Nucleic Acids Research, 2001, Vol. 29, No. 20 4166-4178
© 2001 Oxford University Press
UP element-dependent transcription at the Escherichia coli rrnB P1 promoter: positional requirements and role of the RNA polymerase 
subunit linker
Laboratory of Molecular Microbiology, Division of Genomic Medicine, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK, 1School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UK, 2Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK and 3Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, WI 53706, USA
Received July 5, 2001; Revised and Accepted August 21, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The UP element stimulates transcription from the rrnB P1 promoter through a direct interaction with the C-terminal domain of the RNA polymerase
subunit (CTD). We investigated the effect on transcription from rrnB P1 of varying both the location of the UP element and the length of the subunit interdomain linker, separately and in combination. Displacement of the UP element by a single turn of the DNA helix resulted in a large decrease in transcription from rrnB P1, while displacement by half a turn or two turns totally abolished UP element-dependent transcription. Deletions of six or more amino acids from within the subunit linker resulted in a decrease in UP element-dependent stimulation, which correlated with decreased binding of CTD to the UP element. Increasing the linker length was less deleterious to RNA polymerase function at rrnB P1 but did not compensate for the decrease in activation that resulted from displacing the UP element. Our results suggest that the location of the UP element at rrnB P1 is crucial to its function and that the natural length of the subunit linker is optimal for utilisation of the UP element at this promoter. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Escherichia coli RNA polymerase holoenzyme (RNAP) is a multi-subunit complex consisting of an
subunit homodimer, single ß, ß' and 
subunits, and one of several 
subunit species (1,2). During transcription initiation at many promoters, RNAP containing the major factor, 70, recognises two hexameric promoter elements located 
10 and 35 bp upstream of the transcription start point (+1) (3). Recognition of the –10 and –35 promoter elements requires regions 2.3–2.5 and 4.2 of 70, respectively (4,5). However, a third promoter element, the UP element, is frequently required for full promoter activity (6,7). UP elements interact directly with the C-terminal domain of the subunit of RNAP (CTD) (8,9) in holoenzymes containing 70 or alternative factors (10–12).
UP elements consist of A+T-rich sequences located upstream of the –35 region at many promoters (6,8,13). At the most well-studied UP element-dependent promoter, rrnB P1, the UP element comprises DNA sequences from approximately positions –40 to –60 and is composed of two subsites centred at about –42 and –52 (termed the proximal and distal subsites, respectively), each of which can bind an CTD (6,14–17). The interaction between CTD and the UP element results in an increase in KB, the initial equilibrium constant for RNAP binding to the promoter, but may also affect later steps in the pathway to open complex formation (16,18). The CTD–UP element interaction is responsible for a 30–70-fold increase in in vivo promoter activity at rrnB P1 (8,16,17). The optimised UP element sequence (‘consensus’ UP element) contains alternating A- and T-tracts and is 5-fold more effective than the wild type rrnB P1 UP element (17). UP elements can also be transposed as a separate module from one promoter to another where they retain their ability to stimulate transcription in a factor-independent manner (8,13,16).
Each subunit consists of 329 amino acids organised in two independently folding domains (9,19). The N-terminal domain (NTD; residues 8–231) contains determinants for dimerisation and assembly into RNAP (20,21) and plays a role in transcription activation at some promoters (22,23). CTD (residues 249–329) (24) plays roles in transcription initiation, elongation and termination (25–27). During transcription initiation, CTD provides a contact site for many transcription activators (25,28,29) and is directly involved in UP element recognition (8,30). CTD folds into a (HhH)2 domain (31) in which key residues interacting with DNA reside within the loop and second helix of HhH1 and HhH2, respectively (32). Based on genetic, biochemical and NMR studies of the CTD–DNA interaction, and on the X-ray structure of the RuvA–DNA complex, the (HhH)2 domain is thought to interact in a sequence-specific way with bases in the minor groove and with the DNA backbone across the minor groove (30,32–35).
Several lines of evidence indicate that a flexible linker connects CTD to NTD. First, limited proteolysis studies pointed to an accessible region between amino acids 234 and 249 of E.coli (9,19). Secondly, NMR analysis of an isolated C-terminal fragment of (amino acids 233–329) showed that a region of at least 13 amino acids, extending from D233 to E248, exhibits a high degree of flexibility (36). This is consistent with the crystallographic data for NTD, where the C-terminal limit of helix 3 is assigned to F231 (21). Thirdly, amino acid sequence alignments of subunits from eubacteria and chloroplasts reveal a non-conserved sequence of variable length corresponding to amino acids V237–P251 of the E.coli subunit (37). Consistent with its proposed role as a linker, the introduction of substantial deletions or insertions into this region does not affect the efficiency of assembly of into RNAP, nor do they affect transcription from activator-independent and UP element-independent promoters (37,38). However, deletion of three amino acids from within the linker greatly reduces transcription from a promoter where the activator protein CRP binds to a site centred at –61.5 (Class I), whereas transcription is enhanced from a promoter where CRP binds at –41.5 (Class II). This result, together with the observation that CTD tethered by a shorter linker is still recruited to DNA sequences upstream of the CRP binding site at the Class II promoter, implies that the linker deletion compromises the motional freedom of CTD rather than its inherent ability to bind DNA (37,38).
Although potential UP elements or UP element subsites have been identified in the –40 to –60 region at many promoters, in most cases such searches have not been extended to locations further upstream (6,8,13,17). The recent demonstration that CTD is able to contact DNA near positions –43, –53, –63, –73, –83 and –93 at the lac promoter, unassisted by CRP (39), suggests that the length of the linker is unlikely to restrict access of CTD to UP elements located upstream of –60. However, the question of whether UP elements are able to stimulate transcription efficiently from distant locations has not been satisfactorily addressed.
Gourse and colleagues studied effects of UP element location before the UP element subsites were fully defined (15). They observed that upstream displacement of sequences between positions –47 and –87 by a single turn of the helix did not affect transcription from the rrnB P1 promoter, whereas displacement by two or more turns exerted an inhibitory effect on transcription. However, as the proximal UP element subsite was retained in its normal location in these early experiments, conclusions concerning displacement of the full UP element must be re-evaluated. In cases where UP elements have been identified upstream of the –40 to –60 region, DNA bending by accessory DNA binding proteins such as IHF is also required for full activity (e.g. 12,40,41), presumably to bring binding sequences closer to the core promoter. Therefore, in this work, we have examined the effect of upstream displacement of the full UP element on transcription from the rrnB P1 promoter in the absence of accessory factors. We have also investigated the role of linker length in UP element function at the rrnB P1 promoter and the possibility that lengthening the linker might alleviate the negative effect of UP element displacement. Our results demonstrate that the position of the full UP element is crucial to its function at the rrnB P1 promoter and that the length of the subunit linker is optimised for utilisation of UP elements in their normal position at rrn P1 promoters.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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rrnB P1 promoter fragments
The E.coli strains and plasmids used in this work are listed in Table 1. Standard molecular biology techniques for plasmid isolation and DNA manipulation were used throughout (42). All rrnB P1 promoter derivatives used for transcription assays or single copy lacZ fusion analysis were cloned as EcoRI–HindIII fragments into the transcription vector pRLG770 or
phage system I (16), respectively, and possess a downstream end point at position +52 with respect to the rrnB P1 transcription initiation site. Upstream end points are at position –66 for promoter fragments present in pRLG4238, pRLG3278, pRLG4213, pRLG4214 and pRLG4210, and in the lacZ fusions present in RLG3074, RLG4192, RLG3097 and RLG2263, but contain different sequences from –38 to –59 (6,17). Promoter fragments present in plasmids pRLG4713–pRLG4717, pRLG4719 and pRLG4720 (and the corresponding lacZ fusions present in RLG4721–RLG4727) possess wild type rrnB P1 sequences up to position –37, upstream of which is a consensus UP element located at various positions. These promoter fragments were constructed by amplifying the rrnB P1 core promoter region from pRLG4210 using primer RLG1620 (5'-GCGCTACGGCGTTTCACTTC-3') (13), which anneals downstream of the pRLG770 HindIII site, as the reverse primer in each case and one of a series of upstream primers of the type 5'-GCGCGAATTCGGAAAATTTTTTTTAAAAAAGAX-3' (EcoRI site italicised and UP element emboldened) where ‘X’ corresponds to sequences inserted between the displaced UP element and position –37 of the rrnB P1 promoter (see Fig. 1) plus, in some cases, additional rrnB P1 core promoter sequences (–37 to –16) to allow primer annealing (Table 2). The consensus UP element present in these derivatives is a combination of the individual SELEX selected consensus proximal and distal UP element subsites, and differs from the version constructed by Estrem et al. (6) only by the substitution of a thymine residue for an adenine at position –46 (underlined).
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Subunit purification and reconstitution of RNAP
His-tagged RNAP
subunits containing wild type and modified linkers were overproduced in strain BL21(DE3) harbouring pHTT7f1NH and the pHTT7f1NH derivatives pMGM2, pMGM5–pMGM11, pMGM31 and pMGM34, which encoded subunits with shorter or longer interdomain linkers (37). Following 3 h of induction of the plasmid-borne phage T7 promoter, subunits were purified by Ni2+-affinity chromatography as described previously (30,43). Preparation of inclusion bodies of RNAP ß, ß' and subunits from strains XL1-Blue (pMKSe2), BL21(DE3) (pT7ß') and BL21(DE3) (pLHN12), respectively, and reconstitution of RNAP were performed as described previously (43).
In vitro transcription
Multiple round transcription reactions were performed at 25°C for 20 min in a volume of 25 µl containing 150 mM NaCl, 40 mM Tris–acetate pH 7.9, 10 mM MgCl2, 1 mM dithiothreitol (DTT), 100 µg/ml bovine serum albumin (BSA), 500 µM ATP, 200 µM CTP, 200 µM GTP, 10 µM UTP and 3–5 µCi [-32P]UTP (10 mCi/ml, 800 Ci/mmol). Each reaction also contained 0.6 nM supercoiled plasmid DNA, prepared using the Qiagen plasmid midi kit and purified further by phenol–chloroform extraction. All plasmid templates for transcription assays were derivatives of the transcription vector pRLG770 (44) and, therefore, also result in the production of the RNA-I transcript (108 nucleotides). Transcription originating from rrnB P1 promoter fragments terminate at the rrnB T1T2 terminator present on the vector and give rise to transcripts of 220 nucleotides in length. Reactions were initiated by addition of RNAP and terminated with 25 µl of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol). Samples were fractionated on a 5.5% acrylamide gel containing 7 M urea and transcript abundance was quantified using a PhosphorImager. Native RNAP was a generous gift of R. Landick and was 60 ± 10% active in binding to the PR promoter (45). RNAP reconstituted with the wild type subunit was 55% as active as native RNAP. Reconstituted RNAPs were used at concentrations that resulted in equivalent transcription from the core rrnB P1 promoter present on pRLG4210 (4.4 nM native RNAP and 8–28 nM reconstituted RNAP) (see legend to Fig. 3).
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Hydroxyl radical footprinting
Fragments containing the wild type rrnB P1 promoter were prepared for footprinting by first linearising CsCl-purified pRLG4238 DNA with HindIII, then removing the 5' terminal phosphates with calf intestinal alkaline phosphatase and subsequently making a second cut with AatII. The smaller, 205 bp, DNA fragment was purified from a preparative polyacrylamide gel and was labelled on the 5' hydroxyl at the HindIII terminus with 10 µCi [
-32P]ATP (3000 Ci/mmol) using 0.5–1.0 U T4 polynucleotide kinase for 30 min at 37°C. Unincorporated nucleotides were removed by a pass through a Sephadex G-50 spin column and the purified labelled DNA used for footprinting. Binding reactions contained 10 mM Tris–HCl pH 8.0, 30 mM KCl, 10 mM MgCl2, 1 mM DTT, 100 µg/ml BSA, 500 µM ATP, 50 µM CTP, 1.0–4.0 nM labelled DNA fragment and RNAP reconstituted with wild type (21 nM), 
6 (45 nM), 9 (65 nM), 12 (73 nM) or 235 (72 nM), and were incubated at 37°C for 30 min. Prior to hydroxyl radical treatment, heparin was added to a final concentration of 10 µg/ml for 30 s, whereupon hydroxyl radical treatment was performed as previously described (46). The products of the footprinting reaction were fractionated on 8% polyacrylamide sequencing gels that were calibrated with Maxam–Gilbert DNA sequence ladders and resultant footprinting patterns were analysed using a phosphorimager.
Determination of in vivo promoter activity
Analysis of rrnB P1 promoter activity in vivo was performed using strain NK5031 lysogenic for system I derivatives (16) in which rrnB P1 promoter variants were transcriptionally fused to trpB'A-lacZ. Construction of recombinant phages harbouring fusions of lacZ to rrnB P1 derivatives in which the consensus UP element had been displaced was carried out as described previously (47–49) and monolysogens of NK5031 (i.e. RLG4721–RLG4727) were identified by a combination of a PCR-based assay (17) and ß-galactosidase measurement. Overnight cultures of the lacZ fusion strains were diluted 1:50 into pre-warmed Luria–Bertani broth [containing ampicillin (100 µg/ml) and 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) when harbouring pLAW2 derivatives expressing mutant rpoA alleles] and grown at 30°C with vigorous shaking for three to four generations (OD600 0.4–0.6). ß-Galactosidase activity was determined by the method of Miller (50) using the chloroform–SDS procedure and values were corrected for background ß-galactosidase activity by assaying a lysogen containing a promoter-less lacZ fusion (RLG1336). Results are presented as percentages, with the strain harbouring the consensus rrnB P1–lacZ fusion assigned a value of 100% in experiments utilising untransformed strains, and strains harbouring pLAW2 or pMGM12 assigned a value of 100% when measurements were performed on transformants harbouring pLAW2 derivatives.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of UP element displacement on transcription from the rrnB P1 promoter in vitro and in vivo
To investigate the relationship between UP element location and UP element-dependent stimulation of transcription from the rrnB P1 promoter, a series of rrnB P1 promoter derivatives were constructed in which the full consensus UP element was displaced upstream from the rrnB P1 core promoter by distances corresponding to 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 turns of the DNA helix, i.e. 5, 11, 16, 22, 27 and 33 bp, respectively (Fig. 1). These promoters were cloned into the transcription vector pRLG770 and their activities in vitro were compared with that of an rrnB P1 promoter derivative containing the consensus UP element in the wild type position, and to the core rrnB P1 promoter, in which the UP element is replaced by a sequence devoid of UP element-like activity (17). The results showed that whereas the presence of the consensus UP element in the wild type location resulted in a 29-fold increase in promoter activity relative to the core promoter, displacement of the UP element by one helical turn (11 bp) resulted in only
5-fold activation (Fig. 2A, compare lanes 2 and 4 with lane 1). Upstream displacement of the consensus UP element by 0.5, 1.5, 2, 2.5 or 3 turns of the DNA helix essentially abolished UP element-dependent stimulation (Fig. 2A). These results suggest that the location of the UP element at the wild type rrnB P1 promoter is optimal for UP element-dependent stimulation. The results also confirm the previous observation that activation of rrnB P1 by upstream sequences is face of the helix dependent (15).
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To examine the effect of UP element displacement on transcription from rrnB P1 in vivo, the rrnB P1 promoter derivatives possessing displaced UP elements were cloned upstream of the lacZ gene on a recombinant
phage and monolysogens were constructed (see Table 1). The results from ß-galactosidase assays showed that UP element-dependent stimulation of transcription is maximal when the UP element is present in the wild type location (Fig. 2B). Displacement of the consensus UP element by 11 bp upstream from the wild type location gave rise to >20-fold decrease in transcriptional activity, whereas displacement of the UP element by any other distance, including displacement by only 5 bp, abolished UP element-dependent transcription (Fig. 2B). These results are fully consistent with the data obtained in vitro and emphasise the crucial importance of the position of the UP element to its function at rrnB P1.
Effect of linker length on UP element-dependent transcription in vitro
To test the requirements for the RNAP subunit interdomain linker at the rrnB P1 promoter in vitro, RNAPs were reconstituted using a series of 10 mutant subunits possessing interdomain linkers of different lengths. These mutant derivatives have been described previously (37) and contain deletions of 3, 6, 9 and 12 amino acids (termed 3, 6, 9 and 12), and insertions of 3, 6, 10, 13, 16 and 32 amino acids (termed 
3, 6, 10, 13, 16 and 32). Multiple round transcription assays were performed with template DNA containing the wild type rrnB P1 promoter, including its natural UP element, or rrnB P1derivatives possessing the consensus full UP element or either the consensus proximal or distal UP element subsites, instead of the rrnB P1 UP element (6,17). As transcription from the rrnB P1 core promoter in comparison to a control promoter, the RNA-I promoter, encoded on the same plasmid, was relatively unaffected by alterations to the length of the interdomain linker, i.e. the ratio of transcripts originating from the core rrnB P1 and RNA-I promoters remained constant (Fig. 3A), transcription from the rrnB P1 core promoter was used to normalise the activities of the RNAP preparations containing interdomain linkers of different lengths.
At the wild type rrnB P1 promoter, a progressive decrease in RNAP activity was observed as the size of the deletion introduced into the subunit linker was increased (Fig. 3B). The effect of linker deletion was similar for the rrnB P1 promoter containing the consensus UP element (Fig. 3C), although the extent of the decrease was less marked. Removal of 12 amino acids from the linker resulted in an 80–90% decrease in transcriptional efficiency from the promoters with the wild type and consensus UP elements, almost as large a decrease as was observed following complete removal of the CTD. Transcription from these promoters was much less sensitive to increases in the length of the linker. Thus, insertion of 16 amino acids resulted in only a 20–25% decrease in transcription from both promoters, and RNAP containing a 32 amino acid insertion in the linker retained approximately half of wild type RNAP activity at these promoters.
The effect of altering linker length on transcription from the rrnB P1 promoter containing only the consensus distal subsite or only the consensus proximal UP subsite was broadly similar to the overall pattern observed for the wild type rrnB P1 promoter, although the effect of deletion of three amino acids from the linker was greater on transcription from rrnB P1 containing the proximal UP element subsite than on the other rrnB P1 derivatives (Fig. 3D and E).
Effect of linker length on UP element-dependent transcription in vivo
To investigate the effects of altering the length of the interdomain linker on UP element-dependent transcription in vivo, plasmids expressing each of the mutant rpoA alleles were introduced into NK5031 harbouring single copy transcriptional fusions of the lacZ gene to various rrnB P1 promoter derivatives. ß-Galactosidase activities were measured following induction of mutant synthesis for three to four generations. In general agreement with our observations in vitro, decreasing the number of amino acids in the linker caused a general decrease in the efficiency of transcription from the wild type rrnB P1 promoter, whereas only longer insertions within the linker (16 and 32) decreased expression substantially (Fig. 4A). The overall pattern was similar at the consensus rrnB P1 promoter, although this promoter was less sensitive to changes in linker length than the wild type promoter (Fig. 4B).
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Unlike the results in vitro, deletions of more than three amino acids from within the
linker strongly stimulated transcription from the core rrnB P1 promoter in vivo (Fig. 4C). Thus, cells containing RNAP in which the 12 subunit was incorporated into RNAP gave rise to an 15-fold increase in the ß-galactosidase activity relative to cells expressing only wild type rpoA. Increasing the length of the linker caused a gradual but much less profound increase in transcription from the core promoter. The increase in rrnB P1 core promoter activity in vivo can be interpreted in the context of our understanding of rRNA transcription regulation (58,59); i.e. decreased rRNA transcription (from decreased UP element function) leads to compensating derepression of rrnB P1 core promoter activity to keep total rRNA synthesis constant (see ref. 8 and Discussion). Figure 4D and E illustrates the effects of the altered linkers on UP element function in vivo, adjusted for these feedback effects. The fold increases in transcription resulting from the wild type rrnB P1 UP element (Fig. 4D) or from the consensus UP element (Fig. 4E) in the presence of each of the linker mutants are presented as a percentage of the fold increases in transcription observed in the presence of wild type . In this representation, it is obvious that the linker deletions exert a strong negative effect on UP element function in vivo, consistent with their effects on UP element utilisation in vitro. Likewise, it is apparent that UP element-dependent function at rrnB P1 is also impaired by insertions within the linker, although to a lesser degree.
Effect of shortening the linker on interactions between CTD and the UP element at the rrnB P1 promoter
To determine whether there is a correlation between the effect of shortening the interdomain linker on the activity of RNAP at the rrnB P1 promoter, and the ability of CTD to bind the UP element, we carried out hydroxyl radical footprinting experiments on RNAP:rrnB P1 binary complexes. As previously observed, wild type RNAP protected a continuous region extending from approximately position –10 to +16 (not shown) and short regions around positions –20 and –30 (14) (Fig. 5A, lane 3, and B). In addition, short protected regions were observed around positions –43 and –53, corresponding to the sites of CTD interaction within the proximal and distal UP element subsites (14,17). RNAP reconstituted with the 6 derivative affords a similar degree of protection of the proximal and distal UP element subsites as wild type RNAP (Fig. 5A, lane 4, and B). However, protection of both UP element subsites was greatly diminished following deletion of a further three amino acids from the linker (9 RNAP) and was essentially abolished following deletion of 12 amino acids (12 RNAP), without a concomitant reduction in the protection downstream of –40 (Fig. 5A, lanes 5 and 6, and B). RNAP reconstituted with the 235 subunit failed to protect the UP element subsites from hydroxyl radical attack, as shown previously (8), confirming that the protections observed around –43 and –53 are due to CTD (Fig. 5A, lane 7, and B). The pattern of protection produced in the core promoter region by each of the linker modified RNAPs and the 235 RNAP derivative was essentially identical to that afforded by wild type RNAP. These results indicate that as many as six amino acids can be deleted from the interdomain linker without significantly compromising the ability of CTD to engage the UP element subsites in vitro. The strong correlation between the transcriptional activity of the different RNAP deletion derivatives at rrnB P1 and their ability to protect the UP element subsites suggests that that the observed decrease in in vitro and in vivo transcriptional activity at the wild type rrnB P1 promoter upon shortening the interdomain linker is due to a decreased ability to interact with the UP element.
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Effect of increasing the length of the
interdomain linker on stimulation of transcription by displaced UP elements in vitro and in vivoThe footprinting experiments suggest that the ‘reach’ of
CTD is a function of linker length. Therefore, we used in vitro transcription assays to investigate whether tethering CTD by a longer linker could improve the utilisation of displaced UP elements at rrnB P1. We observed no significant difference between the activity of RNAP reconstituted with the 13 derivative and RNAP reconstituted with wild type at the rrnB P1 promoter containing the UP element either in the normal position (as also shown in Fig. 3B) or when the UP element was displaced by one or two turns of the helix (Fig. 6A). A similar pattern was observed when RNAPs reconstituted with mutants possessing 10 or 16 additional amino acids were compared with wild type RNAP (results not shown). We conclude that extending the linker does not significantly enhance the response of RNAP to displaced UP elements at the rrnB P1 promoter in vitro.
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To explore the effect of lengthening the
interdomain linker on the response of RNAP to displaced UP elements in vivo, the strains harbouring single copy fusions of lacZ to rrnB P1 promoters possessing displaced consensus UP elements were transformed with plasmids expressing wild type rpoA, or the 6 and 13 rpoA mutants. Consistent with our in vitro observations, ß-galactosidase assays indicated that possession of an extended linker does not significantly increase the activity of RNAP at any of the promoters containing displaced UP elements (Fig. 6B).
Previous studies have demonstrated that displacement of the distal UP element subsite by one helical turn, while retaining the proximal subsite in its normal location, does not impair UP element-dependent transcription, whereas displacement of the distal subsite by two or three turns abolishes its contribution (15). To examine whether extending the interdomain linker compensates for the negative effect of displacing the distal UP element subsite alone, we performed ß-galactosidase measurements on a series of strains harbouring single copy fusions of the lacZ gene to rrnB P1 promoter derivatives in which the distal UP element subsite had been displaced by a half, one, two and three turns of the helix (15). Our results showed that the activity of all the promoter variants was unaffected in cells containing 6 or 13 subunits (results not shown). These results suggest that extending the subunit linker also does not compensate for the negative effect on transcription of displacement of the distal UP element subsite.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiments described here provide important information regarding the requirements for productive interactions between
CTD and UP elements. We have shown that upstream displacement of the full UP element by 5 bp or more greatly reduces UP element-dependent stimulation of transcription from rrnB P1. Our results are consistent with previous observations that CTD can sometimes bind to the opposite face of the DNA helix to that occupied by the rest of RNAP, but that such interactions do not stimulate transcription and can in fact induce an inhibitory effect (60,61). As there appears to be a requirement for UP element:CTD interactions to be located on the same face of the DNA helix at rrnB P1 as the rest of RNAP, adjacent to bound at the –35 region, it is possible that optimal UP element function requires contact between CTD and at some promoters. Recent evidence supports the importance of such an interaction at the rrnB P1 promoter containing the proximal UP element subsite (W.E.Ross and R.L.Gourse, unpublished results; 7,32) and at Class I CRP-dependent promoters (23; N.J.Savery, G.Lloyd, S.J.W.Busby, M.S.Thomas, R.H.Ebright and R.L.Gourse, unpublished results). Alternatively, it is possible that insertions between the –35 hexamer and the UP element result in perturbations in DNA structure that interfere with CTD function.
Displacement of the full UP element by two (or more) turns upstream of rrnB P1 (i.e. moving the centres of the proximal and distal subsites to –64 and –75, respectively) essentially abolishes UP element-dependent stimulation, and displacement of the distal UP element subsite was shown previously to decrease the efficiency of CTD binding (15). Conversely, experiments with promoters containing displaced CRP sites have revealed that CTD can bind adjacent to CRP at positions as far as 85 bp upstream of the transcription start site (60,62). CTD is also able to access naturally occurring UP elements located 80 bp upstream of the Pseudomonas putida Pu promoter (12) or 90 bp upstream of the PL promoter (40,41). However, in these cases, the interaction between CTD and more distant DNA binding sites is facilitated by the binding of the transcription factors CRP and IHF, respectively, to upstream sequences. Although CTD can be crosslinked to DNA sites as far as 63, 73, 83 and 93 bp upstream of the lac(ICAP)UV5 promoter in the absence of CRP (39), these presumably transient interactions do not facilitate transcription initiation.
Figures 3–5 indicate that there is a good correlation between linker length, the ability of CTD to access the UP element and the degree of UP element-dependent stimulation at the rrnB P1 promoter. Our observations on the effect of linker deletion on transcription from the rrnB P1 promoter are also broadly similar to the results obtained by Fujita and colleagues (38), although they reported a more pronounced effect of the shorter deletion derivatives on transcription in vitro. In contrast, shortening the subunit interdomain linker exerted a strong stimulatory effect on the core rrnB P1 promoter in vivo (Fig. 4C). This phenomenon was not observed for the UP element-independent lacUV5 promoter (W.Meng, S.J.W.Busby and M.S.Thomas, unpublished results). The decreased efficiency of UP element utilisation by RNAPs containing deletions within the subunit linker would be expected to result in a transient decrease in rRNA synthesis. As reported previously (8), when UP element function is compromised by mutations in rpoA, the activity of an rrnB P1 core promoter–lacZ fusion increases. For reasons that remain unclear, the magnitude of the ‘derepression’ observed in the presence of overexpressed linker modified subunits was greater than the 3-fold increase in core promoter activity measured in the presence of subunits devoid of the C-terminal domain (8). The increase in UP element-independent promoter activity in the seven rrn operons responsible for rRNA biosynthesis in E.coli compensates for the decrease in UP element function and the resulting transient reduction in ribosome synthesis, a phenomenon referred to as ‘rRNA feedback’ (8,58,63). However, the effects of the linker mutants on UP element function were apparent in vivo when the effects of rRNA feedback were taken into consideration (Fig. 4D and E).
Increasing the length of the interdomain linker also impairs transcription from the rrnB P1 promoter, although RNAP appears less sensitive to linker insertions than deletions. This result indicates that the length of the wild type interdomain linker is optimal for efficient utilisation of the UP element at rrnB P1. It is probable that the linker length adopted by bacterial RNAP results, at least in part, from selective pressure for UP element utilisation at rRNA promoters. In this context, it is interesting that chloroplasts contain subunits possessing considerably longer linkers (37), suggesting that chloroplast RNAP is not subject to the same constraints as the bacterial enzyme.
Increasing the length of the linker by 16 amino acids (essentially doubling its length) would be expected to result in extending the ‘reach’ of CTD by 58 Å, enough to allow utilisation of UP elements positioned one or two turns upstream of the normal location (the pitch of B-form DNA is 34 Å per turn). As RNAP containing subunits with linkers lengthened by up to 32 amino acids did not increase utilisation of displaced UP elements, it is possible that the linkers containing insertions are not fully extended. Studies by Fujita and colleagues suggest, in fact, that segments of the linker may adopt an ordered (helical) structure under certain conditions (38). In conjunction with the possible requirement for – interactions and/or DNA structural requirements at the junction of the –35 region and UP element, the possibility that the linker has structure might explain why lengthening the linker does not extend the operational distance of CTD with respect to UP element utilisation.
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ACKNOWLEDGEMENTS |
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M.S.T. would like to thank members of the Gourse and Landick laboratories for their help and advice. This work was supported by project grants 050794 (to M.S.T. and S.J.W.B.) and 052849 (to R.L.G. and S.J.W.B.) from the Wellcome Trust, and by grant RO1 GM37048 from the National Institutes of Health to R.L.G.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +44 114 271 2834; Fax: +44 114 273 9926; Email: m.s.thomas{at}shef.ac.uk Correspondence may also be addressed to Richard L. Gourse. Tel: +1 608 262 2914; Fax: +1 608 262 9865; Email: rgourse{at}bact.wisc.eduPresent address:Wenmao Meng, School of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 Zhang,G., Campbell,E.A., Minakhin,L., Richter,C., Severinov,K. and Darst,S.A. (1999) Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 Å resolution. Cell, 98, 811–824.[ISI][Medline]
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5 Barne,K.A., Bown,J.A., Busby,S.J.W. and Minchin,S.D. (1997) Region 2.5 of the Escherichia coli RNA polymerase 70 subunit is responsible for the recognition of the ‘extended –10’ motif at promoters. EMBO J., 16, 4034–4040.[ISI][Medline]
6 Estrem,S.T., Ross,W., Gaal,T., Chen,Z.W.S., Niu,W., Ebright,R.H. and Gourse,R.L. (1999) Bacterial promoter architecture: subsite structure of UP elements and interactions with the carboxy-terminal domain of the RNA polymerase subunit. Genes Dev., 13, 2134–2147.
7 Gourse,R.L., Ross,W. and Gaal,T. (2000) UPs and downs in bacterial transcription initiation: the role of the alpha subunit of RNA polymerase in promoter recognition. Mol. Microbiol., 37, 687–695.[ISI][Medline]
8 Ross,W., Gosink,K.K., Salomon,J., Igarishi,K., Zou,C., Ishihama,A., Severinov,K. and Gourse,R.L. (1993) A third recognition element in bacterial promoters: DNA binding by the subunit of RNA polymerase. Science, 262, 1407–1413.
9 Blatter,E.E., Ross,W., Tang,H., Gourse,R.L. and Ebright,R.H. (1994) Domain organization of RNA polymerase subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding. Cell, 78, 889–896.[ISI][Medline]
10 Newlands,J.T., Gaal,T., Mecsas,J. and Gourse,R.L. (1993) Transcription of the Escherichia coli rrnB P1 promoter by the heat shock RNA polymerase (E sigma 32) in vitro. J. Bacteriol., 175, 661–668.
11 Fredrick,K., Caramori,T., Chen,Y.F., Galizzi,A. and Helmann,J.D. (1995) Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element. Proc. Natl Acad. Sci. USA, 92, 2582–2586.
12 Bertoni,G., Fujita,N., Ishihama,A. and de Lorenzo,V. (1998) Active recruitment of 54-RNA polymerase to the Pu promoter of Pseudomonas putida: role of IHF and CTD. EMBO J., 17, 5120–5128.[ISI][Medline]
13 Ross,W., Aiyar,S.E., Salomon,J. and Gourse,R.L. (1998) Escherichia coli promoters with UP elements of different strength: modular structure of bacterial promoters. J. Bacteriol., 180, 5375–5383.
14 Newlands,J.T., Ross,W., Gosink,K.K. and Gourse,R.L. (1991) Factor-independent activation of Escherichia coli rRNA transcription. II. Characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase. J. Mol. Biol., 220, 560–583.
15 Newlands,J.T., Josaitis,C., Ross,W. and Gourse,R.L. (1992) Both FIS-dependent and factor-independent upstream activation of the rrnB P1 promoter are face of the helix dependent. Nucleic Acids Res., 20, 719–726.
16 Rao,L., Ross,W., Appleman,J.A., Gaal,T., Leirmo,S., Schlax,P.J., Record,M.T.,Jr and Gourse,R.L. (1994) Factor-independent activation of rrnB P1; an ‘extended’ promoter with an upstream element that dramatically increases promoter strength. J. Mol. Biol., 235, 1421–1435.[ISI][Medline]
17 Estrem,S.T., Gaal,T., Ross,W. and Gourse,R.L. (1998) Identification of an UP element consensus sequence for bacterial promoters. Proc. Natl Acad. Sci. USA, 95, 9761–9766.
18 Strainic,M.G., Sullivan,J.J., Velvis,A. and deHaseth,P.L. (1998) Promoter recognition by Escherichia coli RNA polymerase: effects of the UP element on open complex formation and promoter clearance. Biochemistry, 37, 18074–18080.[Medline]
19 Negishi,T., Fujita,N. and Ishihama,A. (1995) Structural map of the alpha subunit of Escherichia coli RNA polymerase: structural domains identified by proteolytic cleavage. J. Mol. Biol., 248, 723–728.[ISI][Medline]
20 Igarashi,K., Fujita,N. and Ishihama,A. (1991) Identification of a subunit assembly domain in the alpha subunit of Escherichia coli RNA polymerase. J. Mol. Biol., 218, 1–6.[ISI][Medline]
21 Zhang,G. and Darst,S. (1998) Structure of the Escherichia coli RNA polymerase subunit amino-terminal domain. Science, 281, 262–266.
22 Niu,W., Kim,Y., Tau,G., Heyduk,T. and Ebright,R.H. (1996) Transcription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase. Cell, 87, 1123–1134.[ISI][Medline]
23 Busby,S. and Ebright,R.H. (1999) Transcription activation by catabolite activator protein (CAP). J. Mol. Biol., 293, 199–213.[ISI][Medline]
24 Jeon,Y.H., Negishi,T., Shirakawa,M., Yamazaki,T., Fujita,N., Ishihama,A. and Kyogoku,Y. (1995) Solution structure of the activator contact domain of the RNA polymerase subunit. Science, 270, 1495–1497.
25 Ishihama,A. (1993) Protein–protein communication within the transcription apparatus. J. Bacteriol., 175, 2483–2489.
26 Kainz,M. and Gourse,R.L. (1998) The C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase is required for Rho-dependent transcription termination. J. Mol. Biol. 284, 1379–1390.[ISI][Medline]
27 Mah,T.-F., Kuznedelov,K., Mushegian,A., Severinov,K. and Greenblatt,J. (2000) The subunit of E. coli RNA polymerase activates RNA binding by NusA. Genes Dev., 14, 2664–2675.
28 Igarashi,K. and Ishihama,A. (1991) Bipartite functional map of the E. coli RNA polymerase subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP. Cell, 65, 1015–1022.[ISI][Medline]
29 Busby,S. and Ebright,R.H. (1994) Promoter structure, promoter recognition, and transcription activation in prokaryotes. Cell, 79, 743–746.[ISI][Medline]
30 Gaal,T., Ross,W., Blatter,E.E., Tang,H., Jia,X., Krishnan,V.V., Assa-Munt,N., Ebright,R.H. and Gourse,R.L. (1996) DNA-binding determinants of the subunit of RNA polymerase: novel DNA-binding domain architecture. Genes Dev., 10, 16–26.
31 Shao,X. and Grishin,N.V. (2000) Common fold in helix-hairpin-helix proteins. Nucleic Acids Res., 28, 2643–2650.
32 Ross,W., Ernst,A. and Gourse,R.L. (2001) Fine structure of E. coli RNA polymerase-promoter interactions: subunit binding to the UP element minor groove. Genes Dev., 15, 491–506.
33 Murakami,K., Fujita,N. and Ishihama,A. (1996) Transcription factor recognition surface on the RNA polymerase subunit is involved in contact with the DNA enhancer element. EMBO J., 15, 4358–4367.[ISI][Medline]
34 Yasuno,K., Yamazaki,T., Tanaka,Y., Kodama,T.S., Matsugami,A., Katahira,M., Ishihama,A. and Kyogoku,Y. (2001) Interaction of the C-terminal domain of the E. coli RNA polymerase subunit with the UP element: recognizing the backbone structure in the minor groove surface. J. Mol. Biol., 306, 213–225.[ISI][Medline]
35 Ariyoshi,M., Nishino,T., Iwasaki,H., Shinagawa,H. and Morikawa,K. (2000) Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer. Proc. Natl Acad. Sci. USA, 97, 8257–8262.
36 Jeon,Y.H., Yamazaki,T., Otomo,T., Ishihama,A. and Kyogoku,Y. (1997) Flexible linker in the RNA polymerase alpha subunit facilitates the independent motion of the C-terminal activator contact domain. J. Mol. Biol., 267, 953–962.[ISI][Medline]
37 Meng,W., Savery,N.J., Busby,S.J.W. and Thomas,M.S. (2000) The Escherichia coli RNA polymerase subunit: length requirements for transcription activation at CRP-dependent promoters. EMBO J., 19, 1555–1566.[ISI][Medline]
38 Fujita,N., Endo,S. and Ishihama,A. (2000) Structural requirements for the interdomain linker of subunit of Escherichia coli RNA polymerase. Biochemistry, 39, 6243–6249.[Medline]
39 Naryshkin,N., Revyakin,A., Kim,Y., Mekler,V. and Ebright,R.H. (2000) Structural organization of the RNA polymerase-promoter open complex. Cell, 101, 601–611.[ISI][Medline]
40 Giladi,H., Murakami,K., Ishihama,A. and Oppenheim,A.B. (1996) Identification of an UP element within the IHF binding site at the PL1-PL2 tandem promoter of bacteriophage . J. Mol. Biol., 260, 484–491.[ISI][Medline]
41 Giladi,H., Koby,S., Prag,G., Engelhorn,M., Geiselmann,J. and Oppenheim,A. (1998) Participation of IHF and a distant UP element in the stimulation of the phage PL promoter. Mol. Microbiol. 30, 443