Antitermination of transcription from an Escherichia ... - Semantic Scholar

Proc. Nail. Acad. Sci. USA Vol. 81, pp. 6789-6793, November 1984 Genetics

Antitermination of transcription from an Escherichia coli ribosomal RNA promoter (operon fusion/rrnC promoter-leader region/lacZYA genes/insertional polarity)

WILLIAM E. HOLBEN

AND

EDWARD A. MORGAN*

Department of Biology, State University of New York at Buffalo, Buffalo, NY 14260

Communicated by H. E. Umbarger, July 9, 1984

ABSTRACT The Escherichia coli lac and ara promoters and rrnC ribosomal RNA promoter-leader region were fused to lacZYA. Transcription termination signals were introduced into the lac genes of these fusions by Tn9 and ISI insertions. Measurement of lac enzymes from upstream and downstream of the insertions showed that termination signals resulting from these insertions are very efficient when transcription begins at lac or ara promoters but are very inefficient when transcription begins at the rrnC promoter-leader region. The rrnC promoter-leader region must, therefore, modify RNA polymerase to enable it to read through transcription termination signals.

Previous experiments in which transcription termination signals were introduced into ribosomal RNA operons by insertions of TnJO, Tn9, or ISJ (insertion sequence 1) showed that transcription termination is suppressed in ribosomal RNA operons, suggesting that modification of the RNA polymerase at or near the ribosomal RNA promoters is likely (13-15). However, definitive proof that polymerase modification is involved requires elimination of any possible contribution by the sequences surrounding the termination signals. In this paper, we report the use of operon fusions to show that the ability of RNA polymerase to read through termination signals is a special property conferred by the ribosomal RNA promoter-leader region and is independent of the sequences immediately surrounding the termination signals. These observations suggest that RNA polymerase is modified at the ribosomal RNA promoter-leader region to enable read-through of transcription termination signals.

Transcription and translation are functionally coupled in the lac operon and in most bacterial operons that code for translated RNAs. The essence of coupling is that ribosomes translating mRNA immediately behind transcribing RNA polymerase molecules enable RNA polymerase to read through sequences that would terminate transcription in the absence of ribosomes. Coupling therefore confers an advantage to the cell because it allows coding regions to evolve without the need to eliminate certain types of transcription termination signals (and perhaps also signals that cause termination-related events such as pausing). Coupling also may have evolved to allow coupling-dependent processes such as attenuation. The disruption of coupling by premature translation termination unmasks transcription termination sequences frequently present in protein-coding regions and results in premature transcription termination (1-10). The uncoupling of transcription and translation is therefore responsible for some forms of polarity caused by premature nonsense codons in promoter-proximal genes of multicistronic proteincoding operons (1, 2, 4-10). The polar effects resulting from disruption of coupling by premature nonsense codons are dependent on the transcription termination factor p and are relieved by mutations in the gene for p (1, 6-9). Nontranslated operons that are transcribed with negligible natural polarity, such as ribosomal RNA operons (11-15), must not require coupling. Coupling would not be required if RNA polymerase initiating at these promoters is modified to prevent termination. Detailed models for the reduction of termination by modification of RNA polymerase are provided by regulatory events in the bacteriophage X (1, 2, 10, 1619). Coupling would also not be required if the transcript has a primary sequence devoid of termination signals, a secondary or tertiary structure unsuitable for the function of termination signals or termination factors, or if the binding of ribosomal proteins to the nascent transcript prevents premature termination.

MATERIALS AND METHODS Bacterial Strains and Plasmids. EM343 (AlacZYA melB ara Ximm Strr) is an ara derivative of RE16 (20) obtained by ethylmethanesulfonate mutagenesis and ampicillin enrichment. EM350 is a recA derivative of EM343 obtained by trimethoprim selection of a thyA derivative followed by introduction of thyA+ recA after conjugation with KL16-99. EM388 [LaciZYA- thyA36 polAl deoC2 6 (rrnD-rrnE)1 X-F-] was obtained by transposition of Tn9 into lacY of P3478 (21) and subsequent ampicillin enrichment of chloramphenicol-sensitive bacteria followed by genetic and enzymatic screening for a chloramphenicol-sensitive LaciZYAphenotype. ATn9::4.33 is a defective specialized transducing phage that has a Tn9 insertion in lambda DNA near or in int (14). pMC306 (22) is described in Fig. 1. pBH16 and pBH30 are described in Fig. 3 and contain a replication origin of F on an Hpa I fragment derived from pDF42 (23) and a replication origin derived from pUC9 (24). Tn9 consists of a chloramphenicol acetyltransferase gene between direct repeats of IS] (25). Insertions in lac. EM350 containing pMC306 was infected with a helper-depleted preparation of ATn9::4.33. Lac- survivors were detected on lactose tetrazolium agar containing 0.1% arabinose, ampicillin, and chloramphenicol. Transposition is selected for by this procedure because integration of X by site-specific or general recombination is prevented by the recA mutation and by the X repressor present in the cells prior to infection. The location of each Tn9 insertion was determined by use of melibiose and lactose minimal medium and by melibiose, lactose, and 5-bromo-4-chloro-3-indolyl ,& D-thiogalactoside (X-Gal) indicator agar. Purified plasmid DNA was also analyzed by agarose gel electrophoresis after

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Abbreviation: IS, insertion sequence. *To whom reprint requests should be addressed.

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Genetics: Holben and Morgan

Proc. NatL Acad ScL USA 81 1 kb

Ara' Mu' 'trp' 'CBA'

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Z

BomHlSnidI Hind

ing spectrophotometer at 250C. Blank cuvettes contained identical reaction mixtures without ONPG or IPTG. Protein content was measured by dye binding (27).

ApR

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(1984)

rep Col El

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FIG. 1. Linear representation of the structure of the 22-kilobase (kb) plasmid pMC306. Apostrophes indicate that the gene, operon, or region of DNA is partially deleted as a result of the DNA rearrangements involved in formation of the plasmid.

restriction nuclease digestion by EcoRI/BamHI or EcoRI/ Pst I. Determination of the location and orientation of insertions was possible because of a suitably located EcoRI site in lacZ (see Fig. 2) and EcoRI and Pst I sites in Tn9 (25). ISJ insertions in pMC306 were obtained by transforming the Tn9 insertion derivatives of pMC306 into EM343 and subsequently screening for chloramphenicol acetyltransferase-negative colonies on crystal violet indicator agar containing ampicillin (26). Chloramphenicol acetyltransferase is lost when recombination between the flanking direct repeats of IS1 results in loss of the resistance gene and one of the two flanking copies of IS1. The retention of a single IS1 was confirmed by restriction nuclease digestion of purified DNA as described above. The Tn9 and IS1 insertions used in this work have not created any large deletions or duplications as judged by analysis of restriction nuclease fragments and the ability of all insertions to revert to Lac' at low frequency. Assay of lac Enzymes. pMC306 derivatives in EM350 were grown in M63 minimal medium containing 1 ug of thiamine/ ml, 0.4% glycerol, 100 pug of ampicillin/ml, and 0.1% arabinose. pBH16 and pBH30 derivatives in strain EM388 were grown in Luria broth containing 100 jug of ampicillin/ml. Twenty micrograms of chloramphenicol/ml was added when the plasmids contained Tn9. At an OD550 of 0.5, the cultures were chilled on ice for 10 min, washed twice with cold TGTA buffer (50 mM Tris, pH 7.8/2 mM EDTA/50 tiM 2-mercaptoethanol) and then suspended in cold TGTA buffer to an OD550 of 3.5. Three milliliters of this cell suspension was incubated for 5 min at 25TC and then 15 Al of toluene, 300 Al of 10% Triton X-100, and 75 Al of 450 ug of lysozyme/ml were added consecutively with 10 sec of mixing after each addition. The lysates were incubated for an additional 5 min at 250C and chilled on ice prior to enzyme assays. ,/3Galactosidase assay mixtures contained 200 Al of cell lysate and 800 /l of a solution of 10 mg of o-nitrophenyl ,Dgalactoside (ONPG) per ml of TGTA buffer. Thiogalactoside transacetylase assay mixtures contained 100 Al of cell lysate, 100 Al of 0.5 M isopropyl -D-thiogalactoside (IPTG), and 800 ul of a solution of 0.5 mg of 5,5'-dithiobis-2-nitrobenzoic acid per ml of 56 mM Tris HCl, pH 7.8/2.25 mM Na2EDTA/125 mM acetyl-CoA. A3-Galactosidase assays were monitored at 420 nM and thiogalactoside transacetylase assays at 412 nM using a temperature-regulated record-

RESULTS Experiments designed to prove that RNA polymerase transcribing from the ribosomal RNA promoter-leader regions is resistant to termination require that a termination signal in a single sequence context be shown to cause termination with different efficiencies when rrn and other promoters are compared. It is important to measure the efficiency of termination, not just the level of transcription downstream of a termination signal, because the level of transcription downstream of a leaky termination signal is also dependent on promoter strength. The operon fusions we have constructed enable termination efficiency to be measured independently of promoter strength. Insertions in ara-lac. Twenty-nine derivatives of pMC306 were isolated with insertions of Tn9 in the lacZ and lacY genes of the ara-lac fusion operon (Figs. 1 and 2). An IS] insertion was then generated at the site of each Tn9 insertion by homologous recombination between the directly repeated ISI elements of Tn9. The location and orientation of each insertion was determined by genetic and restriction nuclease analysis (see Materials and Methods and Fig. 2). The regional preference for insertions in or near lacY has been observed previously (28). All Insertions in ara-lac Cause Efficient Termination. Enzymatic assays of lacZ and lacA gene products reveal that all insertions in lacZ and lacY reduce lacA expression at least 95% (Table 1, some data not shown). Since lacZ expression is not substantially affected by insertions in lacY, transcription rates and plasmid copy numbers are not significantly affected by the insertions. It is therefore likely that insertions in lacZ also do not substantially affect transcription initiation rates or plasmid copy numbers. It can be concluded that Tn9 and IS1 insertions in many locations and both orientations are at least 95% polar on lacA. Since IS1 insertions cause polarity that is relieved by mutations in p (2931), it follows that Tn9 and IS1 insertions contain or unmask strong p-dependent transcription termination signals. For the purposes of this paper, it is unnecessary to know whether the termination signals are within the insertions or unmasked by the insertions. However, Tn9 and IS insertions are very polar even when located very near the 3' end of lacZ or lacY (Table 1). The strong and universal polarity caused by these insertions is unlike the uneven gradient of generally weaker polarity that is observed when p-dependent termination signals in lac are unmasked by premature nonsense codons (32). It is therefore very likely that Tn9 and ISJ contain strong p-dependent termination signals that function in both orientations. Insertions in rrn-lac Do Not Cause Efficient Termination. HindIII/BamHI fragments carrying lac genes and lac insertion derivatives were cloned from pMC306 into the HindIIII

1 kb trp

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FIG. 2. Locations of Tn9 and IS] insertions in pMC306. L or R indicate that the chloramphenicol acetyltransferase gene of Tn9 is transcribed to the left or right in the Tn9 insertion at each location.

Proc. NatL. Acad Sci USA 81 (1984)

Genetics: Holben and Morgan Table 1. Synthesis of A-galactosidase (lacZ) and thiogalactoside transacetylase (lacA) from ara-lac, rrn-lac, and lac-lac fusion operons Enzyme level lac-lac ara-lac rrnC-lac A A/Z Z A A A/Z A/Z Z Insert Z 6.8 0.48 0.10 2.0 20 0.16 0.33 2.1 None 14 iSI 0.15 0.05 0.33 14L 12 0.0 0.0 0.09 1.6 17 6.1 15L 12 0.0 0.0 0.17 1.0 7.3 0.20 0.00 0.00 16L 8.4 0.0 0.0 0.17 1.2 17R 9.5 0.0 0.0 0.16 0.57 3.6 0.16 0.00 0.00 19R 16 0.0 0.0 0.11 0.63 5.7 0.17 0.00 0.00 20L 17 0.0 0.0 0.12 1.4 11 22R 10 0.0 0.0 0.10 0.71 7.1 0.0 0.0 0.10 0.64 6.4 0.18 0.00 0.00 23R 19 0.11 0.00 0.00 24L 13 0.0 0.0 0.10 1.1 11 0.0 0.0 0.10 1.3 13 26L 12 0.17 0.00 0.00 0.0 0.0 0.06 0.69 12 27R 13 Tn9 0.0 0.0 0.17 0.56 3.3 14L 20 15L 14 0.0 0.0 0.14 0.11 0.79 16L 15 0.0 0.0 0.20 0.26 1.3 0.19 0.00 0.00 17R 18 0.0 0.0 0.12 0.35 2.9 0.16 0.00 0.00 19R 14 0.0 0.0 0.13 0.43 3.3 0.1 0.0 0.09 0.21 2.3 0.17 0.00 0.00 20L 14 0.1 0.0 0.09 0.42 4.7 0.11 0.00 0.00 22R 15 23R 13 0.0 0.0 0.09 0.36 4.0 0.17 0.00 0.00 24L 18 0.0 0.0 0.22 0.51 2.3 0.20 0.00 0.00 One unit of ,B-galactosidase is defined as 1 ,tmol of o-nitrophenyl ,-D-thiogalactoside hydrolized per min per mg of protein. One unit of thiogalactoside transacetylase as defined is 1 lmol of thionitrobenzoic acid produced per mg of protein. A blank in the table indicates that the corresponding plasmid construction was not made. All insertions in lacZ reduced lacA activity by >95% in aralac fusions (they were not examined by lac-lac fusions). In rrn-lac fusions, lacZ insertions 3R and 4L reduced lacA expression to 43% and 17% of that of an rrn-lac fusion without insertions. The only significant variation in the assays arises from culture variability and is an average of 10% of the mean value. The values given are the average of assays performed on at least two independent cultures of cells containing each plasmid construction.

BamHI sites of pBH16 (Fig. 3) and transformed into the polA strain EM388, in which pBH16 replicates from the origin of F. These constructions fuse the rrnC promoter-leader region to the trpB portion of the ara-Mu-trp-lac fusion operon from pMC306. Since trpB is translationally coupled to trpA and a trpA-lacZ fusion protein is produced, this fusion junction results in a low level of synthesis of the trpA-lacZ fusion protein (ref. 33 and Table 1). The low level of translation of the trpB-trpA-lacZ portion of the transcript results in only a 2fold transcriptional polarity on lacA when transcription begins from the ara promoter (33). In the polA bacterial strain EM388, the rrnC-lac fusion plasmids replicate only from the origin of F and therefore probably exist in a single copy per cell. The rrnC-lac fusion plasmids do not severely affect the growth of EM388 and are very stable. In poIA+ strains, the same fusion plasmids exist in multicopy form, are unstable, and severely affect cell growth (unpublished observations). For this reason EM388 was used to assay rrn-lac fusions, whereas ara-lac fusions on pMC306 were assayed using strain EM350. In EM350, pMC306 is a multicopy plasmid that replicates from the ColEl origin. Enzymatic assays using extracts of EM388 containing rrnC-lac fusion plasmids reveal that insertions in lac Y do not significantly affect lacZ expression (Table 1). Therefore, insertions in lacZ or lac Y probably do not affect plasmid copy

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number or transcription initiation. As determined by lacA measurement and lacA/lacZ ratios (Table 1), all insertions tested are substantially less polar when synthesis is directed by the rrnC promoter than when directed by the ara promoter.

When lacA expression downstream of insertions is compared with expression from uninterrupted rrnC-lac fusion operons, it is apparent that IS] insertions very near the carboxyl terminus of lacY or between lacZ and lacY cause little termination in rrnC-lac fusions (Table 1). From this observation it can be concluded that RNA polymerase initiating at the rrnC promoter-leader region efficiently reads through termination signals that cause >95% termination when transcription is initiated at the ara promoter. A gradient of polarity on lacA expression is observed when insertions in a single orientation in the lacY gene are examined (Table 1). This gradient probably occurs because insertions nearer the 5' end of lacY prevent coupling over larger regions and probably unmask more transcription termination signals in lacY coding regions between the insertion and lacA. A gradient of polarity of insertions would then be expected if RNA polymerase initiating at ribosomal promoters is not 100% efficient in reading through termination sequences. Partial termination would also be consistent with the greater polarity of Tn9 insertions (which have two ISJ sequences and may have other termination signals between the ISI elements) than polarity resulting from a single ISI sequence in the same orientation at the same site (Table 1). Insertions in lac-lac Cause Efficient Termination. Differences in polarity observed using the ara and rrnC promoter regions could be due to disruption of translational coupling resulting from use of the trpB HindIII site to construct rrnClac fusions or use of the strain EM388 to assay rrnC-lac fusions. To rule out these possibilities the HindIII/BamHI lac Pst l Hind

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FIG. 3. The structures of pBH16 and pBH30. Both plasmids contain origins of replication derived from F and pUC9. Insertions in the polylinker of pBH30 are transcribed from the lac promoter. Insertions in the polylinker of pBH16 are transcribed from the rrnC promoter. rrnC sequences in pBH16 are represented as thick lines, and flanking non-rrn chromosomal DNA as thin lines. The tandem rrnC promoters are followed by the entire leader region, 16S rRNA gene sequences from positions 1-16 and 613-650, a polylinker with HindIII and EcoRI ends, the rrnC tRNAASP and tRNATTrp genes, and the rrnC termination sequence. A Sma I site was generated within the 16S rRNA sequences between positions 16 and 613 by ligating blunt ends resulting from Bcl I and Xma I termini that had been filled in with the Klenow fragment of DNA polymerase 1. pBH30 contains E. coli chromosomal DNA extending approximately 350 bases upstream of the rrnC P1 promoter and 150 bases downstream of the rrnC termination signal. Upstream Hpa I and downstream Pvu II sites flanking rrnC in pLC22-36 (13) are the boundaries of rrnC and flanking E. coli chromosomal DNA of pBH16. Nucleotide sequence determination has verified that the rrnC promoter-leader region on pBH16 is intact. The origin of F is an Hpa I fragment from pDF42 (23) inserted in a Pvu II site located downstream of lac in pUC9 (24). The lac promoter of pUC9 was removed during construction of pBH16. kb, Kilobases.

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Genetics: Holben and Morgan

fragments of pMC306 and several insertion derivatives were cloned under control of the pBH30 lac promoter (Fig. 3). These constructions result in lacZ'-'trpB-trpA'-'lacZYA fusion operons with a frameshift in the lacZ'-'trpB gene that introduces many translation termination codons beginning in trpB very near the HindIII site (24, 41). Therefore, trpB-trpA translational coupling is prevented in these lac-lac constructions as it is in the rrnC-lac constructions. Synthesis of lac enzymes from the lac-lac fusion operons was assayed using strain EM388, where the lac-lac fusion plasmids replicate from the origin of F. Insertions in lac-lac fusion operons are extremely polar (Table 1). Relief of Natural Termination in lac. When rrnC promoters on pBH16 are fused to lac, the ratio of thiogalactoside transacetylase to /B-galactosidase is 9.6 times greater than that from similarly constructed lac-lac fusions made with pBH30 (Table 1). This observation suggests that substantial termination may occur between lacZ and lacA in lac-lac fusions but not in rrnC-lac fusions.

Proc. Nad Acad ScL USA 81

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gested that these conserved elements are important to rrn transcript termination (39). The termination signal of rrnC functions in vitro without p when purified RNA polymerase initiates at nuclease-generated ends of DNA molecules (38). It remains to be determined which proteins are required for termination at this signal in vivo when transcription initiates at rrn promoter-leader regions or whether all p-independent termination signals can terminate rrn transcripts. Transcription of lac from the rrnC promoter-leader region results in a 9.6-fold greater ratio of thiogalactoside transacetylase to /3-galactosidase compared with transcription from the lac promoter. This observation suggests that 89% of lac transcripts may terminate between lacZ and lacA when polymerase is not modified to prevent termination. Termination in this region may be due to a termination signal located between lacZ and lacY (t). Therefore, the 7:1 molar ratio of 3-galactosidase to thiogalactoside transacetylase synthesized from the wild-type lac operon (40) may be largely due to termination between lacZ and lacA.

DISCUSSION RNA polymerase initiating at the rrnC promoter-leader region can efficiently read through p-dependent transcription termination signals in lac. It is therefore likely that RNA polymerase is modified at the rrnC promoter-leader region to allow read-through of p-dependent transcription termination signals. Modification of polymerase presumably compensates for the lack of transcriptional-translational coupling in rrn operons and allows transcription of rrn operons with little or no polarity. The modification of polymerase may also play an as yet unknown role in regulation of rrn operon transcription. RNA polymerase could be modified during transcription initiation at the rrn promoters or while polymerase traverses the rrn leader region. The X phage antitermination proteins N and Q (16, 19) provide models that suggest how RNA polymerase modification at the rrn promoter-leader region might occur. Modification of polymerase by N and Q proteins requires E. coli nusA protein (16-18). It is therefore significant that a nusA utilization sequence is present in the leader region of all seven rrn operons of E. coli, including the rrnC operon (ref. 34, unpublished data). There are additional nusA utilization sequences just prior to the 23S rRNA genes. These observations suggest that the nusA protein might be involved in antitermination in rrn operons either alone or in cooperation with unknown E. coli proteins. In vitro, the nusA protein causes RNA polymerase to pause in the rrn leader for the unphysiologically long period of 20 sec (34). Perhaps polymerase pauses at this site in vivo until modification by a host antitermination protein allows transcription to continue. This mechanism would ensure that polymerase molecules will not prematurely terminate after entering rRNA genes. Alternatively, the antitermination mechanism may modify polymerase before the pause site to prevent significant pausing from occurring in vivo. It is also possible that the antitermination mechanism and pause site cooperate in a regulatory response that determines transcriptional rates of rRNA genes. rrn operon termination signals might need special structures to terminate rrn transcripts. Interestingly, two of the four sequenced rrn termination signals have tandem stemloop structures (35, 36). However, two operons, including the rrnC operon used here, have a single stem-loop structure (37, 38). Therefore, the tandem arrangement is not critical for termination of rrn transcripts. In four rrn termination regions for which the nucleotide sequence is known, the first (or only) rrn termination signals encountered by polymerase have conserved elements of structure and sequence not found in other bacterial termination signals. It has been sug-

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