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Home , Promoter (genetics), Repressor, RpoB

Proc. Natl. Acad. Sci. USA Vol. 75, No. 8, pp. S89t--895,, August 1978 Cotranscription of for RNA subunits ,3 and I' with genes for ribosomal in Escherichia coli1 (transducing phages/ //RNA nucleotidyltransferase) MASAYUKI YAMAMOTO AND MASAYASU NOMURAt Institute for 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 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 unit described in the legends to the figures. is: , 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 form which confers Rif-R . Therefore, an understanding of the mechanisms 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 synthesis are interconnected. recombination between the hybrid phage 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 () 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 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 . 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. One and the mapping results obtained with the transformation was S159 and the other was its X lysogenic derivative, S159(X). technique (17, 18). Cells were irradiated with UV light to inactivate host gene To study the expression of bacterial genes carried by the functions and then infected with purified transducing phages; Downloaded by guest on October 2, 2021 Genetics: YamamotoandNomura Proc. Natl. Acad. Sci. USA 75 (1978) 3893

Re PI" V-4 r1ft c- Table 1. Synthesis of RNA polymerase subunits and r-proteins o 0 Lfl U) u)Lfl in UV-irradiated host cells %I.. 0 0 0 Promoter for m m m m Host rpoB,C Phage S159 S159(X) Bacterial Phage Xrifdl8 (I, (3', L1, L10, (3, (3', L1, L10, + _[6.8+16] L11, L7/L12 L11, L7/L12 - 18.6 3RP504ix (- - + - 15.4 3RP507i- - - - 12.3 +12.2 3RP51lix - - 3RP517ix (3, L10, L7/L12 (3*, L10, L7/L12 + - iOHF2 - _ i0HF4 ,B*, L7/L12 - - + 6 For ,B*, see the legend to Fig. 4. -5 - 4.4L+4.4R resis followed by autoradiography (Fig. 4). As can be seen from Fig. 4 and Table 1, when S159(X) was the host strain, the rpoB (j3 or its derivative, ,B*; - 2.8 see the legend to Fig. 4) was synthesized only after infection - 2.6 with 3RP517iV phage (lane 10) and not with the other three hybrid phages (lanes 7-9). Thus, only 3RP517ix, and not the other hybrid phages, carries a bacterial promoter for rpoB. FIG. 2. Agarose gel electrophoresis of EcoRI digests of hybrid When the nonlysogenic host, S159, was used, the rpoB gene phages. Bands are labeled with the approximate sizes in % X units. product was synthesized both with 3RP517ix (lane 5) and with Brackets indicate fragments formed from the cohesive joining of left and right ends. The DNA samples are identified above each lane. 3RP504ix (lane 2). Since 3RP504ix stimulated the synthesis of Electrophoresis was on 1% agarose gels, and gels were stained with the rpoB gene product only in the absence of the X ethidium bromide (16). whereas the control, Xrifdl8 (lanes 1 and 6), and 3RP517ix the proteins synthesized after infection were labeled with [35S]. Radioactive proteins were analyzed by sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electropho- l,'e *-756 ± 013w 3RP504 I =- R I~ 3RP507 i -- O 0 _Ep _ i - 18.6 + 10 "747±0.14DI HIN' 20.1 t 0. 8 - - _ _ ,

3RP504

I 3RP507 ,_ 74 ± 1.2 -w L10 MO L10 L11- 3.5+0.2 L7/12- _~ P_ -L7/12 1^ I 3 RP507 1 2 3 4 5 6 7 8 9 10 _~~~~~~~~~ 3RP51 S159 S159 A _- 20.5 + 1.1 ,1- 18.6 +F 0.6 _ FIG. 4. NaDodSO4polyacrylamide gel analysis ofproteins syn- thesized after infection of UV-irradiated S159 cells (lanes 1-5) and S159(A) cells (lanes 6-10) with various hybrid phages: Xrifdl8 (lanes 1 - 17.8 0.4 - 23.1 ± 0g9 1 and 6), 3RP504i0 (lanes 2 and 7), 3RP507ix (lanes 3 and 8), 3RP51lix (lanes 4 and 9), and 3RP517iX (lanes 5 and 10). Experiments were done 3RP511 as described (19) and proteins were labeled with [35SJmethionine. An -,. autoradiogram is shown. Identification of proteins synthesized after 3RP517 infection with Xrifdl8 was described previously (7, 19; and unpub- lished experiments) and their positions on the gel are indicated. ,B* 2.1±0.2 consists oftwo protein bands that moved slightly faster than the (3and FIG. 3. Schematic representation of the structure of heterodu- (3' protein bands. Specific crossreaction of (* with an antiserum plexes between transducing phages. Only the horizontal lines repre- against RNA polymerase was demonstrated (data not shown). Our sent DNA strands. The thin lines represent A (or 080) phage DNA; interpretation is that 3RP517ik does not have a complete rpoB gene boxes represent bacterial DNA. The filled boxes represent and the gene is fused to A DNA (see Fig. 1). This may have caused two DNA which is derived from Charon 3. Distances are given in kilobases; different artificial terminations (in transcription or in ), 0.465 kilobase corresponds to 1% A unit. leading to two protein species. Downloaded by guest on October 2, 2021 3894 Genetics: Yamamoto and Nomura Proc. Natl. Acad. Sci. USA 75 (1978) Eco RI (I) 44L26 6 t tf t p 80 (a) 3RP517 12.5 HindIII ) 4

(b) Charon 10 lac biG

2.6 6 - 40M NI 4 ml -~

(c) IOHF2

6 2.6 PL

(d ) IOHF4 FIG. 5. Structures of hybrid phages 10HF2 (c) and 1OHF4 (d) constructed from Charon 10 (b) and 3RP517 (a). Cloning of the 12.5% HindIll fragment present in 3RP517 was done with Charon 10. Both 3RP517 DNA and Charon 10 DNA were digested with HindIII sep- 4'.,W arately. The digests were mixed and ligated. Transfection and plating -L10 were done with strain KH802 as described in the legend to Fig. 1. The blue plaques that appeared on LB-Xgal plates were picked. The ..Basin...~~~~~~~~~~~~~~~~~~. phages in these plaques were screened for the presence of the rifd al- -L7/12 lele, as described in the legend to Fig. 1, except that a mixture of 0.05 ml each of overnight cultures of KH802 and S159(X) was used as in- dicator . Phages that gave rise to Rif-R colonies were purified and their DNA was analyzed by digestion with HindIII, EcoRI, and 1 2 3 4 5 6 7 8 HindIlI plus EcoRI. Structures of the two hybrid phages, determined by restriction enzyme analysis and by heteroduplex analysis, are S159 S159 X shown in c and d. FIG. 6. NaDodSO4/polyacrylamide gel electrophoresis of proteins synthesized after infection of UV-irradiated S159 and S159(X) cells with various phages: Charon 10 (lanes 1 and 5), 10HF2 (lanes 2 and showed stimulation both in the presence and the absence of the 6), 1OHF4 (lanes 3 and 7), and 3RP517iX (lanes 4 and 8). In S159 cells repressor, we conclude that the DNA segment carried by infected with Charon 10 (lane 1), a faint radioactive protein band can 3RP504ix does not have the intact promoter for rpoB and that be seen at approximately the same position as the 3* bands. Lanes the rpoB gene is fused to a major X promoter, probably X-PL, 3, 4, and 8 clearly show the two ,B* bands. Lane 2 does not appear to in 3RP504ix. This conclusion is consistent with the observation show the ,B* bands. For other details, see the legend to Fig. 4. that 3RP507ix carries the same DNA segment as that in 3RP504ix, but in the opposite orientation, and fails to stimulate of in host in the synthesis p the nonlysogenic (lane 3 Fig. 4). A 12.5% HindIII fragment carrying rpoB and rpll was iso- The bacterial promoter for rpoB must reside on a fragment lated from 3RP517 and inserted into the Charon 10 vector present in 3RP517iX but missing in 3RP5041X. The only such phage (Fig. 5; for Charon 10, see ref 15). Two hybrid phages is is to fragment the 4.4%L EcoRI fragment, which the right with the cloned fragment in different orientations were selected of the segment carried by 3RP504iA (Fig. la, c, and f). The for further analysis. The structures of these two phages are conclusion that the bacterial is on the 4.4%L promoter fragment shown in Fig. Sc and 5d. The structures were determined by is consistent with the finding that the hybrid phage 3RP51 1X restriction enzyme analysis with HindIII, EcoRI, and HindIII (Fig. le), which is identical in structure to 3RP517tX that except plus EcoRI, and by heteroduplex analysis (data not shown). it lacks the 4.4%L fragment, fails to express rpoB in either host (Fig. 4, lanes 4 and 9). UV-irradiated lysogenic and nonlysogenic cells were then in- The EcoRI-sensitive site between the 2.6% and 4.4%L frag- fected with these two transducing phages and proteins syn- ments is inside the L7/L12 gene (rplL) (10; and unpublished thesized after infection were analyzed by NaDodSO4/poly- data); therefore, the promoter for rpoB must be to the right of acrylamide gel electrophoresis. The results are shown in Fig. rplL (in Fig. la). It cannot be to the right of rplA or rplK (in 6 and Table 1. Both rpoB and rpIL gene products were syn- Fig. la) because 3RP517:X cannot stimulate the synthesis of Li thesized only in nonlysogenic host cells that had been infected or LIi, the protein products of these genes (Fig. 4, lanes 5 and with 10HF4 (Fig. 6, lane 3). In lysogenic host cells, lOHF4 10; Table 1). The promoter(s) for the rpIL and rplJ genes are failed to express both rpoB and rplL genes (lane 7). When carried intact by 3RP517iX since this phage can stimulate the lOHF2 was used, the expression of these genes was not observed synthesis of both L7/L12 and L10 (Fig. 4, lanes 5 and 10; Table in either host (lanes 2 and 6; see the legend to Fig. 6). In contrast, 1). Hence, the rpoB promoter is either between rpIL and rplJ the control, 3RP517i0, was able to express these genes in both or between rplJ and rplA. hosts (lanes 4 and 8), as described above. We conclude that the These two promoter locations can be distinguished. Within promoter for the rpoB and rpIL genes is not present on the DNA the 4.4%L EcoRI fragment, there is a HindIII-sensitive site (10) to the right of rplL and to the left of rplA (10). Recent DNA- segment cloned in these phages and that the expression of these sequencing experiments have demonstrated that the site is in genes in lOHF4 is caused by a X promoter to which these genes fact within the rpIJ gene (unpublished experiments). Therefore, are fused. From all the results discussed above, the promoter we asked whether the promoter for rpoB is to the left or right for the rpoB,C genes appears to be located to the right of rplJ of this site. but to the left of the rplA,K genes, as shown in the upper part Downloaded by guest on October 2, 2021 Genetics: Yamamoto and Nomura Proc. Natl. Acad. Sci. USA 75 (1978) 3895

of Fig. 1; that is, the rpoB,C genes appear to be cotranscribed 3. Kirschbaum, J. B. & Scaife, J. (1974) Mol. Gen. Genet. 132, with two neighboring r-protein genes, rplfJand rplL. 19S3-201. 4. Claeys, I. V., Miller, J. H., Kirschbaum, J. B., Nasi, S., Molholt, DISCUSSION B., Gross, G., Fields, D. A. & Bautz, E. K. F. (1976) in Control of Although the present work indicates that the rpoB,C genes Ribosome Synthesis, eds. Kjeldgaard, N. C. & Maale, 0. (Aca- demic Press, New York), pp. 56-65. share a common promoter with two r-protein genes, rplJ and 5. Bachmann, B. J., Low, K. & Taylor, A. L. (1976) Bacteriol. Rev. rplL, regulation of the synthesis of the P and ff subunits appears 40, 116-167. to be different from that of r-proteins L7/L12 and L10. First, 6. Jaskunas, S. R., Burgess, R. R. & Nomura, M. (1975) Proc. Natl. the synthesis of L7/L12 and L10 appears to be under the Acad. Sd. USA 72,5036-5040. stringent control system (20-22), while the synthesis of ft and 7. Lindahl, L., Jaskunas, S. R., Dennis, P. P. & Nomura, M. (1975) ,f is not (22-24). Second, the drug rifampicin induces increased Proc. Natl. Acad. Sd. USA 72,2743-2747. production of ft and fi', but not that of L7/L12 (25). Therefore, 8. Watson, R., Parker, J., Ffil, N. P., Flaks, J. G. & Friesen, J. D. the effects of amino acid starvation or rifampicin addition on (1975) Proc. Natl. Acad. Sd. USA 72,2765-2769. the synthesis of these proteins cannot be explained solely by 9. Bendiak, D. S., Parker, J. & Friesen, J. D. (1977) J. Bacteriol. 129, their possible effects on transcription initiation. More complex 536-59. 10. Lindahl, L, Yamamoto, M., Nomura, M., Kirschbauim, J. B., Allet, mechanisms, such as those involving attenuation, mRNA sta- B. & Rochaix, J.-D. (1977) J. Mol. Biol. 109,23-47. bility, or translational control, may have to be considered. This 11. Williams, B. G., Blattner, F. R., Jaskunas, S. R. & Nomura, M. may perhaps not be surprising, because a similar situation has (1977) J. Biol. Chem. 252, 7344-7354. already been observed regarding the effects of partial amino 12. Yamamoto, M., Lindahl, L. & Nomura, M. (1976) Cell 7,179- acid starvation on the expression of the rpoA gene and on r- 190. protein genes that are cotranscribed with the rpoA gene; the 13. Lindahl, L. & Nomura, M. (1976) in Control ofRibosome Syn- expression of the r-protein genes is under stringent control, but thesis, eds Kjeldgaard, N. C. & Maal*, 0. (Academic Press, New that of the rpoA gene is apparently not (or only weakly) under York), pp. 206-217. stringent control (22, 23). Other previous observations, however, 14. Wood, W. B. (1966) J. Mol. Biol. 16,118-133. support cotranscription or coregulation of RNA polymerase 15. Blattner, F. R., Williams, B. G., Blechl, A. E., Denniston- Thompson, K., Faber, H. E., Furlong, L.-A., Grunwald, D. J., genes and r-protein genes (26-28). In light of the conclusion Kiefer, D. 0., Moore, D. D., Schumm, J. W., Sheldon, E. L. & described in this paper, these and other earlier observations on Smithies, 0. (1977) Science 196, 161-169. the regulation of RNA polymerase subunit synthesis are clearly 16. Shinnick, T. M., Lund, E., Smithies, 0. & Blattner, F. R. (1975) worthy of further investigation. Nucleic Acids Res. 2,1911-1929. Our experiments indicate that the genes for L7/L12 (rplL) 17. Schweitzer, S. M. & Matzura, H. (1977) Mol. Gen. Genet. 155, and L10 (rplJ) are in the same transcription unit. These two 213-217. proteins interact in solution (29, 30; see also ref. 31 for the 18. Collins, J., Fill, N. P., Jorgensen, P. & Friesen, J. D. (1976) in analogous situation in Bacillus stearothermophilus) and Control ofRiasom Synthesis, eds. Kjeldgaard, N. C. & Maaloe, probably also within (see ref. 31). We also note that O. (Academic Press, New York), pp. 356-367. 19. Jaskunas, S. R., Lindahl, L., Nomura, M. & Burgess, R. R. (1975) both proteins are the most acidic 50S r-proteins and probably 257,458-462. correspond to the acidic 50S r-proteins that were reported to 20. Dennis, P. P. & Nomura, M. (1974) Proc. Natl. Acad. Sci. USA stimulate the transcription of phage T4 DNA in vitro (32). 71,3819-43823. Again, further studies are required to establish whether or not 21. Morrissey, J. J., Cupp, L. E., Weissbach, H. & Brot, N. (1976) J. these two r-proteins have any unique role in RNA polymerase Biol. Chem. 251,5516-5521. function or assembly in vivo. 22. Reeh, S., Pedersen, S. & Friesenj J. D. (1976) Mol. Gen. Genet. 149,279-289. Note Added in Proof. After submitting this paper, we learned that 23. Blumenthal, R. M., Lemaux, P. G., Neidhardt, F. C. & Dennis, T. G. Linn and J. Scaife have independently done experiments similar P. P. (1976) Mol. Gen. Genet. 149,291-296. to ours, and have reached the same conclusion, namely, that the rpoBC 24. Maher, D. L. & Dennis, P. P. (1977) Mol. Gen. Genet. 155, genes are cotranscribed with rplJ and rpIL. 203-211. We thank G. D. Strycharz and Nt Dietzman for their assance with 25. Hayward, R. S. & Fyfe, S. K. (1978) Mol. Gen. Genet. 160, experiments, and C. Gross and M. Susman for reading the manuscript. 77-80. We also thank Dr. B. G. Williams for suggesting a method to replace 26. Iwakura, Y., Ito, K. & Ishihama,.A. (1974) Mol. Gen. Genet. 133, the 480 immunity specificity of hybrid phages with A immunity 1-23. specificity. This work was supported in part by the College of Agri- 27. Iwakura, Y. & Ishihama, A. (1975) Mol. Gen. Genet. 142,67- culture and Sciences, University of Wisconsin and by grants from 84. the National Science Foundation (PCM7818490) and the National 28. Dennis, P. P. (1977) J. Mol. Biol. 115,603-625. Institutes of Health (GM-20427). 29. Pettersson, I., Hardy, S. J. S. & Liljas, A. (1976) FEBS Lett. 64, 135-138. 30. Chu, F., Caldwell, P., Samuels, M., Weissbach, H. & Brot, N. 1. Scaife, J (1976) in RNA Polymerase, eds. Losick, R. & Cham- (1977) Biochem. Biophys. Res. Commun. 76,593-601. berlin, M. (Cold Spring Harbor Laboratory, Cold Spring Harbor, 31. Marquis, D. & Fahnestock, S. R. (1978) J. Mol. Biol. 119,557- NY), pp. 207-225. 567. 2. Errington, L., Glass, R. E., Hayward, R. S. & Scaife, J. G. (1974) 32. Leavitt, J. C., Moldave, K. & Nakada, D. (1972) J. Mol. Biol. 70, Nature 249,519-522. 15-40. Downloaded by guest on October 2, 2021