Treatment of sensitive Escherichia coli cells with colicin E3 leads to inactivation of 30S ribosomal subunits. In vitro reconstitution of 30S subunits indicates that the E3-induced defect lies solely in the 16S RNA. 16S RNA from E3-treated cells lacks several T1 RNase oligonucleotides of normal 16S RNA, including the one from the 3'-end of the 16S RNA, as analyzed by the fingerprint technique of Sanger. An RNA fragment about 50 nucleotides long has been isolated from E3-treated cells. This RNA contains the original 3'-terminal oligonucleotide and other oligonucleotides missing in the E3-16S RNA. The results show that colicin E3 treatment causes the cleavage of 16S RNA at a specific position near the 3'-terminus. Preparation of 16S RNA and total 30S proteins, as well as the method of reconstitution of 30S subunits, was described previously (5,8). Ribosomal RNA was analyzed on 3% acrylamide-0.5% agarose column gels or 10% acrylamide slab gels in Tris-EDTA-borate buffer, pH 8.3 (9).The purified RNAs were studied by the fingerprint technique of Sanger et al. (10, 11) after they were digested with T,RNase and alkaline phosphatase. The fingerprints obtained were compared with those obtained by Fellner and his coworkers (12), in order to assign numbers to the various oligonucleotides. The molar yield of each product was determined by counting, in a scintillation counter, the area of paper containing it. Particular oligonucleotides were characterized after elution from the paper by digestion with 5 ul of pancreatic RNase (0.1 mg/ml in 10 mM Tris (pH 7.6)-i mM EDTA plus 2 mg/ml of carrier RNA) for 30 min at 37°C and electrophoresis at pH 3.5 on DEAE-cellulose paper (13). RESULTS Reconstitution of 30S particles with components from E3-inactivated 30S subunits ("E3-30S")We have used the ribosome reconstitution technique (5) to determine which component is responsible for the inactivity of E3-30S. As shown in Table 1
Energy-coupled reactions of the Escherichia coli outer membrane transport proteins BtuB and Cir require the tonB product. Some point mutations in a region of btuB and cir that is highly conserved in TonB-dependent transport proteins led to loss of TonB-coupled uptake of vitamin B12 and colicin Ia, whereas binding was unaffected. Most other point mutations in this region had no detectable effect on transport activity. Mutations in tonB that suppressed the transport defect phenotype of these btuB mutations were isolated. All carried changes of glutamine 165 to leucine, lysine, or proline. The various tonB mutations differed markedly in their suppression activities on different btuB or cir mutations. This allele specificity of suppression indicates that TonB interacts directly with the outer membrane transport proteins in a manner that recognizes the local conformation but not specific side chains within this conserved region. An effect of the context of the remainder of the protein was seen, since the same substitution (valine 1O-*glycine) in btuB and cir responded differently to the suppressors. This finding supports the proposal that TonB interacts with more of the transport proteins than the first conserved domain alone.The outer membrane of Escherichia coli contains several high-affinity, active-transport systems for substrates, such as ferric-siderophore complexes and cobalamins, which are too large to diffuse effectively through the nonspecific porin channels. Transport is mediated by minor outer membrane proteins that bind these substrates with high affinity and specificity and that can serve as receptors for the adsorption and entry of specific bacteriophages and bacteriocins. The BtuB polypeptide is responsible for the uptake of vitamin B12 and other cobalamins, bacteriophage BF23, and colicins A and E. The FepA protein transports ferric enterochelin and colicins B and D. The iron-repressible Cir protein was initially identified as the receptor for colicin I, has been implicated in the uptake of catechol-substituted cephalosporins, and may mediate uptake of ferric complexes of other 2,3-dihydroxybenzoate derivatives (4).Active transport of most of the substrates listed above also requires functioning of the tonB product and is stimulated by the products of exbB and exbD (1, 7-9, 12). In the absence of tonB function, the outer membrane transport proteins are produced in normal or larger amounts and still bind their substrates effectively, but they are unable to carry out the energy-dependent steps of substrate accumulation (7). Analysis of cobalamin uptake in btuC mutants blocked in the cytoplasmic membrane transport system suggested that tonB-dependent active transport across the outer membrane is driven by the proton motive force (23). It was suggested that TonB couples energy from the proton motive force across the cytoplasmic membrane to the outer membrane transporters (7,12,23).The deduced sequences of tonB-dependent transport proteins possess short, highly conserved regions separated by long variable...
The molecular basis for the greatly elevated expression of the cir gene (encoding the colicin I receptor) in cells unable to maintain a critical supply of intracellular iron was investigated by genetic and biochemical means. Deletion analysis of the cloned promoter region allowed delineation of sequences necessary for control of transcription initiating at the two promoters, P1 and P2. Gel retardation assays were used to demonstrate both binding of purified Fur (ferric uptake regulation) protein to the iron control region and lack of binding to DNA fragments which are not involved in cir regulation. An operator sequence spanning 43 to 47 base pairs and completely encompassing the two promoters was identified by DNase I protection experiments (footprinting), with binding occurring in a metal-dependent fashion. Thus, during iron-replete growth, Fur appears to act as a repressor of transcription by blocking formation of a DNA-RNA polymerase complex, analogous to the mechanism previously described for regulation of the aerobactin operon (V. de Lorenzo, S. Wee, M. Herrero, and J. B. Neilands, J. Bacteriol. 169:2624Bacteriol. 169: -2630Bacteriol. 169: , 1987. Characterized and putative Fur recognition sites from several genes were analyzed and classified by statistical methods.
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