Murray P. Deutscher

Degradation of RNA in bacteria: Comparison of mRNA and stable RNA

Degradation of RNA plays a central role in RNA metabolism. In recent years, our knowledge of the mechanisms of RNA degradation has increased considerably with discovery of the participating RNases and analysis of mutants affected in the various degradative pathways. Among these processes, mRNA decay and stable RNA degradation generally have been considered distinct, and also separate from RNA maturation. In this review, each of these processes is described, as it is currently understood in bacteria. The picture that emerges is that decay of mRNA and degradation of stable RNA share many common features, and that their initial steps also overlap with those of RNA maturation. Thus, bacterial cells do not contain dedicated machinery for degradation of different classes of RNA or for different processes. Rather, only the specificity of the RNase and the accessibility of the substrate determine whether or not a particular RNA will be acted upon.

RNase G (CafA protein) and RNase E are both required for the 5' maturation of 16S ribosomal RNA.

In Escherichia coli, rRNA operons are transcribed as 30S precursor molecules that must be extensively processed to generate mature 16S, 23S and 5S rRNA. While it is known that RNase III cleaves the primary transcript to separate the individual rRNAs, there is little information about the secondary processing reactions needed to form their mature 3′ and 5′ termini. We have now found that inactivation of the endoribonuclease RNase E slows down in vivo maturation of 16S RNA from the 17S RNase III cleavage product. Moreover, in the absence of CafA protein, a homolog of RNase E, formation of 16S RNA also slows down, but in this case a 16.3S intermediate accumulates. When both RNase E and CafA are inactivated, 5′ maturation of 16S rRNA is completely blocked. In contrast, 3′ maturation is essentially unaffected. The 5′ unprocessed precursor that accumulates in the double mutant can be assembled into 30S and 70S ribosomes. Precursors also can be processed in vitro by RNase E and CafA. These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5′ end of 16S rRNA, and that CafA protein is a new ribonuclease. We propose that it be renamed RNase G.

Complex of aminoacyl-transfer RNA synthetases☆

Abstract A high molecular weight complex has been isolated from rat liver which contains all the aminoacyl-transfer RNA synthetases and and a large proportion of the tRNA present in the high-speed supernatant fraction. The complex could be partially purified by chromatography on Sephadex G200 and Sepharose 6B. The complex could be disrupted by excess homogenization and freezing and thawing with a concomitant decrease in the activities of the aminoacyl-tRNA synthetases. The results suggest that some of the presumed “soluble” components of the protein synthetic apparatus may be present in highly organized structures within the cell.

Quality control of ribosomal RNA mediated by polynucleotide phosphorylase and RNase R

Despite their overall accuracy, errors in macromolecular processes, such as rRNA synthesis and ribosome assembly, inevitably occur. However, whether these errors are remediated and how this might be accomplished is not known. In previous work, we showed that a double mutant strain lacking both polynucleotide phosphorylase (PNPase) and RNase R activities is inviable. In the course of examining the molecular basis for this phenotype, we found that shifting a temperature-sensitive mutant strain to 42°C led to cessation of growth and loss of cell viability. Northern analysis of RNA isolated from such cells after the temperature shift revealed that fragments of 16S and 23S rRNA accumulated to a high level, and that the amount of ribosomes and ribosomal subunits decreased due to defects in ribosome assembly. rRNA fragments were not detected at 31°C or when single mutant strains were grown at 42°C. Pulse–chase analysis showed that the rRNA fragments appeared within 5 min at 42°C, and that they accumulated before the loss of cell viability. The data are consistent with a model in which PNPase and RNase R mediate a previously unknown quality control process that normally removes defective rRNAs as soon as they are generated. In the absence of these RNases, rRNA fragments accumulate, leading to interference with ribosome maturation and ultimately to cell death.

The vacB Gene Required for Virulence in Shigella flexneri and Escherichia coli Encodes the Exoribonuclease RNase R

vacB, a gene previously shown to be required for expression of virulence in Shigella and enteroinvasive Escherichia coli, has been found to encode the 3′–5′ exoribonuclease, RNase R. Thus, cloning of E. coli vacB led to overexpression of RNase R activity, and partial deletion or interruption of the cloned gene abolished this overexpression. Interruption of the chromosomal copy ofvacB eliminated endogenous RNase R activity; however, the absence of RNase R by itself had no effect on cell growth. In contrast, cells lacking both RNase R and polynucleotide phosphorylase were found to be inviable. These data indicate that RNase R participates in an essential cell function in addition to its role in virulence. The identification of the vacB gene product as RNase R should aid in understanding how the virulence phenotype in enterobacteria is expressed and regulated. On the basis of this information we propose that vacB be renamed rnr.

RNA quality control: degradation of defective transfer RNA.

The distinction between stable (tRNA and rRNA) and unstable (mRNA) RNA has been considered an important feature of bacterial RNA metabolism. One factor thought to contribute to the difference between these RNA populations is polyadenylation, which promotes degradation of unstable RNA. However, the recent discovery that polyadenylation also occurs on stable RNA led us to examine whether poly(A) might serve as a signal for eliminating defective stable RNAs, and thus play a role in RNA quality control. Here we show that a readily denaturable, mutant tRNATrp does not accumulate to normal levels in Escherichia coli because its precursor is rapidly degraded. Degradation is largely dependent on polyadenylation of the precursor by poly(A) polymerase and on its removal by polynucleotide phosphorylase. Thus, in the absence of these two enzymes large amounts of tRNATrp precursor accumulate. We propose that defective stable RNA precursors that are poorly converted to their mature forms may be polyadenylated and subsequently degraded. These data indicate that quality control of stable RNA metabolism in many ways resembles normal turnover of unstable RNA.

Substrate Recognition and Catalysis by the Exoribonuclease RNase R

RNase R is a processive, 3′ to 5′ hydrolytic exoribonuclease that together with polynucleotide phosphorylase plays an important role in the degradation of structured RNAs. However, RNase R differs from other exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3′ overhang is present. Using a variety of specifically designed substrates, we show here that a 3′ overhang of at least 7 nucleotides is required for tight binding and activity, whereas optimum binding and activity are achieved when the overhang is 10 or more nucleotides in length. In contrast, duplex RNAs with no overhang or with a 4-nucleotide overhang bind extremely poorly to RNase R and are inactive as substrates. A duplex RNA with a 10-nucleotide 5′ overhang also is not a substrate. Interestingly, this molecule is bound only weakly, indicating that RNase R does not simply recognize single-stranded RNA, but the RNA must thread into the enzyme with 3′ to 5′ polarity. We also show that ribose moieties are required for recognition of the substrate as a whole since RNase R is unable to bind or degrade single-stranded DNA. However, RNA molecules with deoxyribose or dideoxyribose residues at their 3′ termini can be bound and degraded. Based on these data and a homology model of RNase R, derived from the structure of the closely related enzyme, RNase II, we present a model for how RNase R interacts with its substrates and degrades RNA.

Exoribonucleases and their multiple roles in RNA metabolism

In recent years there has been a dramatic shift in our thinking about ribonucleases (RNases). Although they were once considered to be nonspecific, degradative enzymes, it is now clear that RNases play a central role in every aspect of cellular RNA metabolism, including decay of mRNA, conversion of RNA precursors to their mature forms, and end-turnover of certain RNAs. Recognition of the importance of this class of enzymes has led to an explosion of work and the establishment of significant new concepts. Thus, we now realize that RNases, both endoribonucleases and exoribonucleases, can be highly specific for particular sequences or structures. It has also become apparent that a single cell can contain a large number of distinct RNases, approaching as many as 20 members, often with overlapping specificities. Some RNases also have been found to be components of supramolecular complexes and to function in concert with other enzymes to carry out their role in RNA metabolism. This review focuses on the exoribonucleases, both prokaryotic and eukaryotic, and details their structure, catalytic properties, and physiological function.

Chapter 9 Maturation and Degradation of Ribosomal RNA in Bacteria

Ribosomal RNAs are the major components of ribosomes and are responsible for their catalytic activity. The three bacterial rRNAs (16S, 23S, and 5S) are cotranscribed as a single molecule that must be converted to the mature, functioning species through a series of nucleolytic processing events and base and sugar modifications that occur in the context of the assembling ribosome. One focus of this review is to examine the reactions that lead from the rRNA precursor to the mature species and to describe the ribonucleases (RNases) that carry out these processing reactions. rRNA, although usually stable in growing cells, also can be degraded if its assembly into ribosomes is aberrant or in response to certain stress conditions, such as starvation. The second focus of this review is to describe these degradative reactions, the RNases that carry them out, and the conditions that initiate the turnover process.

Efficient Mammalian Protein Synthesis Requires an Intact F-Actin System

The mammalian protein synthesizing system is highly organized in vivo, and its substrate, tRNA, is channeled throughout the translation process. However, the cellular components responsible for this organization are not known. To examine this question a series of studies was carried out using intact and permeabilized Chinese hamster ovary cells. We show that cold shock dramatically reduces the protein synthetic capacity of these cells by as much as 95%. The loss of activity can be reversed by a short recovery period under conditions that allow energy metabolism to occur; transcription and translation during the recovery period are not needed. While individual components of the translation apparatus are not inactivated by the cold shock, the supramolecular organization of the system appears to be altered and F-actin levels are found to decrease. Resumption of protein synthesis during the recovery period coincides closely with the restoration of F-actin to normal levels. Moreover, disruption of actin filaments, but not microtubules, also leads to a major reduction in translation. These data support the conclusion that the cellular microfilament network plays an important role in the structure and function of the translation system and that perturbations of this network can have profound effects on protein synthesis.