Molecular Microbiology (1992) 6{12), 1655-1661
In vivo characterization of tus gene expression in Escherichia coii Bryan A. RoecMein and Peter L. Kuempel* Department of Molecular. Cellular and Developmental Biology. Campus Box 347. University of Colorado, Boulder. Colorado 80309, USA. Summary The tus gene encodes a DNA-binding protein (Tus) that inhibrts replication forks at specific block-sites within the terminus region of the Escherichia coli chromosome. One of these block-sites, TerB, is adjacent to the tus gene. Using primer extension and a promoter fusion to characterize in vivo expression, we have demonstrated that the tus transcription start site is within TerB, and that Tus protein autoregutates expression at this weak promoter We have also demonstrated that a minority of tus transcripts are initiated from an upstream region that contains two additional open reading frames. This readthrough transcription into tus is reduced in the presence of Tus protein.
Introduction The circular chromosome of Escherichia coli is replicated in a bidirectional fashion, and the replication forks meet in the terminus region at the end of the replication cycle. Although relatively few genes have been identified in this genetically sparse region, a system that blocks replication forks has been characterized {Hill et al., 1987; de Massy ef at.. 1987). This terminator system functions as a replication fork trap, permitting forks to enter the terminus region from either side, but preventing them from exiting it. Consequently, replication forks meet in the terminus region and not elsev^rhere on the chromosome. The presently identified components of the terminator system are six polar terminator sites {TerA-TerF: Hill et al., 1987; 1988b; de Massy et at., 1987; Hidaka et at.. 1988; 1991; Kobayashi etal., 1989; Francois et at.. 1989; B. Sharma and T. Hill, personal communication) which block replication forks travelling in the non-permitted direction, and the Tus protein (Hill ef a/.,1988a; 1989; Kobayashi ef a/., 1989; Sista et at.. 1989). This protein binds to the TerS terminator
Received ISNovemtJW, 1991; revised and accepted 10 March. 1992. 'For correspondence. Tel. (303) 492 7952; Fax (303) 429 7744.
site with a KD of 3.4 x 10"''3M(Gottlieb eta/.,1992),and/n vitro experiments indicate that it functions by inhibiting DNA helicases (Lee et at.. 1989; Khatri ef at., 1989). The tus gene Is located in the terminus region at 35.6 min (Hill ef at., 1988a). and analysis of the DNA sequence revealed that tus and a potential promoter were adjacent to the TerSterminator site (Hill etat., 1989; Hidaka etal., 1989). In addition, it was noted that tus was immediately downstream from an unidentified open reading frame (ORF) (Fig. 1; Hill etat., 1989). This arrangement suggested two ways in which expression of tus might be regulated. First, TerS overlapped the potential, but apparently weak, promoter for the tus gene, if this site actually functioned as the tus promoter, expression was expected to be autoregulated; binding of Tus to TerB would interfere with initiation of transcription by RNA polymerase. In addition, it was possible that tus was cotranscribed with the upstream locus. This could also lead to autoregulation, if binding of Tus to TerB blocked transcription elongation into tus. We report here that Tus regulates its own transcription, primarily by blocking initiation at the weak promoter that overlaps TerB. A small amount of transcription also occurs by readthrough from initiation that occurs further upstream. Interestingly, this transcription is reduced in the presence of Tus. A preliminary report of these results was presented at the EMBO Workshop on The Bacterial Cell Cycle: Structural and Molecular Aspects'. October. 1990 (Roecklein et al., 1991). Natarajan et al. (1991) have also recently reported in vitro results that demonstrate that initiation of tus transcription is autoregulated. Results and Discussion Identification of the tus promoter We used SI endonuclease protection as an initial test for a functional promoter immediately upstream of the tus gene. The potential promoter is shown in Figs 1 and 2, and a DNA probe that extended from nucieotides 58 to 758 with respect to the co-ordinates of Hill ef al. (1989), was prepared by pclymerase chain reaction (Fig. 1). RNA from PK2619, which was tus' due to an insertion in the middle of the gene at bp 1279 produced protected fragments of approximately 160 nuclectides in length (Fig. 3). This was the size expected if transcription was initiated in the region of the indicated promoter. Furthermore, these fragments
1656
B. A. Roecklein and P. L Keumpet (1282)
(1)
T T
uspT
urpT
TerB
T
I)
tus
T
T fumC
primer B ••— primer A S1 probe
were absent when RNA from PK2607 was tested, which lacked the promoter sequences. The fragments were barely visible when RNA from the tus^ strain PK457 was tested (data not shown), which was consistent with autoregulation. Primer extension with two different primers has been used to identify more precisely the start-site cf the tus transcript. The location of primer A is indicated in Fig. 1, and when extended it would yield a product of 85-92 nucleotides if transcription was initiated at the indicated promoter. Figure 4 shows that RNA from PK2619 produced a band of about that size. A band of the same size, although considerably fainter, was also obtained with RNA from PK457 (Roecklein etat., 1991). The exact length of extended primer A was determined by comparing its size with that of sequencing reaction products obtained with the same primer. The results in Fig. 4 indicate that transcription was initiated at the adenine at co-ordinate 602. To confirm this assignment, primer extension and sequencing ladder products were mixed and run in the same Ipne. An extra band was observed at the appropriate position when the extended primer was run with sequencing ladder products that terminated at positions corresponding to Gs in the indicated sequence. Since the extended product itself ended with an A, no extra band was observed when the extended primer was run with sequencing ladder products that terminated at positions corresponding to As in the indicated sequence. Initiation at the A at co-ordinate 602 was also demonstrated using primer B, which was nearer the promoter (Figs 1 and 2). A product of 34 nucleotides was observed, and RNA from PK2619 and PK457 produced extended
products of the same length (Fig. 5). However. PK2619 produced about 30-fold more product than PK457. as determined by phospho-imaging. No product was produced with RNA from the promoter deletion strain PK2607. The RNA from PK2619 also produced small amounts cf other products that were not detected in previous analyses. The n+1 product probably resulted from the non-template-directed addition of an extra nucleotide by avian myeloblastosis virus (AMV) reverse transcriptase (Inoue etat.. 1986). It is visible in Fig. 5 owing to the longer exposure time that was used in order to observe the fainter bands. These results demonstrate that the primary transcription initiation site of tus was immediately upstream from the gene, at the site that was previously identified as a possible promoter (Hill ef ai, 1989; Hidaka ef al., 1989). However, this promoter only has weak overall homology to the consensus promoter sequence (Hoopes and McClure, 1987) and only low levels of expression were observed, even in the absence of Tus repressor. The - 3 5 sequence for the tus promoter is TGGTCA. which contains two alterations from the consensus TTGACA. There are two possible - 1 0 sequences within this region, but neither fits the consensus pattern very well. One of these (TATAAA; Pribnow 1). lacks the almost invariable T found at the 3' end, the other (TAAAAT; Pribnow 2). lacks the internal T and is beyond the ideal spacing with respect to the - 3 5 region. Regardless of which of these - 1 0 sequences is actually used, the adenine at position 602 is within the typical range (5-9 bp) for Initiation from E coti promoters (Hoopes and McClure, 1987). The transcription start-site for tus has also been studied
primer B [rOCG*CTOTOCT*T*AA*T*AGT *T OTTGT*ACT
•M
Pribl
OOTTAATATTATOG CO CGTTACQ ATCTC
Srt)
TerB
Fig. 1. tus. 7erS and flanking genes in the 4.95 kb EcoRi (•) fragment at physical co-ordinates 1698 to1703(Koharae(a/.. 1987).TiieS1 probe was used to locate the tus promoter {tusp] by SI endonuciease protection of tus mRNA; primer A and primer B were oiigonucleotides used fcr prirr>er extension. u/pTand uspTare open reading frames upstream of tus (our unpubiished data); 7. indicates the predicted promoter. fumC encodes fumarase C (Woods ef al.. 1936), EcoRV sites (•) et co-ordinates 1 and 1282 indicate positions of kanamycin inserts in strains PK2703 and PK26I9. respectively. This EcoRV fragment and the 0.35kb fragment upstream were replaced with a kanamycin insert in strain PK2607.
MET
Fig, 2. The tus promoter region including the TerB termination site. Co-ordinates are from Hiii et al. (1989). The stop codon for the (JspTopen reading frame is indicated at position 550. Pribl and Pnb2 indicate two alternative - 1 0 promoter sequences (Pribnow boxes). Primer B was the primer used to detect readthrough transcription and £is an additional confirmationof the (us 5' end.
Autoregutation of tus expression
analysis and in vitro run-off transcription. It is possible that precise initiation from the weak tus promoter is perturbed when it is removed from its normal chromosome location, and that promoter function is affected by factors such as context, copy number or supercoiling.
-310 281 27i -234
Autoregutation of tus
-194
-Tus
•
1657
-lie
Fig. 3. Identification of the 5' end of tus by SI endonuciease protection. Ttie location of the SI probe is indicated in Fig. 1. RNA from PK2619 yielded bands (Tus) of approximately 160 nucieotides that were absent in RNA from strain PK2607. Lane M contained Has ill-digested 0X174 as molecular weight markers.
by Natarajan ef at. (1991). They observed a number of start-sites, most of which were outside the typical range for initiation from Pribnow 1 and Pribnow 2. In contrast, we have consistently detected only a single 5' end for the tus transcript within this region, A possible explanation for this difference is that we studied the in vivo expression of the chromosomal tus gene, whereas Natarajan et at. (1991) used subcloned promoter fragments for in vivo RNA
— 589
The experiments described above demonstrated that the 5'-end of the tus transcript was within the Tus-binding site at TerB. This suggested that fus expression was autoregulated. Consistent with this, both the SI endonuciease protection and primer extension experiments indicated that more tus RNA was present in PK2619 {tus) than in PK457 (tus*). In a preliminary report we have also demonstrated that expression from the chromosomal tus promoter was silenced in PK2619 if Tus was overproduced from a plasmid (Roecklein etal.. 1991). As an additional test of autoregulation, we have subcloned the tus promoter and fused it to the gene for the a-fragment of taoZ. This reporter gene provided a more quantitative determination of the range of tus autoregulation, as well as an estimation of the strength of the promoter. To construct this fusion plasmid (pROlOO), the 105bp Bgl\-Ssp\ fragment of tus (co-ordinates 519 to 624) was used as the source for the tus promoter and this was cloned intc pLBU3 (Gentz etal., 1981). The transcription initiation site of fus was consequently 170bp upstream from the lac operator region, and this intervening region contained stop codons in all reading frames. Since the operator site was still present, binding of lac repressor would be expected to inhibit transcription elongation from an upstream promoter (Deuschleef a/., 1986). It should be noted that the tacZ promoter was not functional because of the absence of the - 3 5 region, although the - 1 0 site was present. Expression from the tus promoter, in tenns of p-galactosidase activity, was tested in tus* and tus' strains (Table 1). As expected, in the absence of IPTG, pROlOO did not produce measurable levels of p-galactosidase activity. In
A*-602 Table 1. p-galactosidase activity in tus* (PK2744) and tus (PK2901) strains. Activity —611 Fig. 4. Locaiization of the 5' end of tus RNA by comparison with sequencing products. Primer A was used for primer extension and to generate sequencing reaction products. The middle iane shows primer extension of tus RNA from PK2619. Lanes T. G. A and C indicate dideoxy sequencing reaction products that terminated at the indicated bases in the template strand. A ' indicates the 5' end of tus at position 602 (Fig. 2). G+PK2619 and A+PK2619 refer to mixtures of the extended primer and indicated sequencing reaction products.
Plasmid pROlOO pROICX) pLBU3 pLBU3 pEMBL9 None (PK2801)
Tus
IPTG + 0.19 2.28 0.06 0.06 130
In vivo characterization of tus gene expression in Escherichia coii Bryan A. RoecMein and Peter L. Kuempel* Department of Molecular. Cellular and Developmental Biology. Campus Box 347. University of Colorado, Boulder. Colorado 80309, USA. Summary The tus gene encodes a DNA-binding protein (Tus) that inhibrts replication forks at specific block-sites within the terminus region of the Escherichia coli chromosome. One of these block-sites, TerB, is adjacent to the tus gene. Using primer extension and a promoter fusion to characterize in vivo expression, we have demonstrated that the tus transcription start site is within TerB, and that Tus protein autoregutates expression at this weak promoter We have also demonstrated that a minority of tus transcripts are initiated from an upstream region that contains two additional open reading frames. This readthrough transcription into tus is reduced in the presence of Tus protein.
Introduction The circular chromosome of Escherichia coli is replicated in a bidirectional fashion, and the replication forks meet in the terminus region at the end of the replication cycle. Although relatively few genes have been identified in this genetically sparse region, a system that blocks replication forks has been characterized {Hill et al., 1987; de Massy ef at.. 1987). This terminator system functions as a replication fork trap, permitting forks to enter the terminus region from either side, but preventing them from exiting it. Consequently, replication forks meet in the terminus region and not elsev^rhere on the chromosome. The presently identified components of the terminator system are six polar terminator sites {TerA-TerF: Hill et al., 1987; 1988b; de Massy et at., 1987; Hidaka et at.. 1988; 1991; Kobayashi etal., 1989; Francois et at.. 1989; B. Sharma and T. Hill, personal communication) which block replication forks travelling in the non-permitted direction, and the Tus protein (Hill ef a/.,1988a; 1989; Kobayashi ef a/., 1989; Sista et at.. 1989). This protein binds to the TerS terminator
Received ISNovemtJW, 1991; revised and accepted 10 March. 1992. 'For correspondence. Tel. (303) 492 7952; Fax (303) 429 7744.
site with a KD of 3.4 x 10"''3M(Gottlieb eta/.,1992),and/n vitro experiments indicate that it functions by inhibiting DNA helicases (Lee et at.. 1989; Khatri ef at., 1989). The tus gene Is located in the terminus region at 35.6 min (Hill ef at., 1988a). and analysis of the DNA sequence revealed that tus and a potential promoter were adjacent to the TerSterminator site (Hill etat., 1989; Hidaka etal., 1989). In addition, it was noted that tus was immediately downstream from an unidentified open reading frame (ORF) (Fig. 1; Hill etat., 1989). This arrangement suggested two ways in which expression of tus might be regulated. First, TerS overlapped the potential, but apparently weak, promoter for the tus gene, if this site actually functioned as the tus promoter, expression was expected to be autoregulated; binding of Tus to TerB would interfere with initiation of transcription by RNA polymerase. In addition, it was possible that tus was cotranscribed with the upstream locus. This could also lead to autoregulation, if binding of Tus to TerB blocked transcription elongation into tus. We report here that Tus regulates its own transcription, primarily by blocking initiation at the weak promoter that overlaps TerB. A small amount of transcription also occurs by readthrough from initiation that occurs further upstream. Interestingly, this transcription is reduced in the presence of Tus. A preliminary report of these results was presented at the EMBO Workshop on The Bacterial Cell Cycle: Structural and Molecular Aspects'. October. 1990 (Roecklein et al., 1991). Natarajan et al. (1991) have also recently reported in vitro results that demonstrate that initiation of tus transcription is autoregulated. Results and Discussion Identification of the tus promoter We used SI endonuclease protection as an initial test for a functional promoter immediately upstream of the tus gene. The potential promoter is shown in Figs 1 and 2, and a DNA probe that extended from nucieotides 58 to 758 with respect to the co-ordinates of Hill ef al. (1989), was prepared by pclymerase chain reaction (Fig. 1). RNA from PK2619, which was tus' due to an insertion in the middle of the gene at bp 1279 produced protected fragments of approximately 160 nuclectides in length (Fig. 3). This was the size expected if transcription was initiated in the region of the indicated promoter. Furthermore, these fragments
1656
B. A. Roecklein and P. L Keumpet (1282)
(1)
T T
uspT
urpT
TerB
T
I)
tus
T
T fumC
primer B ••— primer A S1 probe
were absent when RNA from PK2607 was tested, which lacked the promoter sequences. The fragments were barely visible when RNA from the tus^ strain PK457 was tested (data not shown), which was consistent with autoregulation. Primer extension with two different primers has been used to identify more precisely the start-site cf the tus transcript. The location of primer A is indicated in Fig. 1, and when extended it would yield a product of 85-92 nucleotides if transcription was initiated at the indicated promoter. Figure 4 shows that RNA from PK2619 produced a band of about that size. A band of the same size, although considerably fainter, was also obtained with RNA from PK457 (Roecklein etat., 1991). The exact length of extended primer A was determined by comparing its size with that of sequencing reaction products obtained with the same primer. The results in Fig. 4 indicate that transcription was initiated at the adenine at co-ordinate 602. To confirm this assignment, primer extension and sequencing ladder products were mixed and run in the same Ipne. An extra band was observed at the appropriate position when the extended primer was run with sequencing ladder products that terminated at positions corresponding to Gs in the indicated sequence. Since the extended product itself ended with an A, no extra band was observed when the extended primer was run with sequencing ladder products that terminated at positions corresponding to As in the indicated sequence. Initiation at the A at co-ordinate 602 was also demonstrated using primer B, which was nearer the promoter (Figs 1 and 2). A product of 34 nucleotides was observed, and RNA from PK2619 and PK457 produced extended
products of the same length (Fig. 5). However. PK2619 produced about 30-fold more product than PK457. as determined by phospho-imaging. No product was produced with RNA from the promoter deletion strain PK2607. The RNA from PK2619 also produced small amounts cf other products that were not detected in previous analyses. The n+1 product probably resulted from the non-template-directed addition of an extra nucleotide by avian myeloblastosis virus (AMV) reverse transcriptase (Inoue etat.. 1986). It is visible in Fig. 5 owing to the longer exposure time that was used in order to observe the fainter bands. These results demonstrate that the primary transcription initiation site of tus was immediately upstream from the gene, at the site that was previously identified as a possible promoter (Hill ef ai, 1989; Hidaka ef al., 1989). However, this promoter only has weak overall homology to the consensus promoter sequence (Hoopes and McClure, 1987) and only low levels of expression were observed, even in the absence of Tus repressor. The - 3 5 sequence for the tus promoter is TGGTCA. which contains two alterations from the consensus TTGACA. There are two possible - 1 0 sequences within this region, but neither fits the consensus pattern very well. One of these (TATAAA; Pribnow 1). lacks the almost invariable T found at the 3' end, the other (TAAAAT; Pribnow 2). lacks the internal T and is beyond the ideal spacing with respect to the - 3 5 region. Regardless of which of these - 1 0 sequences is actually used, the adenine at position 602 is within the typical range (5-9 bp) for Initiation from E coti promoters (Hoopes and McClure, 1987). The transcription start-site for tus has also been studied
primer B [rOCG*CTOTOCT*T*AA*T*AGT *T OTTGT*ACT
•M
Pribl
OOTTAATATTATOG CO CGTTACQ ATCTC
Srt)
TerB
Fig. 1. tus. 7erS and flanking genes in the 4.95 kb EcoRi (•) fragment at physical co-ordinates 1698 to1703(Koharae(a/.. 1987).TiieS1 probe was used to locate the tus promoter {tusp] by SI endonuciease protection of tus mRNA; primer A and primer B were oiigonucleotides used fcr prirr>er extension. u/pTand uspTare open reading frames upstream of tus (our unpubiished data); 7. indicates the predicted promoter. fumC encodes fumarase C (Woods ef al.. 1936), EcoRV sites (•) et co-ordinates 1 and 1282 indicate positions of kanamycin inserts in strains PK2703 and PK26I9. respectively. This EcoRV fragment and the 0.35kb fragment upstream were replaced with a kanamycin insert in strain PK2607.
MET
Fig, 2. The tus promoter region including the TerB termination site. Co-ordinates are from Hiii et al. (1989). The stop codon for the (JspTopen reading frame is indicated at position 550. Pribl and Pnb2 indicate two alternative - 1 0 promoter sequences (Pribnow boxes). Primer B was the primer used to detect readthrough transcription and £is an additional confirmationof the (us 5' end.
Autoregutation of tus expression
analysis and in vitro run-off transcription. It is possible that precise initiation from the weak tus promoter is perturbed when it is removed from its normal chromosome location, and that promoter function is affected by factors such as context, copy number or supercoiling.
-310 281 27i -234
Autoregutation of tus
-194
-Tus
•
1657
-lie
Fig. 3. Identification of the 5' end of tus by SI endonuciease protection. Ttie location of the SI probe is indicated in Fig. 1. RNA from PK2619 yielded bands (Tus) of approximately 160 nucieotides that were absent in RNA from strain PK2607. Lane M contained Has ill-digested 0X174 as molecular weight markers.
by Natarajan ef at. (1991). They observed a number of start-sites, most of which were outside the typical range for initiation from Pribnow 1 and Pribnow 2. In contrast, we have consistently detected only a single 5' end for the tus transcript within this region, A possible explanation for this difference is that we studied the in vivo expression of the chromosomal tus gene, whereas Natarajan et at. (1991) used subcloned promoter fragments for in vivo RNA
— 589
The experiments described above demonstrated that the 5'-end of the tus transcript was within the Tus-binding site at TerB. This suggested that fus expression was autoregulated. Consistent with this, both the SI endonuciease protection and primer extension experiments indicated that more tus RNA was present in PK2619 {tus) than in PK457 (tus*). In a preliminary report we have also demonstrated that expression from the chromosomal tus promoter was silenced in PK2619 if Tus was overproduced from a plasmid (Roecklein etal.. 1991). As an additional test of autoregulation, we have subcloned the tus promoter and fused it to the gene for the a-fragment of taoZ. This reporter gene provided a more quantitative determination of the range of tus autoregulation, as well as an estimation of the strength of the promoter. To construct this fusion plasmid (pROlOO), the 105bp Bgl\-Ssp\ fragment of tus (co-ordinates 519 to 624) was used as the source for the tus promoter and this was cloned intc pLBU3 (Gentz etal., 1981). The transcription initiation site of fus was consequently 170bp upstream from the lac operator region, and this intervening region contained stop codons in all reading frames. Since the operator site was still present, binding of lac repressor would be expected to inhibit transcription elongation from an upstream promoter (Deuschleef a/., 1986). It should be noted that the tacZ promoter was not functional because of the absence of the - 3 5 region, although the - 1 0 site was present. Expression from the tus promoter, in tenns of p-galactosidase activity, was tested in tus* and tus' strains (Table 1). As expected, in the absence of IPTG, pROlOO did not produce measurable levels of p-galactosidase activity. In
A*-602 Table 1. p-galactosidase activity in tus* (PK2744) and tus (PK2901) strains. Activity —611 Fig. 4. Locaiization of the 5' end of tus RNA by comparison with sequencing products. Primer A was used for primer extension and to generate sequencing reaction products. The middle iane shows primer extension of tus RNA from PK2619. Lanes T. G. A and C indicate dideoxy sequencing reaction products that terminated at the indicated bases in the template strand. A ' indicates the 5' end of tus at position 602 (Fig. 2). G+PK2619 and A+PK2619 refer to mixtures of the extended primer and indicated sequencing reaction products.
Plasmid pROlOO pROICX) pLBU3 pLBU3 pEMBL9 None (PK2801)
Tus
IPTG + 0.19 2.28 0.06 0.06 130
