RNase III, an Escherichia coli double-stranded endoribonuclease, is known to be involved in maturation of rRNA and regulation of several bacteriophage and Escherichia coli genes. Clones of the region of the E. coli chromosome containing the gene for RNase III (rnc) were obtained by screening genomic libraries in A with DNA known to map near rnc. A phage clone with the rnc region was randomly mutagenized with a ATnJO element, and the insertions were recombined onto the chromosome, generating a series of strains with ATn10 insertions in the rnc region. Two insertions that had Rnc-phenotypes were located. One of them lay in the rnc gene, and one was in the rnc leader sequence. Polarity studies showed that rnc is in an operon with two other genes, era and recO. The sequence of the recO gene beyond era indicated it could encode a protein of -26 kilodaltons and, like rnc and era, had codon usage consistent with a low level of expression. Experiments using antibiotic cassettes to disrupt the genes rnc, era, and recO showed that era is essential for E. coli growth but that rnc and recO are dispensable.RNase III is an Escherichia coli endoribonuclease that cleaves specific double-stranded RNA structures (12,17). It is known to initiate maturation of rRNA from 30S precursor RNA (11,49) and has been implicated in the posttranscriptional processing of the mRNA transcripts of the early genes of bacteriophage T7 (15) Auxotrophs were screened on M56 glucose minimal plates. Each plate was spread with 100 ,ul of hypoxanthine (7.5 mg/ml; purl), glycine (1%; glyA), or nicotinic acid (0.25%; nadB), as appropriate. Antibiotics were used in the following final concentrations (micrograms per milliliter):ampicillin, 50; kanamycin, 25; chloramphenicol, 10; and tetracycline, 12.5. For growth of tetracycline-dependent (Tetd) strains in liquid medium, tetracycline was used at 3.0 ,ug/ml. Development of lambda clones and recombinants. Two different lambda phage libraries of the E. coli genome
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.
The mini-TnlO transposon (A16A17TnJO) confers tetracycline resistance. When inserted between a gene and its promoter, it blocks transcription and prevents expression of that gene. Tetracycline in the medium induces divergent transcription of the tetA and tetR genes within the transposon, and this transcription extends beyond the transposon in both directions into the bacterial genes. If the mini-TnlO inserts between an essential bacterial gene and its promoter, th,e insertion mutation can cause conditional growth which is dependent on the presence of tetracycline. Two essential genes in adjacent operons of Escherichia coli have been detected by screening for tetracycline dependence among tetracycline-resistant insertion mutants. These essential genes are the era gene in the rnc operon and the dpj gene in the adjacent pdxJ operon. The pdxJ operon has not been described previously. It consists of two genes, pdxJ and dpj. Whereas the dpj gene is essential for E. coli growth in all media tested, pdxJ is not essential. The pdxJ gene encodes a protein required in the biosynthesis of pyridoxine (vitamin B6).Conditional mutations facilitate the study of bacterial genes in two ways. They allow detection of physiological changes under nonpermissive conditions and provide the opportunity to select suppressor mutations. In addition to conditional lethal mutations of the thermosensitive type, conditional detects dependent on expression from heterologous promoters are also valuable for study. Chow and Berg (6) designed a transposition vector, TnStacl, to detect essential genes in Escherichia coli as conditionally lethal transposition mutations. Mutants generated depend on induction of the tac promoter from the transposon to express essential genes when their normal expression has been blocked by insertion of the transposon. We show here that a TnJO derivative can also be used to isolate a class of conditionally lethal mutations.We have defined three genes of the mc operon: mc, era, and recO (31). These genes encode the proteins RNaseIII, a double-strand-dependent endoribonuclease (7); Era, a GTPbinding protein (5, 21); and RecO, required for DNA repair and RecF pathway recombination (25). Of these, only era is essential for bacterial growth (14, 31). We previously isolated an E. coli mutant in which a mini-TnlO (A16A17TnJO; 33) inserted in the leader sequence of the mc operon caused the cell to become conditionally dependent on tetracycline for growth. This insertion mutation, mc-40, caused era expression to be defective in the absence of tetracycline presumably because the mini-TnlO element was blocking normal transcription from the mc promoter. We proposed that tetracycline, which induces the divergent promoters for the tetA and tetR genes of the mini-TnlO transposon (13), allowed transcription of these genes to continue beyond the TnlO element and into the era gene. In this study, we have examined expression of lacZ fused to the mc operon both with and without the mc-40 mini-TnJO insertion (A40). We have also isolated and ...
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