1 Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Division of Protein Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
Correspondence
Kristine B. Arnvig
karnvig{at}nimr.mrc.ac.uk
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ABSTRACT |
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*Present address: Indian Institute of Science, Bangalore 560012, India.
Present address: Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, Canada V5Z 3J5.
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INTRODUCTION |
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In Escherichia coli the intrinsic strength of rrn promoters is greatly increased by the presence of an upstream activating region (UAR) (Condon et al., 1995). The E. coli UAR consists of two distinct components: the UP element which is factor independent (Newlands et al., 1991
) and an element consisting of between three and five binding sites for the transcription factor Fis (Hirvonen et al., 2001
). Both mechanisms require the C-terminal domain (CTD) of the RNA polymerase alpha subunit (RNAP
) for activation (Bokal et al., 1997
; Ross et al., 1993
). However, a major difference between the two lies in the positioning requirements relative to the core promoter. Factor-dependent activation, i.e. activation by Fis (or other class I transcription factors), is face-of-the-helix dependent; in other words the activator binding sites can be separated from the core promoter and still retain most of their activation potential, provided that they remain on the same face of the DNA helix (Bokal et al., 1997
; Gaston et al., 1990
; Ishihama, 1993
; Newlands et al., 1992
). In contrast, factor-independent activation involving the UP element requires a contact between the promoter proximal alpha subunit and the sigma factor: the element must be immediately upstream of, as well as in phase with, the 35 box (Hirvonen et al., 2001
; Meng et al., 2001
). Little is known about bacterial rrn regulatory elements other than those of E. coli, although a number of rrn operons from other species have been sequenced and/or investigated. Most of these appear to harbour UP elements, as shown either by function or by the presence of characteristic AT-rich tracts immediately upstream of the 35 region (Aiyar et al., 2002
; Amador et al., 1999
; Dryden & Kaplan, 1993
; Garnier et al., 1991
; Helmann, 1995
; La Fontaine & Rood, 1996
; Zahn et al., 2001
). Mycobacterial rrn operons do not contain phased AT-rich tracts characteristic of UP elements (Gonzalez-y-Merchand et al., 1997
). However, in the thermophile Thermus thermophilus, sequence-specific interactions between RNAP
and a putative UP element with a different nucleotide composition have been reported, suggesting that a high AT content is not a universal requirement (Wada et al., 2000
).
The objective of the present study was to dissect the promoter region of the rrnB operon of Mycobacterium smegmatis in order to identify and characterize activating or otherwise regulatory regions in this operon. Using reporter gene fusions, we have characterized the intrinsic strength of the P1B promoter and shown that its upstream region contains activating elements. We demonstrate that the promoter activation is face-of-the-helix dependent, indicating that the rrnB UAR does not contain an UP element, which makes it significantly different from the E. coli rrn paradigm.
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METHODS |
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Bacterial cell extracts.
Cell extracts were prepared as described by Papavinasasundaram et al. (2001). An aliquot of each extract was used to determine the total protein concentration using a BCA protein assay kit (Pierce).
-Galactosidase (
-gal) activity was determined as described by Miller (1972)
, normalized to total protein concentration and expressed as units (mg protein)1. The promoter activities were assayed in at least three individual cultures for each strain.
Expression and purification of M. tuberculosis RNAP.
The plasmid encoding M. tuberculosis rpoA (pKA) was transformed into E. coli BL21 (DE3) pLysS. The cells were grown in Terrific Broth (Sambrook et al., 1989
) and induced with 1 mM IPTG when the OD600 reached 0·6. After a further 6 h growth at 30 °C, the culture was harvested by centrifugation for 30 min at 3000 r.p.m. RNAP
of M. tuberculosis was highly soluble. The His-tagged protein was purified by affinity chromatography on a cobalt resin column (Talon, Clontech). Briefly, the cells were resuspended in buffer A (50 mM phosphate buffer, 250 mM NaCl, pH 8·0) and sonicated. Post-lysis, the cell extract was cleared by centrifugation, and the proteins in the supernatant were bound to the Talon resin pre-equilibrated in buffer A. The Talon column was washed with 10 column volumes of buffer B (50 mM phosphate buffer, 250 mM NaCl, 5 mM imidazole, pH 7·5) and the protein was subsequently eluted from the column in buffer C (50 mM phosphate buffer, 250 mM NaCl, 300 mM imidazole, pH 7·0). The eluted protein was about 85 % pure and was subjected to size exclusion chromatography on a Pharmacia S-75 column in 50 mM Tris, 250 mM NaCl, pH 7·5.
Electrophoretic mobility shift assays (EMSAs).
The entire region of RNAP known to interact with the UP element is 100 % conserved between M. tuberculosis and M. smegmatis and so the M. tuberculosis RNAP
was used in all experiments. The binding reaction was performed at room temperature in 20 µl reaction buffer: 20 mM HEPES, pH 7·5, 0·2 mM EDTA, 10 mM (NH4)2SO4, 1 mM DTT, 15 mM MgCl2, 15 mM KCl, 50 µg BSA ml1 and 2 µg poly dI : dC ml1. The samples were electrophoresed on native 8 % acrylamide gels in 0·5x TBE, dried and exposed to a phosphor imager screen.
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RESULTS |
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Initially we wanted to determine the intrinsic strength of a P1B minimal promoter fragment as our point of reference. The region spanning the 10 and 35 hexamers (i.e. 36 to 8 relative to the transcription start point) was inserted into the promoter probe vector, and the resulting reporter construct gave rise to a -gal activity of 24 units (mg protein)1. For comparison, we made a construct with the M. tuberculosis rrnA PCL1 promoter fragment covering the same region, i.e. 36 to 8. This construct gave rise to a
-gal activity of almost 200 units (mg protein)1, i.e. approximately eight times the activity of the corresponding P1B promoter fragment (data not shown). Next we compared our minimal promoter construct with constructs that had been extended downstream to either +1 or +10. Both of these 3'-end additions gave rise to a small increase in
-gal activity relative to the minimal construct (see Fig. 1
). Thus, extending the promoter insert to include the native transcription start point increased activity 1·5-fold, while a further extension to +10 relative to the transcription start point increased activity another twofold (Fig. 1
). These results demonstrate that during exponential growth the P1B minimal promoter gives rise to a relatively low
-gal activity, compared to that of the M. tuberculosis PCL1 minimal promoter fragment, and moreover that the region downstream of 8 supports only a limited increase in
-gal activity.
The rrnB upstream activating region
Our initial experiments indicated that the activity of the P1B promoter was significantly increased by including the region upstream of the 35 hexamer (K. B. Arnvig, unpublished data). Therefore a detailed investigation of the P1B UAR was carried out in order to identify putative activating elements. A series of P1B constructs was made in which the 3' end was maintained at 8, as in the minimal promoter construct, while the UAR was gradually extended to 200 relative to the transcription start point. The results, shown in Fig. 1, demonstrate how the promoter activity of P1B increases significantly as the UAR is extended towards 200, reaching a maximum
-gal activity of 8758 units (mg protein)1, which corresponds to more than 350-fold activation of the minimal construct. The most significant increase in activity (18-fold) is observed as the UAR is extended from 39 to 52. Interestingly, this fragment contains a highly conserved sequence found in a number of mycobacterial rrn operons, indicated as UR1 in Fig. 1
(Gonzalez-y-Merchand et al., 1997
). The region between 52 and 70, which contains the tyrS stop codon as well as a second UR element (UR2), increases
-gal activity another three- to fourfold, whereas no increase, but in fact a slight decrease, is observed between 70 and 80. Finally, there is another activating region between 80 and 200, which increases
-gal activity approximately fivefold. However, the increase from 140 to 200 is minor (1·1-fold), indicating that most of the activating sequences of the rrnB operon are located within the first 100 nt of the UAR. As a control, the UAR was cloned into pEJ414, but showed no detectable promoter activity on its own in either orientation (data not shown).
The activity of the P1B UAR was subsequently determined in constructs with the 3' end at +1 or +10 in order to establish if the region between 8 and +10 affected UAR activity or vice versa, i.e. if they act independently. The results, taken from the activities in Fig. 1, indicate that the UAR (140 to 37) activates the P1B promoter 320-fold with the 3' end at 8, 450-fold with the 3' end at +1 and 360-fold with the 3' end at +10. Conversely, the contribution from the region between 8 and +10 varies from 2·8-fold (with the 5' end at 36) to 3·1-fold (with the 5' end at 140). We regard these variations as minor and therefore conclude that the two regions act independently.
Lack of sequence-specific interaction between the rrnB UAR and RNAP
A number of bacterial promoters, such as E. coli rrn promoters, contain a third element, in addition to the 10 and 35 boxes, located immediately upstream of the 35 box (Newlands et al., 1991; Rao et al., 1994
). This so-called UP element consists of AT-rich tracts in phase with the 35 box, and it has been shown to interact with RNAP
in a sequence-specific manner (Estrem et al., 1998
; Rao et al., 1994
; Ross et al., 1993
). The M. smegmatis P1B UAR does not contain an apparent UP element sequence, but it is possible that an alternative sequence may function as a UP element in mycobacteria, analogous to the situation in T. thermophilus (Wada et al., 2000
). A series of EMSAs was performed to establish whether or not the mycobacterial RNAP
interacts in a sequence-specific manner with the P1B UAR. Results of EMSAs performed with 104 bp P1B UAR (from 140 to 37) and various concentrations of purified RNAP
are shown in Fig. 2(a)
. The results demonstrate that there is complex formation between the P1B UAR and RNAP
at protein concentrations starting at 20 µM RNAP
, and that 75 µM RNAP
is required to shift more than 50 % of the probe. For comparison, the E. coli UP element DNA displays 100 % complex formation at 4 µM RNAP
(Ross et al., 2001
), and we therefore consider the P1B UAR interaction to be of low affinity. Fig. 2(b)
illustrates how the RNAP
UAR complex can be disrupted equally well by the addition of excess unlabelled UAR DNA and non-UAR DNA (compare lanes 35 with lanes 68). To ensure that the non-UAR competitor did not contain UAR-like determinants by coincidence, the experiment was repeated with another non-UAR DNA (a 147 bp PvuIFspI pUC19 DNA fragment) but, as before, the non-UAR and UAR DNA fragments competed to the same extent (data not shown). These results strongly suggest that the interaction between the UAR and RNAP
is not sequence specific. The ability of RNAP
to interact with DNA independent of sequence was further supported by EMSAs in which non-UAR and UAR DNA formed complexes with RNAP
equally well (data not shown). The results demonstrate the absence of sequence-specific interaction between the rrnB UAR and the RNAP
subunit, which in turn suggests that there is no UP element in the mycobacterial rrnB UAR.
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DISCUSSION |
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The region that conferred the highest increase in promoter activity is located between 41 and 52. This segment contains an element, TCTGACCT/AGGG, which is repeated exactly two helical turns further upstream (see Fig. 1). A similar repeat is present in the rrnB operon of Mycobacterium fortuitum, although the sequence has been changed slightly from TCTGACCAGGG-N9-TCTGACCTGGG in M. smegmatis to TCTGACCTGCG-N9-AATGACCTGGC in M. fortuitum. Moreover, the core sequence, PuACCNG, is also found in a single copy immediately upstream of most, if not all rrnA promoters (Gonzalez-y-Merchand et al., 1997
). We therefore propose this to be a likely recognition site for a transcriptional activator common to both rrnA and rrnB and thus to all mycobacteria. We did not identify a similar sequence in the region from 8 to 140. Due to the different relative positions of tyrS and P1B in M. smegmatis and M. fortuitum, the location of this element differs so that it overlaps with the tyrS coding sequence in M. smegmatis but not in M. fortuitum. Whether this has implications for the function of the element has not been determined, although it is clear that such an overlap puts some constraints on the sequence. This type of arrangement, a promoter region within the coding region of an upstream gene, is not unknown and has been described before in both E. coli and cyanobacteria (Bognar et al., 1989
; Grundstrom & Jaurin, 1982
; Plansangkate et al., 2004
).
The overall conclusion from these experiments is that one or more transcriptional activators are likely to be responsible for the more than 300-fold activation seen in the rrnB operon. As a comparison, factor-dependent activation is less than tenfold in E. coli, whereas factor-independent activation accounts for the majority of the total transcriptional activation in the seven rrn operons (Hirvonen et al., 2001). In a number of bacterial species, the rrn operons contain either functional UP elements (Aiyar et al., 2002
; Ross et al., 1993
; Wada et al., 2000
) or phased AT-rich tracts, i.e. putative UP elements upstream of their rrn promoters (Amador et al., 1999
; Dryden & Kaplan, 1993
; Garnier et al., 1991
; Helmann, 1995
; La Fontaine & Rood, 1996
; Zahn et al., 2001
). Mycobacteria appear to rely exclusively on factor-dependent activation to achieve maximal promoter activity, and this is the first characterization of a UAR that differs significantly from the existing paradigm.
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ACKNOWLEDGEMENTS |
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Received 31 August 2004;
revised 12 October 2004;
accepted 14 October 2004.
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