Specific Inactivation of 16S Ribosomal RNA Induced by Colicin E3 In Vivo

Abstract

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

The sequence of the 3′‐OH end of the 16 S RNA ofEscherichia coli

“…This report deals with the sequence of these 2 bands. Our data are almost in complete agreement with the components found by Bowman et al [2], in the colicin E3 released fragment but not with the sequence reported by Ehresmann et al [3] , for the terminal 38 nucleotides in the 3'-OH end of the 16 S RNA.…”

“…Fhresmann et al have not reported on the purity of their 3'-OH fragment, so it is not possible to judge whether this may account for the difficulties. The possibility remains that there are strain differences, since there are minor differences between our results and those of Bowman et al [2] on the composition of the E3 fragment.…”

“…2a. Both E3 fragments contain all the components reported by Bowman et al [2] except UC and AC. From the data in fig.…”

“…Five methyl groups are found here [2] ,4 of them on adjacent adenylic acid residues in oligonucleotide 71 [8]. The presence or absence of these 4 methyl groups determines kasugamycin sensitivity or resistance of 30 S ribosomes [9].…”

“…Among these pieces is a fragment from the 3'-OH end of the 16 S RNA which contains 26 nucleotides (band 3') [I] . When colicin E3 reacts with 70 S ribosomes, either in vivo or in vitro, it releases a 50 nucleotide fragment from the 3'-OH end of the 16 S RNA [2]. This report deals with the sequence of these 2 bands.…”