Russian Journal of Genetics, Vol. 38, No. 5, 2002, pp. 501–509. Translated from Genetika, Vol. 38, No. 5, 2002, pp. 613–621. Original Russian Text Copyright © 2002 by Evdokimova, Eremina, Errais, Mironov.
GENETICS OF MICROORGANISMS
Formation of an Additional Promoter in the Regulatory Region of the Escherichia coli udp Gene and Its Structural and Functional Characterization A. A. Evdokimova, S. Yu. Eremina, L. L. Errais, and A. S. Mironov State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, 113545 Russia; fax: (095) 315-05-01; e-mail:
[email protected] Received July 27, 2000
Abstract—Structural and functional organization of the mutant udpP18 promoter generated after the spontaneous deletion of the G base in the –79 position relative to the start site of transcription from the main (P1) promoter within the regulatory region of the udp gene was studied. In this mutant, a new, functionally active promoter (P2) with the start site of transcription in the –64 position that contained the typical motif 5'-TG-3' located in front of the Pribnow sequence was formed. The data presented suggest that the expression of the P2 promoter, unlike that of P1, is not subjected to regulation with participation of the CytR protein and the cAMP–CRP complex. Results of mutational analysis of the P2 promoter showed that substitutions of the nucleotide G in the –14 position and nucleotide T in the –15 position significantly diminish the level of transcription from the P2 promoter. On the basis of these data, it is concluded that the P2 promoter could be assigned with respect to its characteristics to a group of promoters with an extended –10 region. The synergistic effect of P1 and P2 promoters on total expression of the udp gene in the mutant udpP18 was detected.
INTRODUCTION The structural udp gene that controls the synthesis of uridine phosphorylase is located at 86 min of the Escherichia coli K12 chromosomal map [1]. The udp gene, together with other genes that are responsible for the transport and catabolism of nucleosides, belongs to the CytR regulon [2] and is negatively regulated by the CytR repressor protein and positively regulated by the transcription-activating cAMP–CRP complex. The regulatory region of the udp gene contains two tandemly located binding sites for the CRP protein, namely, CRP1 and CRP2 with coordinates –41.5 and –93.5, respectively, relative to the start site of transcription [3]. An imperfect inverted repeat 5'-TGCAA-N5TTGCA-3' was localized in the region between CRP1 and CRP2 sites. According to in vitro data [3] and results of mutational analysis [4], this repeat is the main binding site for the CytR protein. The CytR repressor protein belongs to the family LacI of DNA-binding proteins. The efficiency of its binding to the promoter drastically increases in the presence of the cAMP–CRP complex [5], which is a distinct feature of this repressor protein. The cooperative binding of the CytR and CRP proteins to the promoter results from the direct protein–protein interaction [6, 7]. Under defined conditions, uridine phosphorylase is the only enzyme ensuring reversible phosphorolysis of
thymidine. This feature of uridine phosphorylase was used to obtain mutations that ensured the ability of thymine auxotrophs to grow on a medium with thymine and led to enhanced expression of the udp gene. One obtained mutation (udpP18) was unique in completely abolishing both the negative and positive control of udp gene expression [8]. Sequencing of the mutant udpP18 promoter revealed that a new, functionally active promoter with the start site of transcription in the –64 position appeared in the regulatory region of the udp gene owing to the spontaneous deletion of the G base in the –79 position relative to the start site of transcription of the main (P1) promoter [3]. In the present work, this promoter was termed P2. The formation of the new P2 promoter may be caused by the appearance of the T base in the –15 position and the G base in the –14 position relative to the new start site of transcription. In addition, the deletion of the G base in the –79 position ensures the optimal distance (17 nucleotides) between the –10 (5'-TAaAtT-3') and –35 (5'-gTGAtt-3') regions of the new promoter (Fig. 1). Thus, the newly arisen P2 promoter resembles in structure the promoters that have been described in the literature as promoters with an “extended –10” region, which contain an additional motif 5'-TG-3' located at a distance of one nucleotide upstream of the Pribnow sequence [9–12]. This work is devoted to studying the effect of different nucleotide substitutions in the –15 and –14 positions of the mutant P2 promoter on the functional activ-
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(b)
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Fig. 1. Nucleotide sequence of the fragment of the regulatory region of the E. coli udp gene: (a) the wild-type udpP+ promoter; (b) mutant udpP18 promoter generated by a deletion of the G/C nucleotide pair in the –79 position within the GGG triplet of the wild-type promoter (underlined by a double line in the a position) and containing the active promoter P1 and promoter P2 with its variants carrying nucleotide substitutions in the –15 and –14 positions; (c) the mutant udpP18 promoter with an inactivated P1 promoter (the nucleotide substitution –11 A G) and its variants carrying nucleotide substitutions in the –15 and –14 positions; (d) a truncated variant of the mutant udpP18 promoter with a deletion of the P1 promoter. The –35 and –10 regions of the P1 and P2 promoters are underlined. The solid line denotes the TG motif that forms an extended –10 region of the P2 promoter. Solid letters indicate nucleotide substitutions and nucleotides that correspond to transcription initiation sites. Arrows indicate the direction of transcription.
EVDOKIMOVA et al.
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Table 1. Bacterial strains, plasmids, and phages used in this study Strain
Genotype
Source
E. coli K12 TG1
thi supE hsd∆5 ∆(lac-proAB)/F' tra∆36 proAB+ lacI(q) lacZ ∆ M15
Collection of the State Research Institute of Genetics and Selection of Microorganisms
AM2009
F– thi1 thr1 leuB6 lacY1 tonA21 supE44 ∆(pro-lac) ∆(met-udp)
Laboratory collection
AM2010
As AM2009 but cytR–
The same
AM2011
As AM2009 but cya–
″
AM2012
As AM2010 but cya–
″
RZ1032
Hfr KL16 PO/45 lyzA(61–62) dut1 ung1 thi1 relA1 zbd-279::Tn10 sup E44 Collection of the State Research Institute of Genetics and Selection of Microorganisms
Plasmids pJEL250
Low-copy number vector*, AmpR
P. Valentin-Hansen (Denmark)
pAM276
pJEL250 containing a 332-bp (from –276 to +56) EcoRI-BamHI fragment of the udpP+ promoter
This work
pAM1276
As pAM276 but containing the –11A
pAM279
As pAM276 but containing the udpP18 mutation (deletion –79 G)
″
pAM1279
As pAM279 but containing the –11A
G substitution in the P1 promoter
″
pAM280
As pAM279 but containing the –14G
A substitution in the P2 promoter
″
pAM1280
As pAM280 but containing the –11A
G substitution in the P1 promoter
″
pAM281
As pAM279 but containing the –14G
C substitution in the P2 promoter
″
pAM1281
As pAM281 but containing the –11A
G substitution in the P1 promoter
″
pAM282
As pAM279 but containing the –14G
T substitution in the P2 promoter
″
pAM1282
As pAM282 but containing the –11A
G substitution in the P1 promoter
″
pAM283
As pAM279 but containing the –15T
A substitution in the P2 promoter
″
pAM1283
As pAM283 but containing the –11A
G substitution in the P1 promoter
″
pAM284
As pAM279 but containing the –15T
C substitution in the P2 promoter
″
pAM1284
As pAM284 but containing the –11A
G substitution in the P1 promoter
″
pAM285
As pAM279 but containing the –15T
G substitution in the P2 promoter
″
pAM1285
As pAM285 but containing the –11A
G substitution in the P1 promoter
″
pAM2279
As pAM279 but containing a 220-bp (from –276 to –56) truncated variant of the udpP18 promoter
Phages
M13 mp18
G substitution in the P1 promoter
″ Laboratory collection
* The structure of plasmid pJEL250 is described in Materials and Methods.
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Table 2. Structure of primers used in this study Coordinates* Primer
Sequence (from 5' to 3') 5'
3'
–276
–259
A
TTTTGAATTCGAAGCGAGAGGCATG
C
TTTTGGATCCATCAGACTTGGACATATA
+56
+42
D
ATAAATTTACCNCCAAAAGTG
–62
–89
E
ATAAATTTACNACCAAAAGTG
–62
–89
F
CAAATGCGTTGCATAAATTTAC
–56
–77
M
GCAAAAAGACACTTCACCGAAGGG
+4
–20
H
CACGAGGCCCTTTCGTCT
6346
6363
K
TTCCCAGTCACGACGTTG
6531
6514
U
TGAAAACGACGGCCAGT (universal primer for the phage M13mp18)
R
CAGGAAACAGCTATGAC (reverse primer for the phage M13mp18)
* Coordinates of primers A, C, D, E, F, and M are given in agreement with the numbering of nucleotides adopted for the regulatory region of the udp gene presented in Fig. 1. Recognition sites for enzymes EcoRI (GAATTC) and BamHI (GGATCC) are underlined. For primers D and E used in site-specific mutagenesis experiments, the letter N refers to each of four nucleotides. Primer M contains the nucleotide substitution G C indicated by letters in bold type. Coordinates of primers 2461 and 2462 are given in agreement with the physical map of plasmid pJEL250 (P. Valentin-Hansen, personal communication).
ity of this promoter. In addition, we evaluated the relative contribution of the additional (P2) and main (P1) promoters to the total expression of the udp gene in the udpP18 mutant. MATERIALS AND METHODS Bacterial strains, plasmids, and phages used in this work and their genetic characteristics are described in Table 1. The pJEL250 vector is a truncated variant of the R1 plasmid [13] with genes responsible for plasmid copy number (copA and copB) being under the control of the λ bacteriophage PR promoter regulated by the heatlabile repressor cI857. When plasmid pJEL250-carrying bacteria are grown at the low temperature of 30°C, the plasmid copy number is one to two per cell, whereas at 42°C it amounts to 1000 copies per cell [13]. In addition, the pJEL250 vector contains a structural gene (lacZ) devoid of its own promoter but carrying the SD sequence. In front of the SD sequence, a polylinker, which allows the cloning of DNA fragments at unique restriction sites for EcoRI and BamHI enzymes, is located. Therefore, cloning of promoter sites in this polylinker region allows the examination of their regulation in assays of β-galactosidase activity. Media. The composition of media used and the concentrations of additives were described in [4]. MacConkey (Difco) indicator medium was used to determine the Lac+ phenotype.
DNA techniques. Isolation of plasmid and singlestranded DNA, cloning, transformation, and analysis of recombinant plasmids were conducted by standard methods described in [14]. The following enzymes were used: restriction endonucleases EcoRI and BamHI, DNA ligase of phage T4, T4 polynucleotide kinase, Klenow fragment (Fermentas), and heat-stable Taq polymerase (USB). Polymerase chain reaction (PCR). We used PCR with intact E. coli cells as described in [3]. The procedure was conducted in a TC480 (Perkin-Elmer Cetus) thermocycler. The temperature rate depended on the length of the amplified fragment and the length and composition of the primers used. The structure and coordinates of primers used in this work are presented in Table 2. PCR products were isolated and purified using the GeneClean (Bio 101) kit. DNA sequencing was conducted by the Sanger method [15]. Site-specific mutagenesis was accomplished by the Kunkel method [16] with the strain RZ1032 and primers D, E, and M (Table 2). Construction of recombinant plasmids. After sitespecific mutagenesis of promoter fragments cloned into the phage M13mp18, the nucleotide sequence of single-stranded DNAs was analyzed for the purpose of selecting the desired mutants. In these mutants, DNA fragments were amplified with primers U and R (Table 2). The obtained fragments were purified, digested with restriction enzymes EcoRI and BamHI, and cloned into vector pJEL250 (treated with the same enzymes). The mixture obtained after ligation was used to transform
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strain AM2009. Transformants carrying insertions of the desired fragments were selected as variants with red pigmentation of colonies on the MacConkey indicator medium containing 50 µg/ml ampicillin. The presence of the corresponding insertions was confirmed by PCR with primers H and K specific for DNA sequences of the vector pJEL250 flanking the polylinker. To construct plasmids, the following primer combinations were used in PCR: A and F for plasmid pAM2279; A and C for all other plasmids. Transcription in vitro. Start sites of transcription from examined promoters were determined by a method described in [9] using the preparation of E. coli RNA-polymerase holoenzyme (Amersham-Pharmacia). Transcription was accomplished on PCR fragments amplified with primers H and K using DNA preparations isolated from various plasmid pAM derivatives that contained mutant variants of the udpP18 promoter. The reaction mixture (25 µl) contained 20 nM of a DNA fragment and 200 nM of the RNA-polymerase holoenzyme in the transcriptional buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 250 µg/ml of bovine serum albumin). After incubation for 20 min at 37°C, samples of ATP, GTP, CTP, and UTP (0.2 mM each), 10 µCu [α-32P] UTP and also heparin (20 mM) were added to the mixture. After incubation for 5 min, the reaction was terminated with an equivalent volume of 40 mM EDTA, the obtained RNA was precipitated with alcohol, and electrophoresis was conducted in 10% PAAG with 8 M urea. Measurements of β-galactosidase activity were made according to Miller [17]. Bacterial culture was grown to the exponential growth phase at 30°C in a liquid minimal medium with the supplements as required and glycerol or glucose (0.4% each) as a sole carbon source. The level of enzymatic activity was calculated in arbitrary units as
505
udpP18 promoter with an inactivated P1 promoter (Fig. 1). Before site-specific mutagenesis, a 332-bp (from –276 to +56) fragment of the mutant udpP18 promoter was amplified in PCR with primers A and C (Table 2) and cloned into the phage M13mp18 (at restriction sites EcoRI and BamHI). Site-specific mutagenesis was conducted by the Kunkel method (see Materials and Methods). Substitutions of the G base in the –14 position were obtained with primer D, and substitutions of the T base in the –15 position, with primer E (Table 2). With primer M, we isolated mutant variants of the udpP18 promoter with the P1 promoter inactivated as a result of the –11 A G substitution. In addition, a truncated variant of the udpP18 promoter carrying a deletion of the P1 promoter region was amplified and cloned with primers A and F (Fig. 1d). As a result of site-specific mutagenesis experiments, we obtained two series of mutant promoters. The first series of these promoters were constructed based on the intact regulatory region of the udpP18 mutant (i.e., containing two sites of transcription initiation: the original P1 start site of udp gene transcription in position +1 and a new P2 start in position –64); these mutant promoters included substitutions of the G and T bases in positions –14 and –15 relative to the new start site of transcription (Fig. 1b). The second series of mutant promoters contained the same nucleotide substitutions as mutants of the first series, but in the background of the P1 promoter inactivated as a result of a substitution of A with G in position –11 relative to the original start site of transcription from the udp gene promoter (Fig. 1c). To examine the effect of these nucleotide substitutions on the expression of mutant promoters, the appropriate fragments were cloned into the expression vector pJEL250 containing the reference gene lacZ devoid of its own promoter.
E = OD420 /OD450/ml/min. Expression of the Mutant Promoter udpP18 and Its Derivatives in Strains of Different Genotypes with Respect to the Regulatory cytR and cya Loci
RESULTS AND DISCUSSION Site-Specific Mutagenesis of the Regulatory Region of the udpP18 Mutant and Cloning the Isolated Variants of the Mutant udpP18 Promoter into the Low-Copy-Number Expression Vector pJEL250 Proceeding from the currently available data on the structure of promoters with an extended –10 region, it was of interest to examine the importance of nucleotide location in the –14 and –15 positions for providing the efficient functioning of the mutant udpP18 promoter. For this, we conducted the directional site-specific mutagenesis of the appropriate fragment of the udpP18 promoter. To evaluate the relative contribution of the new (P2) and original (P1) promoters to the total expression of the mutant udpP18 promoter, we simultaneously conducted site-specific mutagenesis of the –14 and –15 nucleotides on a modified variant of the mutant RUSSIAN JOURNAL OF GENETICS
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The obtained plasmids carrying hybrid operons udpP–lacZ were transformed into a series of isogenic strains (AM2009, AM2010, AM2011, and AM2012; Table 1), which differed in the allelic state of the cytR and cya genes. The activity of β-galactosidase was estimated in these strains (Table 3). Principally, the data in Table 3 indicate that lacZ gene expression controlled by the udpP18 promoter (plasmid pAM279) in cells of the strain AM2009 (cytR+cya+) exceeds that under the control of the wildtype udpP+ promoter (plasmid pAM276) by a factor of 10 on a medium with glucose and nearly by a factor of 20 on a medium with glycerol. In the genome of cytR– bacteria (strain AM2010), the udpP18 and udpP+ promoters manifested approximately equal activity levels, and the activity of the udpP18 promoter was even lower 2002
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Table 3. Activity of β-galactosidase expressed from the hybrid operon udpP-lacZ in strains of different genotype with respect to the cytR and cya genes depending on the structure of P2 and P1 promoters Activity of β-galactosidase in strains with the genotype*
Promoter structure
cytR+ cya+
Plasmid P2
cytR– cya+
cytR+ cya–
cytR– cya–
P1 glucose
glycerol
glucose
glycerol
glucose
glucose
pAM276
–
P1+
0.15
0.15
0.4
1.6
0.02
0.02
pAM279
P2+
P1+
1.4
2.96
0.65
2.64
0.64
0.68
pAM1276
–
–11G
0.02
0.02
0.03
0.04
0.02
0.02
pAM1279
P2+
–11G
0.55
0.32
0.54
0.41
0.28
0.32
pAM280
–14A
P1+
0.88
3.77
0.84
2.81
0.27
0.34
pAM1280
–14A
–11G
0.38
0.31
0.37
0.34
0.53
0.53
pAM281
–14C
P1+
0.5
2.27
0.66
2.45
0.06
0.06
pAM1281
–14C
–11G
0.11
0.13
0.11
0.17
0.08
0.08
pAM282
–14T
P1+
0.72
3.61
0.84
2.81
0.33
0.25
pAM1282
–14T
–11G
0.11
0.13
0.11
0.14
0.07
0.11
pAM283
–15A
P1+
0.78
3.23
0.98
2.81
0.32
0.31
pAM1283
–15A
–11G
0.38
0.24
0.37
0.26
0.29
0.26
pAM284
–15C
P1+
0.75
2.87
0.83
2.68
0.11
0.17
pAM1284
–15C
–11G
0.26
0.22
0.26
0.24
0.12
0.12
pAM285
–15G
P1+
0.71
3.1
0.9
2.79
0.27
0.19
pAM1285
–15G
–11G
0.32
0.24
0.31
0.26
0.31
0.3
pAM2279
P2+
–
0.26
0.12
0.22
0.1
0.23
0.24
* Bacteria were grown at 30°C on a minimal medium with glycerol or glucose (strains AM2009 cytR+ cya+ and AM2010 cytR– cya+) and with glucose (strains AM2011 cytR+ cya– and AM2012 cytR– cya–) as a sole carbon source at 30°C. Enzymatic activity was characterized using arbitrary units as OD420 /OD450 /ml/min. Data given in the table are mean numbers from four independent experiments. Deviations from mean numbers did not exceed 10%.
in the absence of the functionally active cytR protein. More significant differences in respect to the nature of expression of mutant and wild-type promoters were observed in the background of the damaged cAMP– CRP complex (strains AM2011 and AM2012): the activity of the udpP18 promoter decreased 3 to 4 times, compared to that in cya+, whereas the activity of the udpP+ promoter is completely blocked in the background of the cya mutation. On the basis of these data, it is concluded that owing to a deletion of one G base in the –79 position within the regulatory region of the udp gene, a new, additional promoter (P2) was actually formed and that its expression, unlike that of the original P1 promoter, is not subjected to regulation by the CytR repressor protein and the cAMP–CRP complex. This conclusion has been confirmed by results of mea-
suring the activity of promoter constructs containing an additional –11A G substitution in the Pribnow sequence of the P1 promoter, which, as seen in Table 3 (plasmid pAM1276), in fact completely inactivates the wild-type promoter. The introduction of this substitution into the mutant udpP18 promoter (plasmid pAM1279) led to a decrease in its activity, but the expression of the udpP18 promoter remained at a sufficiently high level and was three to four times higher than the basal level of udpP+ promoter expression. The expression of the udpP18 promoter that contains an additional 11G mutation in the genome of cya– bacteria seems to be a reflection of the actual capacity of the autonomously functioning P2 promoter. This conclusion agrees with results obtained by measuring the
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Percentage of the maximum activity of the P2 promoter
FORMATION OF AN ADDITIONAL PROMOTER IN THE REGULATORY REGION 100 90 80 70 60 50 40 30 20 10 0
G A C T
1
2
3
507
4
281 257 242 226 –14
–15
Fig. 2. The relationship between the maximal level of P2 promoter expression and the nucleotide composition in the –14 and –15 positions in a background of the damaged P1 promoter (mutation –11 A G). The activity of the original P2 promoter (–14G–15T) in the plasmid pAM1279 was taken as 100% activity.
enzyme activity in the construct (pAM2279) with a deleted P1 promoter (Table 3, the lower line). Interestingly, the total effect of the two functioning promoters (the original P1 and new P2) on the expression of β-galactosidase activity seems to have the cooperative nature. Thus, the activity of the hybrid operon containing both promoters significantly exceeds the sum of activities of each promoter. We are inclined to believe that the P2 promoter, being not strong enough, may in some way stimulate transcription initiation from the P1 promoter. A possible explanation for the synergistic effect of the two promoters is that the advancement of the transcriptional complex with the start at the P2 promoter leads to a more efficient excision of RNA polymerase molecules stuck in the P1 promoter (“promoter-clearance”). Moreover, RNA polymerase may be involved with higher efficiency in repeated rounds of transcription initiation at the P1 promoter. It is also noteworthy that the autonomously functioning P2 promoter manifested a higher expression level on a medium with glucose than on a medium with glycerol, unlike the original P1 promoter whose activity is inhibited on a medium with glucose. Table 3 also summarizes results of experiments on the effect of particular nucleotide substitutions in the –15 and –14 positions relative to the start site of transcription from the P2 promoter on the nature of its expression. The data obtained indicate that nucleotide substitutions in the –15 and –14 positions in a background of two functioning promoters (P2 and P1) had virtually no effect on the level of mutant udpP18 promoter expression. A different expression pattern was observed in constructs with an inactivated P1 promoter (Table 3 and Fig. 2). Thus, substitutions of the T nucleotide in the –15 position with A, G, and C decreased the expression of the P2 promoter by 30, 57, and 60%, respectively. Almost the opposite effect was observed upon RUSSIAN JOURNAL OF GENETICS
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Fig. 3. In vitro transcription from the udpP18 promoter (a DNA fragment amplified in plasmid pAM279), depending on the presence (lane 2) or absence (lane 1) of the cAMP– CRP complex in the transcription mixture. Lanes 3 and 4 present DNA marker fragments obtained after digesting plasmid pUC19 with restriction enzyme MspI (lane 3) and DNA of plasmid pBR322 with AluI (lane 4). On the right are sizes of marker fragments indicated in bp.
substitutions of the G nucleotide in the position –14: a minimal decrease in the activity was observed in the –14A variant (by 31%), whereas substitutions with C and T result in a fivefold decrease of promoter P2 expression (Fig. 2). Thus, it is concluded that the most optimal configuration of an extended –10 region (from the viewpoint of expression) is the presence of G and T bases in positions –14 and –15, because all substitutions in these positions led to a decrease in promoter activity, and the maximum decrease in promoter activity was observed upon substitution of the G nucleotide in the position –14 with T or C. In vitro Transcription from the udpP18 Promoter and Its Mutant Variants To estimate the efficiency of transcription from various mutant udpP18 promoters and determine the size of transcripts that started from the P2 and P1 promoters, we conducted in vitro experiments using the preparation of RNA-polymerase holoenzyme and purified PCR fragments of the regulatory region of the udp gene containing various promoter mutations. Fragments amplified with primers H and K (see Materials and Methods) were 517 bp in size, and the distance between transcription starts of P2 and P1 promoters and the 3' end of these fragments must be 276 and 212 bp, respectively. As seen in Fig. 3, the synthesis of transcripts of this exact size proceeds on a template isolated from plasmid 2002
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2
3
4
5
regulatory region of the udp gene, could be assigned to a group of promoters with an extended –10 region.
6
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ACKNOWLEDGMENTS We are grateful to P. Valentin-Hansen for providing plasmid pJEL250 and to G.I. Sergeeva and V.A. Muratova for technical assistance. This work was supported by the Howard Hughes Medical Institute (grant no. 75195-546401).
257 242 226
REFERENCES 1. Pritchard, R.H. and Ahmad, S.I., Fluorouracil and the Isolation of Mutants Lacking Uridine Phosphorylase in Escherichia coli: Location of the Gene, Mol. Gen. Genet., 1971, vol. 111, pp. 84–88.
190 Fig. 4. Effect of nucleotide substitutions in –15 and –14 positions on the efficiency of transcription from the mutant udpP18 promoter. The following templates were used for transcription: a DNA fragment amplified in plasmid pAM285 containing the P2 promoter with substitutions –15G –14G (lane 1); plasmid pAM281 with substitutions –15T –14C (lane2); plasmid pAM280 with substitutions –15T –14A (lane3); and plasmid pAM279 carrying the original variant of the udpP18 promoter –15T –14G (lane 4). Lanes 5 and 6 present DNA marker fragments obtained after digesting DNA of plasmid pUC19 with restriction enzyme MspI (lane 5) and DNA of plasmid pBR322 with AluI (lane 6). On the right are sizes of marker fragments indicated in bp.
2. Hammer-Jespersen, K., Nucleoside Catabolism, Metabolism of Nucleotides, Nucleosides, and Nucleobases in Microorganisms, Munch-Petersen, A.L., Ed., London: Academic, 1983, pp. 203–258. 3. Brikun, I., Suziedelis, K., Stemmann, O., et al., Analysis of CRP–CytR Interactions at the Escherichia coli udp Promoter, J. Bacteriol., 1996, vol. 178, pp. 1614–1622. 4. Domakova, E.V., Errais, L.L., Eremina, S.Yu., and Mironov, A.S., Mutation Analysis of the Major CytRBinding Site in the Regulatory Region of the E. coli udp Gene, Genetika (Moscow), 1999, vol. 35, no. 2, pp. 181– 186.
pAM279, provided that the reaction mixture contains, apart from RNA-polymerase, the CRP protein (Fig. 3, 2). Note that RNA with the start at the P2 promoter (in a 281-bp marker region) showed the prominent band with an intensity stronger than that of the transcript synthesized from the P1 promoter (in a region above the 190-bp marker). According to in vivo data showing that P1 is a CRP-dependent promoter, only one upper transcript synthesized from the P2 promoter is formed in the absence of the PCR protein in the transcription mixture (Fig. 3, lane 1). The introduction of nucleotide substitutions in the –14 and –15 positions of the udpP18 promoter markedly attenuated the intensity of a 276-bp transcript (Fig. 4, lanes 1–3), compared to that of the original promoter containing –14G and –15T nucleotides (Fig. 4, lane 4). The strongest repression of transcription was observed for the mutant promoter isolated from plasmid pAM281 and containing the –14G C substitution (Fig. 4, lane 2), which again agrees with results of the assay of this promoter activity in vivo. Thus, data in Table 3 indicate that this exact mutant promoter in the plasmid pAM281 manifests the lowest level of β-galactosidase activity in bacterial cells containing the damaged cAMP–CRP complex. Thus, results of experiments on transcription confirm the earlier conclusion that according to their structural and functional characteristics, the P2 promoter, which we identified in the
5. Pedersen, H., Sogaard-Andersen, L., Holst, B., and Valentin-Hansen, P., Heterologous Cooperativity in Escherichia coli: The CytR Repressor Both Contacts DNA and the cAMP Repressor Protein When Binding to the deoP2 Promoter, J. Biol. Chem., 1991, vol. 266, pp. 17 804– 17 808. 6. Sogaard-Andersen, L., Pedersen, H., Holst, B., and Valentin-Hansen, P., A Novel Function of the cAMP–CRP Complex in Escherichia coli: cAMP–CRP Functions as an Adaptor for the CytR Repressor in the deo Operon, Mol. Microbiol., 1991, vol. 5, pp. 969–975. 7. Sogaard-Andersen, L., Mironov, A.S., Pedersen, H., et al., Single Amino Acid Substitution in the cAMP Receptor Protein Specifically Abolishes Regulation by the CytR Repressor in Escherichia coli, Proc. Natl. Acad. Sci. USA, 1991, vol. 88, pp. 4921–4925. 8. Mironov, A.S. and Sukhodolets, V.V., Promoter-like Mutants with Increased Expression of the E. coli Uridine Phosphorylase Structural Gene, J. Bacteriol., 1979, vol. 137, pp. 802–810. 9. Ponnambalam, S., Webster, C., Bingham, A., and Busby, S., Transcription Initiation at the Escherichia coli Galactose Operon Promoters in the Absence of the Normal –35 Region Sequences, J. Biol. Chem., 1986, vol. 261, pp. 16 043–16 048. 10. Keilty, S. and Rosenberg, M., Constitutive Function of a Positively Regulated Promoter Reveals New Sequences Essential for Activity, J. Biol. Chem., 1987, vol. 262, pp. 6389–6385.
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FORMATION OF AN ADDITIONAL PROMOTER IN THE REGULATORY REGION 11. Kumar, A., Malloch, R.A., Fujita, N., et al., The Minus 35 Recognition of Escherichia coli Sigma 70 Is Inessential for Initiation of Transcription at an “Extended-10” Promoter, J. Mol. Biol., 1993, vol. 232, pp. 406–418. 12. Burns, H., Belyaeva, T., Busby, S., and Minchin, S., Temperature Dependence of Open Complex Formation at Two E. coli Promoters with Extended –10 Sequences, Biochem. J., 1996, vol. 317, pp. 305–311. 13. Love Larsen, J.E., Gerdes, K., Light, J., and Molin, S., Low-Copy-Number Plasmid-Cloning Vectors Amplifiable by Derepression of an Inserted Foreign Promoter, Gene, 1984, vol. 28, pp. 45–54.
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14. Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York: Cold Spring Harbor Lab., 1989, 2nd ed. 15. Sanger, F., Nicklen, S., and Coulson, A.R., DNA Sequencing with Chain-Terminating Inhibitors, Proc. Natl. Acad. Sci. USA, 1977, vol. 74, pp. 5463–5467. 16. Kunkel, T.A., Rapid and Efficient Site-Specific Mutagenesis without Phenotypic Selection, Proc. Natl. Acad. Sci. USA, 1985, vol. 82, pp. 488–492. 17. Miller, J.H., Experiments in Molecular Genetics, Cold Spring Harbor, New York: Cold Spring Harbor Lab., 1972.
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