stable and may persist through several rounds of cell division. Individual rRNAs are stabilized when they are assembled into ribosomal particles (see below), while tRNAs are stabilized because they are generally present in a complex with their cognate aaRS or with translation elongation factor Tu (EF-Tu) (see below). In contrast, most bacterial mRNAs are very unstable, with an average half-life in E. coli of 1 to 3 minutes; this term refers to the time required for the amount of an RNA to decrease to 50% of its initial level. The short half-life of mRNAs in bacteria contrasts with the situation in eukaryotes, where mRNAs are often very stable (with a half-life of hours). Efficient mRNA degradation is important for gene regulation and also releases nucleotides for use in new rounds of transcription. A variety of RNases participate in mRNA degradation, and the profiles of RNases vary somewhat in different groups of bacteria.
RNases
There are two major classes of RNases (Table 2.1 and Figure 2.18). Endoribonucleases cleave the sugar-phosphate backbone of the RNA within the RNA chain to generate two smaller RNA products, one with a 3′ hydroxyl and the other with a 5′ monophosphate. Exoribonucleases digest the RNA processively, removing 1 nucleotide at a time, starting at a free end. Most organisms have multiple endo- and exoribonucleases, and some subtypes of these enzymes are found in many different organisms, while others are present in only certain groups of organisms. In E. coli, all exoribonucleases that have been identified bind to the 3′ end of an RNA substrate and digest the RNA in a 3′-to-5′ direction. In contrast, Bacillus subtilis and a number of other organisms contain both 3′-5′ exoribonucleases and 5′-3′ exoribonucleases. At least one of the B. subtilis 5′-3′ RNases (RNase J2) has both exoribonucleolytic and endoribonucleolytic activities (Table 2.1).
MODULATION OF RNase ACTIVITY
The susceptibility of an RNA to different RNases can be affected by structural features of the RNA. RNA 3′ ends generated by termination of transcription at a factorindependent terminator contain an RNA hairpin, which inhibits binding of 3′-5′ exoribonucleases (Figure 2.18). Degradation of RNAs of this type is often initiated by endonucleolytic cleavage, which removes the 3′ end of the RNA and allows the 5′ region of the molecule to be degraded. Degradation of the 3′ fragment can be initiated by polyadenylation of the 3′ end of the RNA by polyadenylate [poly(A)] polymerase, encoded by the pcnB gene. Addition of the poly(A) tail provides a “landing zone” for 3′-5′ exoribonucleases, which can initiate degradation of the poly(A) sequence and then continue to move through the terminator hairpin. This may be facilitated by colocalization of poly(A) polymerase and polynucleotide phosphorylase (PNPase; Table 2.1), one of the major 3′-5′ exonucleases, with other RNases into a complex called the degradosome. Note that polyadenylation of an mRNA in eukaryotes generally results in stabilization of the mRNA, while polyadenylation of an RNA in bacteria results in rapid degradation. Degradation of the 3′ fragment generated by endonucleolytic cleavage can also be directed by 5′-3′ exoribonucleases in organisms like B. subtilis that have this activity (see Condon, Suggested Reading). It is interesting to note that the 5′ ends of transcripts newly synthesized by RNA polymerase contain a triphosphate (from the initiating nucleotide), whereas the 5′ ends of RNAs generated by endonuclease cleavage contain monophosphates. The presence of a triphosphate protects the RNA, and this triphosphate can be removed by a dedicated enzyme, designated RppH, which enhances susceptibility to degradation by 5′-3′ exonucleases (see Hui et al., Suggested Reading).
Susceptibility to degradation can be used as a mechanism to regulate gene expression, because rapid degradation of an mRNA results in reduced synthesis of its protein product. Modulation of RNA stability can occur through changes in the RNA structure that affect RNase binding by binding of a regulatory protein to the RNA or by binding of a regulatory RNA. Mechanisms of this type are discussed in chapter 11.
Table 2.1 Enzymes involved in mRNA processing and degradation
| Enzyme | Substrate(s) | Description |
| RNase E | mRNA, rRNA, tRNA | Endonuclease, highly conserved in all Proteobacteria and some Firmicutes (not B. subtilis) |
| RNase III | rRNA, polycistronic mRNA | Endonuclease, cleaves double-stranded RNA in some stem-loops; found in most bacteria |
| RNase P | Polycistronic mRNA, tRNA precursors | Ribozyme, necessary to process 5′ ends of tRNAs |
| RNase G | 5′ end of 16S rRNA, mRNA | Endonuclease, replaces RNase E in some bacteria |
| RNases J1 and J2 | mRNA, rRNA | 5′-3′ exonuclease, endonuclease; found in most Firmicutes and some Proteobacteria bacteria (not E. coli) |
| Poly(A) polymerase | mRNA | Found in most bacteria |
| PNPase | mRNA, poly(A) tails | 3′-5′ exonuclease, found in all bacteria |
Figure 2.18 Pathways for RNA degradation. RNA transcripts that are generated by termination at a factor-independent terminator contain a hairpin at the 3′ end, which inhibits degradation by 3′-5′ exoribonucleases. Degradation is often initiated by cleavage by an endonuclease, followed by rapid exonucleolytic digestion from the new 3′ end. The stable 3′ fragment (which retains the terminator hairpin) can be cleaved again by an endoribonuclease or can be degraded by a 5′-3′ exoribonuclease in organisms that have this class of enzyme. Alternatively, poly(A) polymerase can add a poly(A) tail to the 3′ end of the RNA, which allows binding of a 3′-5′ exoribonuclease and degradation.
The Structure and Function of Proteins
Proteins do most of the work of the cell. While there are a few RNA enzymes (ribozymes), most of the enzymes that make and degrade energy sources and make cell constituents are proteins. Also, proteins contribute to much of the structure of the cell. Because of these diverse roles, there are many more types of proteins than there are types of other cell constituents. Even in a relatively simple bacterium, there are thousands of different types of proteins, and most of the DNA sequences in bacteria are dedicated to genes that encode proteins.
Protein Structure
Unlike DNA and RNA, which consist of a chain of nucleotides held together by phosphodiester bonds between the sugars and phosphates, proteins consist of chains of 20 different amino acids held together by peptide bonds (see Figure 2.19). The peptide bond is formed by joining the amino group (NH2) of one amino acid