Analysis of mRNA decay and rRNA processing in Escherichia coli in the absence of RNase E-based degradosome assembly

We demonstrate here that the assembly of the RNase E-based degradosome of Escherichia coli is not required for normal mRNA decay in vivo. In contrast, deletion of the arginine-rich RNA binding site (ARRBS) from the RNase E protein slightly impairs mRNA decay. When both the degradosome scaffold region and the ARRBS are missing, mRNA decay is dramatically slowed, but 9S rRNA processing is almost normal. An extensive RNase E truncation mutation (rnedelta610) had a more pronounced mRNA decay defect at 37 degrees C than the temperature-sensitive rne-1 allele at 44 degrees C. Taken together, these data suggest that the inviability associated with inactivation of RNase E is not related to defects in either mRNA decay or rRNA processing.     

RNase III cleavage of Escherichia coli beta-galactosidase and tryptophan operon mRNA.

Purified RNase III of Escherichia coli cleaved the initial 479-nucleotide sequence of lac operon mRNA at four specific sites and also gave limited cleavage of trp operon mRNA. This action explains the inactivation of mRNA coding capacity by RNase III in vitro.     

Escherichia coli recBC deletion mutants.

Mutants of Escherichia coli with deletions of the recB and recC genes were obtained by two methods using transposable DNA elements. The phenotypes of these mutants are similar to those of mutants with recBC point mutations. These results indicate that the RecBC gene products, exonuclease V, is not essential for the growth of E. coli but is important for DNA repair and recombination.     

RNase III controls the degradation of corA mRNA in Escherichia coli

In Escherichia coli, the corA gene encodes a transporter that mediates the influx of Co(2+), Mg(2+), and Ni(2+) into the cell. During the course of experiments aimed at identifying RNase III-dependent genes in E. coli, we observed that steady-state levels of corA mRNA as well as the degree of cobalt influx into the cell were dependent on cellular concentrations of RNase III. In addition, changes in corA expression levels by different cellular concentrations of RNase III were closely correlated with degrees of resistance of E. coli cells to Co(2+) and Ni(2+). In vitro and in vivo cleavage analyses of corA mRNA identified RNase III cleavage sites in the 5'-untranslated region of the corA mRNA. The introduction of nucleotide substitutions at the identified RNase III cleavage sites abolished RNase III cleavage activity on corA mRNA and resulted in prolonged half-lives of the mRNA, which demonstrates that RNase III cleavage constitutes a rate-determining step for corA mRNA degradation. These findings reveal an RNase III-mediated regulatory pathway that functions to modulate corA expression and, in turn, the influx of metal ions transported by CorA in E. coli.     

Novobiocin-dependent topA deletion mutants of Escherichia coli.

Previous reports of the transduction of topA deletions in Escherichia coli suggested that delta top A transductants grow normally only if they acquire spontaneous mutations that compensate for the topoisomerase I defect. We show that P1-mediated transduction of delta topA in the presence of sublethal concentrations of novobiocin, an inhibitor of the DNA gyrase B subunit, yields uncompensated Top- isolates which are dependent on novobiocin for optimum growth. In the absence of novobiocin these delta topA strains grow slowly, indicating that topA deletions are deleterious but not lethal to the cell. We propose that inhibitors of DNA gyrase B, presumably by lowering intracellular levels of DNA supercoiling, can phenotypically suppress a topoisomerase I defect in E. coli.     

The role of Escherichia coli RNase E and RNase III in the processing of the citQRP operon mRNA from Lactococcus lactis biovar diacetylactis

Citrate transport in Lactococcus lactis biovar diacetylactis (L. diacetylactis) is catalyzed by citrate permease P (CitP), which is encoded by the plasmidic citP gene. Two partial overlapping open reading frames citQ and citR are located upstream of citP. These two genes, together with citP, constitute the citQRPoperon. In this report it was shown that in L. diacetylactis and Escherichia coli, cit mRNA is subject to the same specific cleavages at a complex secondary structure which includes the central region of citQ and the 5'-end of citR. The role of ribonucleases in the fate of the cit mRNA processing was investigated in E. coli RNase mutant strains. The results obtained indicate that both endoribonucleases RNase E and RNase III are involved in the generation of mRNA processed species. RNase E is responsible for the major cleavages detected within citQ and upstream of citR, whereas RNase III cleaves citR within its ribosomal binding site. Preliminary results indicate the existence of a RNaselll-like enzyme in L. diacetylactis. Based on these results, a model for the role of cit mRNA processing in the expression of citP is presented.     

Ectopic RNase E sites promote bypass of 5'-end-dependent mRNA decay in Escherichia coli

In Escherichia coli, 5'-terminal stem-loops form major impediments to mRNA decay, yet conditions that determine their effectiveness or the use of alternative decay pathway(s) are unclear. A synthetic 5'-terminal hairpin stabilizes the rpsT mRNA sixfold. This stabilization is dependent on efficient translational initiation and ribosome transit through at least two-thirds of the coding sequence past a major RNase E cleavage site in the rpsT mRNA. Insertion of a 12-15 residue 'ectopic' RNase E cleavage site from either the rne leader or 9S pre-rRNA into the 5'-non-coding region of the rpsT mRNA significantly reduces the stabilizing effect of the terminal stem-loop, dependent on RNase E. A similar insertion into the rpsT coding sequence is partially destabilizing. These findings demonstrate that RNase E can bypass an interaction with the 5'-terminus, and exploit an alternative 'internal entry' pathway. We propose a model for degradation of the rpsT mRNA, which explains the hierarchy of protection afforded by different 5'-termini, the use of internal entry for bypass of barriers to decay, 'ectopic sites' and the role of translating ribosomes.     

Autoregulation of RNase III operon by mRNA processing.

RNase III has been implicated in the control of gene expression by the processing of mRNA. We have found that the rnc operon is autoregulated; rnc- mutant strains oversynthesize the operon's mRNA and protein products. A site in the 5'-noncoding region of the operon's message is cleaved by RNase III. This site-specific cleavage appears to be the initial step in the functional inactivation of the message, since the half-life of the cut message is dramatically shorter than that of the uncut message.     

RNase I*, a form of RNase I, and mRNA degradation in Escherichia coli.

A previously unreported endoRNase present in the spheroplast fraction of Escherichia coli degraded homoribopolymers and small RNA oligonucleotides but not polymer RNA. Like the periplasmic endoRNase, RNase I, the enzyme cleaved the phosphodiester bond between any nucleotides; however, RNase I degraded polymer RNA as fast as homopolymers or oligomers. Both enzymes migrated as 27-kDa polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and could not be separated by various chromatographic procedures. In rna insertion mutants, both enzymes were completely missing; the spheroplast enzyme is called RNase I*, since it must be a form of RNase I. The two forms could be distinguished by physical treatments. RNase I could be activated by Zn2+, while RNase I* was inactive in the presence of Zn2+. RNase I was inactivated very slowly at 100 degrees C over a wide pH range, while RNase I* was inactivated slowly by heat at pH 4.0 but much more rapidly as the pH was increased to 8.0. In the presence of a thiol-binding agent, the inactivation at the higher pH values was much slower. These results suggest that RNase I*, but not RNase I, has free sulfhydryl groups. RNase I* activity in the cell against a common substrate was estimated to be several times that of RNase I. All four 2',3'-phosphomonoribonucleotides were identified in the soluble pools of growing cells. Such degradative products must arise from RNase I* activity. The activity would be suited for the terminal step in mRNA degradation, the elimination of the final oligonucleotide fragments, without jeopardizing the cell RNA. An enzyme with very similar specificity was found in Saccharomyces cerevisiae, suggesting that the activity may be widespread in nature.     

Mutator mutants of Escherichia coli carrying a defect in the DNA polymerase III tau subunit

To investigate the possible role of accessory subunits of Escherichia coli DNA polymerase III holoenzyme (HE) in determining chromosomal replication fidelity, we have investigated the role of the dnaX gene. This gene encodes both the tau and gamma subunits of HE, which play a central role in the organization and functioning of HE at the replication fork. We find that a classical, temperature-sensitive dnaX allele, dnaX36, displays a pronounced mutator effect, characterized by an unusual specificity: preferential enhancement of transversions and -1 frameshifts. The latter occur predominantly at non-run sequences. The dnaX36 defect does not affect the gamma subunit, but produces a tau subunit carrying a missense substitution (E601K) in its C-terminal domain (domain V) that is involved in interaction with the Pol III alpha subunit. A search for new mutators in the dnaX region of the chromosome yielded six additional dnaX mutators, all carrying a specific tau subunit defect. The new mutators displayed phenotypes similar to dnaX36: strong enhancement of transversions and frameshifts and only weak enhancement for transitions. The combined findings suggest that the tau subunit of HE plays an important role in determining the fidelity of the chromosomal replication, specifically in the avoidance of transversions and frameshift mutations.     

Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants

The endoribonuclease RNase E of Escherichia coli is an essential enzyme that plays a major role in all aspects of RNA metabolism. In contrast, its paralog, RNase G, seems to have more limited functions. It is involved in the maturation of the 5′ terminus of 16S rRNA, the processing of a few tRNAs, and the initiation of decay of a limited number of mRNAs but is not required for cell viability and cannot substitute for RNase E under normal physiological conditions. Here we show that neither the native nor N-terminal extended form of RNase G can restore the growth defect associated with either the rne-1 or rneΔ1018 alleles even when expressed at very high protein levels. In contrast, two distinct spontaneously derived single amino acid substitutions within the predicted RNase H domain of RNase G, generating the rng-219 and rng-248 alleles, result in complementation of the growth defect associated with various RNase E mutants, suggesting that this region of the two proteins may help distinguish their in vivo biological activities. Analysis of rneΔ1018/rng-219 and rneΔ1018/rng-248 double mutants has provided interesting insights into the distinct roles of RNase E and RNase G in mRNA decay and tRNA processing.     

Arabinose-leucine deletion mutants of Escherichia coli B-r

The control of ara gene expression was studied in mutants of Escherichia coli B/r containing deletions which fused the l-arabinose gene complex with the leucine operon (the normal gene order being araDABIOC...leuDCBAO). Complementation experiments with stable merodiploids showed that expression of ara genes cis to araC-leu deletions was controlled by the trans-acting product of the araC gene. Expression of ara genes cis to araB-leu deletions was under leucine control. These studies confirm the existence of a region between genes araC and araB essential for normal activator controlled expression of the ara structural genes. One deletion was characterized as an araO-leu deletion. Its effect on ara gene expression was unique in that ara genes were susceptible to potential regulation by both l-arabinose and leucine. These experiments suggest that two different species of messenger ribonucleic acid (mRNA) may be produced for the ara-leu region as a result of this deletion. One, under l-arabinose-activator control, is initiated in the l-arabinose region; the other, under leucine control, is initiated in the leucine region. The latter indicates that araI can be transcribed. Whether araI is transcribed in the former instance (mRNA made under activator control) remains to be established.