leader sequences in structure formation and correct ribosome biogenesis.