Abstract
Ribonucleases play essential roles in RNA metabolism. In Escherichia coli, the extensive degradation of RNAs that are defective or no longer required by the cell is carried out by one of three processive, 3' to 5' exoribonucleases. Relatively unstructured mRNAs are typically degraded by RNase II or PNPase. In contrast, mRNAs containing extensive secondary structure, and the highly structured rRNA and tRNA molecules, are degraded by PNPase and/or RNase R. However, RNase R differs from other exoribonucleases in that it is able to degrade through these structured RNAs without the aid of a helicase activity. Consequently, its mechanism of action is of great interest. In this dissertation, using a variety of specifically designed substrates, I show that a single-stranded overhang, which must be at least 5 nucleotides in length, is required for tight binding and subsequent degradation of double-stranded RNA by RNase R. Moreover, this overhang must be 3' to the duplex indicating that an RNA substrate must thread into the enzyme with 3' to 5' polarity. Using a series of truncated RNase R proteins, I show that the cold-shock domains and the S1 domain contribute to substrate binding. The cold-shock domains appear to play a role in substrate recruitment, while the S1 domain is required to position substrates for efficient catalysis. Furthermore, the nuclease domain alone is sufficient to bind and degrade structured RNAs. This is a unique property of the nuclease domain of RNase R since this domain in RNase II, a paralogue of RNase R, stalls as it approaches a duplex. RNase R binds RNA more tightly within its nuclease domain than RNase II. Through mutagenesis studies, I identify one amino acid, R572, within the nuclease domain of RNase R that contributes to this tight binding and the ability to degrade double-stranded RNA. Furthermore, I found that degradation of structured RNA is strongly dependent on temperature. Based on these data I propose that tight binding allows RNase R to capitalize on the natural thermal breathing of an RNA duplex to degrade structured RNA.