Molecular cloning of the gene for the RNA-processing enzyme RNase III of Escherichia coli.

Abstract

A ColEl plasmid from the Clarke and Carbon collection [Clarke, L. & Carbon, J. (1976) CeU 9, 91-99] that contains a 14.4-kilobase Escherichia coli DNA insert complements the rnc-105 mutation, which destroys the activity of the RNA-processing enzyme RNase III. This insert and smaller restriction endonuclease fragments derived from it were cloned into the plasmid pBR329. A number of these recombinant plasmids complemented the rnc-lOS mutation in a recA genetic background. The smallest cloned fragment that compensated for the rnc-105 mutation was 1.3 kilobase in size. This fragment led to the synthesis of two polypeptides. One of these polypeptides was 25,300 daltons and corresponded in size to the subunit of RNase HI. Fragments cloned in opposite orientations led to synthesis of RNase III, indicating that the cloned fragments contained an endogenous promoter. Extracts of an rnc' E. coli strain containing an rnc' plasmid had at least 10 times more RNase III activity than did an analogous strain containing the pBR329 plasmid.Within the past decade the importance of RNA processing has emerged in a wide variety of biological systems (1-3). The cloning of genes for precursor RNAs and RNA-processing enzymes could greatly assist the study of RNA processing. Although the genes for many RNA precursors have been cloned, thus far cloning has contributed only marginally to the study of the proteins that mediate many of the RNAprocessing reactions that occur in the cell (4-6). Since the endonuclease RNase III introduces specific cleavages in a number of RNA (rRNA, tRNA, and mRNA) precursors in Escherichia coli and bacteriophages that parasitize it (1-3), we set out to clone the gene for this enzyme. The work described here depended on the existence of the rnc-105 mutation (7) and was aided by the previous purification of RNase III (8,9) and the demonstration that this enzyme is a dimer of two homologous polypeptides, 25,000 daltons each (9). MATERIALS AND METHODSBacterial Strains. All strains were E. coli K-12. JC10240 was srlC30::TnlO, recA56 obtained from Alvin J. Clark (10). X1411 (11) was a minicell-producing strain provided by Roy Curtiss III. N2076 was wild type for RNase III and has been described (12). N2100 was an rnc-105, arg41, purI66, str-9 strain prepared similarly to the previously described N2077 (12) with selection for NAD independence (Nad+). N3589 was an rnc-105, rne-3071 double mutant constructed similarly to N3520 (13). N3441 was constructed by a prolonged mating between an rne-3071 mutant, N3431 (14), and N7060 (12), with selection for streptomycin resistance (Strr) and tyrosine independence (Tyr'). N4021 was an recA56, rnc-105 strain constructed as follows. A Pl-mediated transduction (15) was carried out with JC10240 as donor and N2100 as recipient with selection for tetracycline resistance (Tetr). A resulting Rec-recombinant, N4019, was then used as a recipient in a P1-mediated transduction with D10 (16) as a donor and selection for sorbitol independence (Srl+). A resulting recombinant which wa...

A GTP-binding protein (Era) has an essential role in growth rate and cell cycle control in Escherichia coli

“…This possibility e out because none of the PAM or JFL strains 4 Table 1 are recA and they still show the era phe selection of recombinants with pNG187. The fin amount of RNase III (encoded by rnc) and Era present in wild-type E. coli K-12 is small compared with that of other proteins is in agreement with the conclusion that Era is a regulatory protein (3,8,15,22).…”

Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III

“…One distinct polypeptide band whose synthesis was directed by the mc+ insert of pBK13 was identified (lane 2). The apparent molecular mass of this polypeptide is 27 kDa, in close agreement with the reported 26-kDa molecular mass that was observed for RNase III (54 (Fig. 1A, lane 1) but not in the lane corresponding to the mutant strain (SK7621) (Fig.…”

“…The plasmids used in this work are described in Table 2. Plasmid pBK2 was constructed by subcloning the 4.3-kbp EcoRI fragment containing mc from pLC7-47 (54) into the EcoRI site of pLG339 (43). Plasmid pBK12 was made by subcloning the 4.3-kbp EcoRI fragment containing mc from pLC7-47 into the EcoRI site of pBR325 (35).…”

Genetic analysis of the rnc operon of Escherichia coli

“…The rnc gene has been cloned previously, and the sequence of a -5-kilobase-pair (kbp) segment of DNA contiguous to rnc has been reported (1,37,38,43,55,58). Genetic studies reported here show that rnc, era, and recO form an operon.…”