Genetic analysis of the role of ribonuclease E in RNA metabolism in Escherichia coli K-12
Ow, Maria Cristina
MetadataShow full item record
In E. coli, one mechanism for regulating gene expression involves the rate at which mRNAs are degraded or processed. A large body of work has shown that the principal enzyme involved in this process is ribonuclease E (RNase E). This dissertation describes research undertaken to broaden our understanding of the role of RNase E in RNA metabolism.|The carboxyl-terminus of RNase E is the scaffold for the assembly of a protein complex called the degradosome. To investigate the importance of the degradosome in RNA metabolism, we constructed truncated RNase E mutants lacking the scaffold region. In these mutants (rne 225 and rne 374) mRNA decay was not affected. However, another RNase E mutant (rne 610), extensively truncated at the C-terminus, showed a dramatic increase in the longevity of mRNAs. By comparing the phenotypic properties of rne 610 with the temperature-sensitive rne-1 allele, it was possible to demonstrate that RNase E must have another essential function besides mRNA decay and rRNA processing.|The isolation of a rne 610 temperature-resistant revertant, rne 645, has helped to delineate what this additional function might be. Specifically, examination of tRNA processing showed that RNase E is also required for the processing of the 5' and 3' ends of tRNA precursors. The rne 645 strain processed full-length polycistronic and monocistronic tRNA transcripts 2-3-fold faster than its rne 610 progenitor. These results suggest that the essential function of RNase E may be the processing of tRNA precursors.|We also examined the regulation of RNase E synthesis in the cell. The rne gene is transcribed from three independent promoters, p1, p2, and p3. The p1 promoter accounts for most of the rne transcription followed by p3 and p2. Each promoter alone was sufficient to maintain cell viability. Deletion of two of the three promoters resulted in a significant decrease of RNase E protein level. Remarkably, the large decreases in RNase E protein level were not proportionally reflected in increased mRNA decay rates. The existence of three promoters and the phenotypes associated with deleting two of the three promoters clearly indicate that the regulation of this important ribonuclease is far more complex than previously thought.