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Cotranscription of Genes for RNA Polymerase Subunits ,3 and I' With Proc. Natl. Acad. Sci. USA Vol. 75, No. 8, pp. S89t--895,, August 1978 Genetics Cotranscription of genes for RNA polymerase subunits ,3 and I' with genes for ribosomal proteins in Escherichia coli1 (transducing phages/gene cloning/rifampicin/RNA nucleotidyltransferase) MASAYUKI YAMAMOTO AND MASAYASU NOMURAt Institute for Enzyme Research, Departments of Genetics and Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 Contributed by Masayasu Nomura, May 24,1978 ABSTRACT The Xrifdi8 transducing phage carries genes (cI857). This was done in the following way. Strain Ymel was for RNA polymerase (nucleosidetriphosphate:RNA nucleoti- infected with one of the hybrid phages and XplacS; progeny dyltransferase; EC 2.7.7.6) subunits ft and , (MpoBC) and genes for four ribosomal proteins (rplK for Lii, ipM for LI, rpIJ for phages were plated on a 080 lysogen of strain Ymel using the LIO, and rplL for L7/L12) DNA segments of various sizes, lac indicator plate, LB-Xgal (11). Colorless (Lac-) plaques were which cover the rif'd allele of the rpoB gene, were cloned into picked and the structures of recombinant phages were verified X vector phages. The hybrid phages were then analyzed for their by EcoRI restriction enzyme digestion and by heteroduplex abilit* to express the rpoB gene and neighboring ribosomal analyses. The experiments were carried out according to the protein genes in ultraviolet-irradiated X-lysogenic and nonly- National Institutes of Health guidelines, which recommend P1 sogenic bacterial hosts. The results show that the rpoB gene is cotranscribed with two neighboring ribosomal protein genes physical and EK1 biological containment. Other methods are and that the order of the genes in the rpoBC transcription unit described in the legends to the figures. is: promoter, rplJ, rpIL, rpoB, and zpoC. RESULTS RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl- The Xrifdl8 phage carries both the rpoB and the rpoC genes transferase; EC 2.7.7.6) in Escherichsa coli is responsible for and their promoter (3, 12). The rpoB (rpoB3) gene carried by transcription of genetic information encoded in the bacterial the transducingphage is a mutant form which confers Rif-R chromosome. Therefore, an understanding of the mechanisms phenotype and is dominant over the rifampicin-sensitive wild involved in the regulation of biosynthesis of RNA polymerase type. Our previous studies have shown the approximate loca- is important (for a review, see ref. 1). RNA polymerase consists tions of four r-protein genes and the rpoB,C genes on the of a core enzyme with the subunit structure a2flf', which to- Xrifdl8 chromosome (10, 13; see Fig. 1). In order to define the gether with the a factor comprises the holoenzyme. The genes location of the promoter for the rpoB,C genes in relation to for ,3 and f3' (rpoB and rpoC, respectively) belong to a single neighboring r-protein genes, we partially digested Xrifdl8 DNA transcription unit (2-4) and map at 88.5 min on the revised E. with EcoRI restriction endonuclease and cloned DNA frag- coli genetic map (5). The gene for the a subunit (rpoA) maps ments carrying the rifd (or rpoB3) allele into a X vector phage at 72 min and appears to be cotranscribed with at least three Charon 3 (see ref. 15). The presence of the rifd allele was first ribosomal protein (r-protein) genes (rpsK, rpsD, and rplQ, confirmed by the ability of the constructed hybrid phages to coding for SlI, S4, and L17, respectively) and probably with confer Rif-R character on a rifampicin-sensitive bacterial strain an additional gene (rpsM coding for S13) (6). This suggests that by transduction. Rif-R transductants can arise either by lyso- the regulatory system for RNA polymerase synthesis and that genization of the recipient strain with the hybrid phages or by for ribosome synthesis are interconnected. recombination between the hybrid phage genome and the Previous studies have shown that there are four r-protein bacterial chromosome. We then asked whether the rpoB gene genes mapped close to the rpoB,C genes (7-10; see Fig. 1). on the cloned DNA fragment has a bacterial promoter for its Therefore, we explored the possibility that rpoB,C genes are expression, or if it has fused to the X promoter for its expression, cotranscribed with some or all of these neighboring r-protein or if it cannot be expressed directly because of the absence of genes, as is the case with rpoA. In this paper, we describe ex- promoters. This approach is formally analogous to deletion periments that show that the major promoter for the rpoB,C mapping of promoters. transcription unit (operon) is between rplA and rplJ and the Fig. 1 shows locations of several restriction enzyme-sensitive order of the genes is: promoter, rplJ, rplL, rpoB, and rpoC. sites on Xrifdl8 DNA (Fig. la), the structure of the Charon 3 Thus, genes for all subunits of core polymerase are cotranscribed vector phage (Fig. lb), and the structures of four hybrid phages with r-protein genes. relevant.to the present discussion (Fig. 1 c-f). The structures of the hybrid phages were established by restriction enzyme MATERIALS AND analysis of their DNA by EcoRI (Fig. 2), Sal I, and Sal I plus METHODS EcoRI (data not shown; see Fig. 1) and by DNA-DNA hetero- Details of the cloning methods used have been described (11), duplex analysis (Fig. 3). Several EcoRI fragments in each of the and are outlined in the legends to Figs. 1 and 5. The Charon 3 four DNA segments cloned in the hybrid phages are contiguous phage has 080 immunity specificity, and the four hybrid phages and correspond to an "intact" segment of the Arifdl8 genome derived from it (see Fig. 1) were crossed with Xplac5 to replace whose size varies depending on the hybrid phages. Since all the the 480 immunity specificity with X immunity specificity hybrid phages with the rifd allele (the four shown in Fig. 1 and The costs of publication of this article were defrayed in part by the Abbreviations: r-protein, ribosomal protein; Rif-R, rifampicin-resistant payment of page charges. This article must therefore be hereby marked or rifampicin-resistance; NaDodSO4, sodium dodecyl sulfate. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate t This is paper number 2244 from the Laboratory of Genetics. this fact. t To whom requests for reprints should be addressed. 3891 Downloaded by guest on October 2, 2021 3892 Genetics: Yamamoto and Nomura Proc. Natl. Acad. Sci. USA 75 (1978) p- rp -R ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~- rp rpoC rpo8 L J A K --Nb MM - -. .l .M EcoRI(t) 16.0 5 2.8 6 2.6 4.4 L 18.6 i 4 i SaI (4) 9.3 6.4 5.5 # 2.5 + 23.0 Ho HindIIIdO) Eco RI (f ) 16.0 5 2.8 6 2.6 4.4L 18.6 - 4.4 R 12.3 15.4 12.2 6.8 4'i (a) Xrif 18 lac P 80 L (b) Charon 3 5 2.8 6 2.6 (c) 3RP504 .0 2.6 6 2.8 5 (d) 3RP507 2.6 6 (e) 3RP511 4.4L2.6 6 (f ) 3RP517 No FIG. 1. Structures of the Xrifdl8, Charon 3, and hybrid phages used in this work. Lines represent (or 480) DNA; boxes represent bacterial DNA. All the EcoRI-sensitive sites are shown, while Sal I- and HindIII-sensitive sites are shown only on the expanded part of the Xrifdl8 chro- mosome. Lengths of DNA are shown in % X units. 1% X unit is 465 base pairs. Locations of the genes and promoters shown above the Xrifdl8 chromosome are based on the present and previous work (10; see text). The right end of the 4.4%L EcoRI fragment is within the gene for L11 (rplK; unpublished data) and the order of the four r-protein genes is now unambiguously determined. Hybrid phages were constructed as follows. Charon 3 DNA was digested completely with EcoRI enzyme. Xrifdl8 DNA was digested with one-fourth of the minimum amount of EcoRI enzyme necessary for complete digestion. After inactivation of the EcoRI enzyme with diethylpyrocarbonate, both DNAs were mixed and ligated with T4 DNA ligase, and transfection was carried out with E. coli strain KH802 (11, 14). Plating was done on the lac indicator plate, LB-Xgal (11). Colorless (Lac-) plaques were picked and the ability of phages to confer rifampicin resistance (Rif-R) on 480 phage lysogens was tested. One-tenth milliliter of overnight culture of KH802 was infected with 480 (multiplicity 3) and then spread on LB plates. Individual phage stabs were made on these plates and the plates were incubated at 370 for 3 hr. Soft agar containing rifampicin (1.5 mg per plate) was then overlaid on the plates and incubation was continued. Phages that gave rise to Rif-R colonies were purified and their DNA was analyzed by digestion with restriction enzymes. The structures of four hybrid phages are shown. Arrows indicating the directions of transcription are used to show the orientation of the cloned fragments. others not shown) contain the original 6% EcoRI fragment (but hybrid phages, we first replaced the immunity region of the not necessarily the 2.6% fragment), the rifd allele must be lo- hybrid phages by that of XcI857 by genetic cross and'used the calized on this EcoRI fragment. This is consistent with the re- resultant iX derivatives of these phages for the experiments. Two sults of our previous work on the physical mapping of the host strains were used to analyze the expression of the rpoB gene rpoB,C genes using the in vtro protein synthesis technique (10) and the other bacterial genes carried by the hybrid phages.
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