- The degradosome is a complex of bacterial enzymes, including RNAase and helicase activities, which may be involved in degrading mRNA.
- The overall direction of degradation of bacterial mRNA is 5–3.
- Degradation results from the combination of exonucleolytic cleavages followed by endonucleolytic degradation of the fragment from 3–5.
Bacterial mRNA is constantly degraded by a combination of endonucleases and exonucleases (for review see Grunberg-Manago, 1999). Endonucleases cleave an RNA at an internal site. Exonucleases are involved in trimming reactions in which the extra residues are whittled away, base by base from the end. Bacterial exonucleases that act on single-stranded RNA proceed along the nucleic acid chain from the 3 end.
The way the two types of enzymes work together to degrade an mRNA is shown in Figure 5.20. Degradation of a bacterial mRNA is initiated by an endonucleolytic attack. Several 3 ends may be generated by endonucleolytic cleavages within the mRNA. The overall direction of degradation (as measured by loss of ability to synthesize proteins) is 5–3. This probably results from a succession of endonucleolytic cleavages following the last ribosome. Degradation of the released fragments of mRNA into nucleotides then proceeds by exonucleolytic attack from the free 3–OH end toward the 5 terminus (that is, in the opposite direction from transcription). Endonucleolytic attack releases fragments that may have different susceptibilities to exonucleases. A region of secondary structure within the mRNA may provide an obstacle to the exonuclease, thus protecting the regions on its 5 side. The stability of each mRNA is therefore determined by the susceptibility of its particular sequence to both endo- and exonucleolytic cleavages.
There are ~12 ribonucleases in E. coli. Mutants in the endoribonucleases (except ribonuclease I, which is without effect) accumulate unprocessed precursors to rRNA and tRNA, but are viable. Mutants in the exonucleases often have apparently unaltered phenotypes, which suggests that one enzyme can substitute for the absence of another. Mutants lacking multiple enzymes sometimes are inviable (for review see Caponigro and Parker, 1996).
RNAase E is the key enzyme in initiating cleavage of mRNA. It may be the enzyme that makes the first cleavage for many mRNAs. Bacterial mutants that have a defective ribonuclease E have increased stability (2-3 fold) of mRNA. However, this is not its only function. RNAase E was originally discovered as the enzyme that is responsible for processing 5 rRNA from the primary transcript by a specific endonucleolytic processing event.
The process of degradation may be catalyzed by a multienzyme complex (sometimes called the degradosome) that includes ribonuclease E, PNPase, and a helicase (Miczak et al., 1996). RNAase E plays dual roles. Its N-terminal domain provides an endonuclease activity. The C-terminal domain provides a scaffold that holds together the other components (Vanzo et al., 1998). The helicase unwinds the substrate RNA to make it available to PNPase. According to this model, RNAase E makes the initial cut and then passes the fragments to the other components of the complex for processing.
Polyadenylation may play a role in initiating degradation of some mRNAs in bacteria. Poly(A) polymerase is associated with ribosomes in E. coli, and short (10-40 nucleotide) stretches of poly(A) are added to at least some mRNAs. Triple mutations that remove poly(A) polymerase, ribonuclease E, and polynucleotide phosphorylase (PNPase is a 3–5 exonuclease) have a strong effect on stability. (Mutations in individual genes or pairs of genes have only a weak effect.) Poly(A) polymerase may create a poly(A) tail that acts as a binding site for the nucleases. The role of poly(A) in bacteria would therefore be different from that in eukaryotic cells (O'Hara et al., 1995).