Bacterial ribonucleases and their roles in RNA metabolism

Abstract

Ribonucleases (RNases) are mediators in most reactions of RNA metabolism. In recent years, there has been a surge of new information about RNases and the roles they play in cell physiology. In this review, a detailed description of bacterial RNases is presented, focusing primarily on those from Escherichia coli and Bacillus subtilis, the model Gram-negative and Gram-positive organisms, from which most of our current knowledge has been derived. Information from other organisms is also included, where relevant. In an extensive catalog of the known bacterial RNases, their structure, mechanism of action, physiological roles, genetics, and possible regulation are described. The RNase complement of E. coli and B. subtilis is compared, emphasizing the similarities, but especially the differences, between the two. Included are figures showing the three major RNA metabolic pathways in E. coli and B. subtilis and highlighting specific steps in each of the pathways catalyzed by the different RNases. This compilation of the currently available knowledge about bacterial RNases will be a useful tool for workers in the RNA field and for others interested in learning about this area.

Keywords: RNases; RNA processing; RNase function; RNase mechanism; RNase regulation; RNase structure; mRNA decay.

Figure 1.

Escherichia coli and Bacillus subtilis RNases. RNases present in both E. coli and B. subtilis are shown above the RNA schematic. RNases unique to one or the other organism are shown below the RNA schematic; E. coli enzymes in gray, B. subtilis enzymes in italics. Not shown are several other E. coli toxin RNases; only those discussed in the text are listed (MazF, HicA, and RelE).

Figure 2.

rRNA processing. Processing of 16S, 23S, and 5S rRNA in (A) E. coli and (B) B. subtilis. Mature rRNA, in black, is depicted as a schematic of its secondary structure. Precursor rRNA not present in the mature rRNA is in gray. Location of endonucleolytic cleavages and direction of 5′ or 3′ exonucleolytic processing are indicated by arrows. In B. subtilis, ribosomal proteins L3 and L18 are required for efficient 16S and 5S rRNA processing, respectively.

Figure 3.

tRNA processing. Two pathways for tRNA processing are shown, for tRNAs that do (left) or do not (right) have an encoded −CCA sequence in the genome. In E. coli, all tRNAs have an encoded −CCA sequence; in B. subtilis, only a third of tRNAs have an encoded −CCA sequence. For all tRNAs, RNase P cleaves endonucleolytically to give the mature 5′ end. For 3′ maturation, tRNAs with an encoded −CCA sequence follow the exonucleolytic pathway. Primarily RNase PH removes precursor nucleotides starting from either the native 3′ end or, in E. coli, from a site generated by RNase E cleavage. Other E. coli RNases (T, D, and II; shown above the tRNA 3′ extension) can participate in 3′ processing in the absence of RNase PH. B. subtilis does not have these three RNases, but other 3′ exonucleases (PNPase, RNase R, YhaM, and an as yet unidentified enzyme; shown in italics below the tRNA 3′ extension) can produce the mature 3′ end. For tRNAs without a −CCA sequence (in B. subtilis), the endonucleolytic pathway is followed in which RNase Z cleaves adjacent to the discriminator nucleotide (N). The −CCA sequence is added by nucleotidyl transferase.

Figure 4.

Mechanism of E. coli mRNA decay. An mRNA is depicted as a wavy line, showing 5′ triphosphate and 3′ hydroxyl ends, ribosome binding site (RBS), start and stop codons, and the 3′-proximal transcription termination structure. Enzymes acting on the mRNA are stylized traces of structures from the Protein Data Bank. Initiation of mRNA decay in E. coli is primarily via endonuclease cleavage by RNase E. (A, B) RNase E can access its internal target either by first binding to a monophosphorylated 5′ end and then scanning in the 3′ direction (A) or directly (B). When RNase E activity occurs by the 5′-end-dependent pathway, efficient binding of RNase E to the 5′ end requires conversion of the 5′ nucleoside triphosphate to a nucleoside monophosphate by the action of RppH. RNase E cleavage in the body of the message generates an upstream fragment with a free 3′ hydroxyl end, and a downstream fragment with a 5′ monophosphate end. (C) Subsequent RNase E cleavage, enhanced by the monophosphate 5′ end of the downstream fragment, generates additional mRNA decay intermediates. (D) The upstream products of RNase E cleavage are susceptible to 3′-to-5′ exonucleolytic decay by RNase II, PNPase, or RNase R. (E) 3′-terminal fragments that contain the Rho-independent transcription terminator structure are degraded either by PNPase, likely in conjunction with an RNA helicase, or by RNase R, which has intrinsic helicase activity. (F) Alternatively, the 3′-terminal fragment can be made vulnerable to decay by the iterative action of poly(A) polymerase and 3′ exonucleolytic attack. (G) In all cases of 3′ exonuclease degradation, a limit oligonucleotide product of ≤5 nts remains. These are degraded by the oligoribonuclease, Orn. Nucleotides affected by Orn activity are indicated by the double asterisks.

Figure 5.

Mechanism of B. subtilis mRNA decay. (A) B. subtilis endonuclease RNase Y is likely the functional homolog of E. coli RNase E. An internal cleavage in the body of an mRNA by RNase Y initiates decay in the same way as described for RNase E. It is thought that RNase Y can bind directly to an internal cleavage site. (B, C) In an alternative decay initiation pathway, the 5′ triphosphate end is converted to a monophosphate nucleotide by the action of RppH. RNase Y binds to the 5′ end and accesses an internal site. (D) A single RNase Y cleavage in the body of an mRNA can result in complete turnover of the mRNA via 3′ exonucleolytic decay of the upstream fragment (primarily by PNPase) and 5′ exonucleolytic decay of the downstream fragment by RNase J1. There is likely also another (yet unidentified) 3′ exonuclease that can degrade the upstream fragment. (E) The 3′ exonucleases leave an oligonucleotide limit product (≤5 nts), which is degraded to mononucleotides primarily by the oligoribonucleases, NrnA and NrnB. YhaM may also act as an oligoribonuclease. (F) RNase Y-independent pathway in which, subsequent to generation of a 5′ monophosphate end by RppH, RNase J1 degrades the entire mRNA in the 5′-to-3′ direction.