The Bacteriophage T4 Inhibitor and Coactivator AsiA Inhibits Escherichia coli RNA Polymerase More Rapidly in the Absence of 70 Region 1.1: E
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《细菌学杂志》
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 8 Room 2A-13, Bethesda, Maryland 20892-0830
ABSTRACT
The N-terminal region (region 1.1) of 70, the primary subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of 70 regions 2 and 4. Region 1.1 prevents the interaction of free 70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of 70-dependent transcription from promoters that require an interaction between 70 region 4 and the –35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with 70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that 70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter PuvsX. However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with 70 that lacks region 1.1 is less stable than polymerase with full-length 70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free 70 and then the AsiA/70 complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free 70 plus core, yielding more free 70 at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free 70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free 70 arises through an indirect effect.
INTRODUCTION
RNA synthesis in Escherichia coli is accomplished by an RNA polymerase holoenzyme consisting of a core of five subunits (two 's, , ', and ) and a specificity factor (reviewed in references 15 and 30). Core polymerase contains RNA synthesizing activity, but the specific start site for transcription initiation is determined by the DNA binding specificity of the particular factor. In E. coli, 70 is the primary factor that is responsible for initiation of transcription during exponential growth. Sequence and structural analyses of various proteins from many prokaryotes indicate that primary factors share four main regions of conservation (regions 1 to 4) (15, 23). Sequence recognition capability in 70 is provided by alpha helices within regions 2 and 4, which interact directly with a promoter's –10 and –35 DNA elements, respectively.
Although most family members share conservation in regions 2 through 4, the sequence of region 1.1, the N-terminal, highly negatively charged portion, is conserved only among primary proteins (15, 23, 30). Within 70, region 1.1 is composed of the first 100 residues. There is no evidence to suggest that region 1.1 interacts directly with promoter DNA. Nonetheless, its presence has a major impact on 70-DNA interactions. In free 70, the presence of region 1.1 prevents both specific and nonspecific binding to DNA (9). Early work suggested region 1.1 prevents this binding by directly interacting with region 4. However, subsequent work using nuclear magnetic resonance (NMR) spectroscopy of segmental, isotopically labeled A of Thermotoga maritima, a 70 analog, has failed to detect an interaction between regions 1.1 and 4.2 (5).
Besides its function in free 70, the presence of region 1.1 also modulates the ability of RNA polymerase holoenzyme to form stable, open complexes with certain promoters. Given the particular promoter, the presence of region 1.1 can either inhibit or promote the formation of the stable pretranscription complex (39, 40). Thus, region 1.1 provides important functions both when 70 is free and when it is present within RNA polymerase. However, how region 1.1 works is not yet clear.
The bacteriophage T4 AsiA protein has two functions. It inhibits 70-dependent transcription from promoters that require an interaction between 70 region 4 and the –35 DNA element and it coactivates transcription from T4 middle promoters that use 70 containing RNA polymerase and the T4 transcription activator MotA (reviewed in reference 18). The MotA protein binds to a DNA motif (a MotA box) centered at position –30 of middle promoter DNA, and, in addition, both AsiA and MotA interact with 70 region 4. A recent NMR structure of the AsiA/70 region 4 complex shows that AsiA contacts multiple 70 residues, including residues that make direct contact with base moieties in the –35 element when RNA polymerase binds to a typical 70-dependent promoter (22).
Because the presence of region 1.1 modulates the interaction of 70 with host promoter DNA and AsiA interacts with 70 residues that contact the –35 element, we wondered whether 70 region 1.1 was important for MotA/AsiA activation and AsiA inhibition. We show here that region 1.1 is not required for MotA/AsiA activation at a T4 middle promoter. However, RNA polymerase lacking region 1.1 is more rapidly inhibited by AsiA and more rapidly activated by MotA/AsiA. Our previous work indicated that AsiA binds efficiently to free 70 but poorly, if at all, to holoenzyme (19). Our evidence supports the idea that AsiA inhibits more rapidly when region 1.1 is absent, because the absence of region 1.1 destabilizes the interaction of 70 with core, resulting in more available free 70 at any given time.
MATERIALS AND METHODS
Proteins and buffers. MotA and AsiA were purified as described previously (16). The N-terminal His6-tagged fl, which contains the sequence MRGSHHHHHHGSSGLVPRGSELGTRL fused to the start of the 70 protein, and the N-terminal His-tagged 1.1, which contains the sequence MRGSHHHHHHTDPHASSVP fused to residue 100 of the 70 protein, were purified from the fl and 100 plasmids (40), respectively, as described previously (39). Transcription assays indicated that either saturated core at a ratio of 2:1 (I. Hook-Barnard and D. M. Hinton, unpublished data). Our sequencing of the genes within these plasmids has revealed that fl also contains the substitution V36L and that both fl and 1.1 contain the substitution N149D. (V36 is missing in the N-terminal deletion in 1.1.) We have found no detectable difference in activity between fl and purified wild-type 70 (13; data not shown). N-terminal His6-tagged mutants with N-terminal deletions of 50 (50), 75 (75), or 133 (133) residues were the generous gifts of A. Dombroski and C. W. Bowers. E. coli RNA polymerase core was purchased from Epicenter Technologies.
Incubation buffer I contained 23 mM Tris-Cl, pH 7.9; 37 mM Tris-acetate, pH 7.9; 37 mM NaCl; 22% glycerol; 0.56 mM EDTA; 0.18 mM dithiothreitol (DTT); 0.004% Triton X-100; 139 mM potassium glutamate; 3.7 mM magnesium acetate; 93 μg/ml bovine serum albumin (BSA); and 0.09 mM 2-mercaptoethanol. Incubation buffer II contained 34 mM Tris-Cl, pH 7.9; 26 mM Tris-acetate, pH 7.9; 42 mM NaCl; 34% glycerol; 0.17 mM DTT; 0.71 mM EDTA; 0.006% Triton X-100; 97 mM potassium glutamate; 2.6 mM magnesium acetate; and 65 μg/ml BSA. Incubation buffer III contained 29 mM Tris-Cl, pH 8; 26 mM Tris-acetate, pH 7.9; 38 mM NaCl; 28% glycerol; 0.65 mM EDTA; 0.15 mM DTT; 0.005% Triton X-100; 97 mM potassium glutamate; 2.6 mM magnesium acetate; 65 μg/ml BSA; and 0.06 mM 2-mercaptoethanol. DNA buffer I contained 7.4 mM Tris-Cl, pH 7.9; 51 mM Tris-acetate, pH 7.9; 57 mM NaCl; 0.66 mM EDTA; 0.13 mM DTT; 2.1% glycerol; 190 mM potassium glutamate; 5.1 mM magnesium acetate; 130 μg/ml BSA; 425 μM each of ATP, GTP, and CTP; and 110 μM [-32P]UTP (1 x 104 dpm/pmol). DNA buffer II was identical to DNA buffer I except that it lacked ribonucleoside triphosphates (rNTPs). DNA buffer III contained 4.2 mM Tris-Cl, pH 7.9; 63 mM Tris-acetate, pH 7.9; 0.58 mM EDTA; 0.16 mM DTT; 240 mM potassium glutamate; 6.3 mM magnesium acetate; 160 μg/ml BSA; 525 μM each of ATP, GTP, and CTP; and 13 μM [-32P]UTP (1 x 105 dpm/pmol). DNA buffer IV contained 5.4 mM Tris-Cl, pH 7.9; 52 mM Tris-acetate, pH 7.9; 16 mM NaCl; 0.6% glycerol; 0.61 mM EDTA; 0.13 mM DTT; 190 mM potassium glutamate; 5.2 mM magnesium acetate; 130 μg/ml BSA; 0.06 mM 2-mercaptoethanol; 525 μM each of ATP, GTP, and CTP; and 13 μM [-32P]UTP (1 x 105 dpm/pmol). NTP solution I contained 2 mM each of ATP, CTP, and GTP and 0.5 mM [-32P]UTP (1 x 104 dpm/pmol). Tris-borate-EDTA (TBE) (1x) contained 2.5 mM EDTA and 89 mM Tris-borate, pH 8.3.
DNA. pDKT90 (25) contains the promoters PuvsX and Pminor in the vector pTZ19U (United States Biochemical Co.). pPuvsX/sigma (19), which was derived from pDKT90, contains the canonical 70 –35 region TTGACA located 16 bp upstream of the PuvsX –10 region (TATAAT). pPwt, which was derived from pPuvsX/sigma, is identical to pPuvsX/sigma except for an extra T immediately downstream of the –35 element that results in a 17-bp spacer between the –10 and –35 regions. Plasmid DNA was isolated as described previously (17) and then purified by centrifuging to equilibrium in an ethidium bromide-cesium chloride gradient. Linear templates for in vitro transcription reactions, obtained after digestion with restriction enzymes, were purified by phenol extraction followed by ethanol precipitation.
The pBR-70 chimera plasmid (11) and its derivative pBR-70 (D581G) (21), used for the two-hybrid assays, have been described previously. The -70 chimera proteins from these plasmids are composed of the N-terminal domain of (amino acids 1 to 248) fused in frame to the C-terminal region (amino acids 528 to 613) of wild-type 70 or 70 containing the substitution D581G. The pACcI32 plasmid, used to construct pcI-1.1, has been described previously (20). The 70 region 1.1 fragment (corresponding to amino acids 1 to 100) cloned into pACcI32 was obtained as a PCR product of pLNH12 (29), which contains the wild-type 70 gene, by using PfuTurbo DNA polymerase (Stratagene) and primers that contained the necessary NotI or BglII sites to allow ligation with pACcI32 that had been previously cleaved with NotI and BglII. The PCR product was cloned into pACcI32 by use of standard techniques. DNA sequence analysis of pcI-1.1, performed by MGW Biotech, confirmed the expected sequence.
In vitro transcription assays. Transcription reactions were assembled as indicated in the figure legends. Upon addition of rNTPs, reactions were incubated at 37°C. Multiple-round transcription reactions were incubated for 7.5 min. Single-round transcription reactions were treated with 150 ng of rifampin 20 s after the addition of rNTPs and then incubated for an additional 7 min. Reactions were collected on dry ice. Gel load solution (1x TBE, 7 M urea, 0.1% bromophenol blue, 0.1% xylene cyanol FF) was added at a volume of five times that of the reaction aliquots, and the solution was heated at 95°C for 2 min before electrophoresis on 4% polyacrylamide-7 M urea denaturing gels run in 0.5x TBE. After autoradiography, films were scanned using a Desktop Plus scanner and quantified using Diversity One software from Protein Databases, Inc., or using a Powerlook 2100XL densitometer and Quantity One software from Bio-Rad, Inc.
Two-hybrid assay. -Galactosidase assays were performed as described previously (11), using the strain KS1 (11) and the desired pBR- and pcI plasmids, except that in some cases cultures were grown in M9 medium plus Casamino Acids (plus appropriate antibiotics) rather than LB media.
RESULTS
Region 1.1 of 70 is not required for MotA/AsiA-dependent activation of transcription from PuvsX.. Previously, portions of region 4 of 70 (amino acids 551 to 563 and 580 to 598) as well as the far C terminus of the protein (amino acids 604 to 613) have been shown to be important for the interaction of 70 with AsiA and MotA, respectively (reviewed in reference 18). To investigate the effect of region 1.1 of 70 on MotA/AsiA activation, we examined transcription from the T4 middle promoter PuvsX by using polymerases containing either the full-length 70 (fl) or 70 with various N-terminal deletions. Like other T4 middle promoters, PuvsX has an excellent match to a 70 –10 element, but it has a poor match to the 70 –35 element. Instead, it has a MotA box at position –30. When using polymerase with fl (Efl) in multiple-round transcription reactions, we observed a pattern of PuvsX transcription that was similar to that previously documented for wild-type holoenzyme (16). Transcription, which was seen with polymerase alone (Fig. 1, lane 1), was not significantly affected by MotA (Fig. 1, lane 2) but was greatly inhibited by AsiA (Fig. 1, lane 3). The presence of both MotA and AsiA resulted in a higher level of PuvsX transcription (Fig. 1, lane 4).
The amount of PuvsX RNA obtained with polymerase containing a 70 with an N-terminal deletion of 50 (50) (Fig. 1, lanes 5 to 8), 75 (75) (Fig. 1, lanes 9 to 12), or 99 (1.1) (Fig. 1, lanes 13 to 16) residues was less than that observed with Efl. However, all of these polymerases were activated by the addition of MotA and AsiA (Fig. 1, lanes 8, 12, and 16). The level of activated PuvsX transcription (observed in the presence of polymerase, MotA, and AsiA) relative to the level observed with polymerase alone was quite similar, ranging from 1.6 to 2.0, and the level of activated PuvsX transcription relative to that observed with polymerase plus AsiA was 10-fold or greater in each case. Thus, the presence of region 1.1 is not required for MotA/AsiA activation of PuvsX. Polymerase containing a deletion of 133 amino acids from the N terminus (E133), which removes region 1.2 as well as region 1.1, did not produce PuvsX RNA either in the absence or in the presence of MotA and AsiA (data not shown). This result is consistent with the very poor activity of E133 observed with other promoters (40).
Although removal of residues within region 1.1 did not affect the increase in the level of PuvsX RNA with MotA/AsiA relative to that seen with polymerase alone, it did significantly affect transcription from another promoter, Pminor (Fig. 1, lanes 1, 5, 9, and 13). Our previous investigations have indicated that the relative level of Pminor RNA is greater with E1.1 than with Efl because, in the absence of region 1.1, polymerase forms stable pretranscription complexes at this promoter more rapidly than it does when region 1.1 is present (39).
AsiA inhibits and MotA/AsiA activates more rapidly in the absence of 70 region 1.1. Transcription from 70-dependent promoters that require an interaction with the –35 region of promoter DNA is significantly inhibited when RNA polymerase is associated with AsiA (7, 31, 33, 36). Previous work has indicated that this association of AsiA with RNA polymerase occurs efficiently by a two-step process: first AsiA binds to 70 and then the AsiA/70 complex binds to core (19). Thus, AsiA inhibition of holoenzyme is a process that depends on the dynamic equilibrium between holoenzyme and free 70 plus core.
As seen in Fig. 2A, this association of AsiA with polymerase can be monitored by assaying the level of transcription when AsiA is incubated with free fl before the addition of core versus the level when AsiA is incubated directly with holoenzyme. In this assay, we used the PuvsX/sigma promoter, which is derived from the T4 middle promoter PuvsX but contains a canonical 70 –35 sequence rather than a MotA box (39). Thus, PuvsX/sigma models an excellent 70-dependent promoter. Incubation of AsiA with free fl at either 4°C or 30°C before the addition of core or incubation of AsiA with Efl for 15 min at 30°C completely inhibited the subsequent single round of PuvsX/sigma transcription, indicative of the formation of the AsiA-bound polymerase (Fig. 2A, lanes 3, 4, and 8). However, incubation of AsiA with Efl for 15 min at 4°C did not inhibit transcription (Fig. 2A, lane 7).
As seen with fl, incubation of AsiA with 1.1 before the addition of core also completely inhibited PuvsX/sigma transcription (Fig. 2B, lanes 3 and 4). However, in contrast to the results seen with Efl, incubation of AsiA with E1.1 at 4°C reduced the levels of PuvsX/sigma and Pminor RNAs to 29% and 11%, respectively, of those seen without AsiA (Fig. 2B, lane 7 versus lane 5). Thus, in the absence of region 1.1, the AsiA-associated polymerase forms much more readily at 4°C.
To quantify the effect of region 1.1 on the formation of AsiA-associated polymerase, we performed time course experiments in which we incubated AsiA with holoenzyme for various times at either 4°C or 30°C (Fig. 3) before a single round of transcription using PuvsX in the absence of MotA. Our results indicated that at 4°C about half of the E1.1 was inhibited after an incubation with AsiA for 10 min, whereas there was no detectable inhibition of Efl at this temperature. At 30°C, E1.1 was inhibited about two times more rapidly than was Efl. Thus, in the absence of region 1.1, the rate of AsiA inhibition of polymerase is significantly increased at either 4°C or 30°C. To determine whether the rate of MotA/AsiA activation was also increased in the absence of region 1.1, we repeated the time course experiments using both the T4 middle promoter PuvsX and the 70-dependent promoter Pwt and either Efl or E1.1 (Fig. 4). We found that the absence of region 1.1 increases the rates of inhibition and activation similarly.
Region 1.1 does not interact with 70 region 4 in a two-hybrid assay. We investigated whether 70 regions 1.1 and 4 interact directly by performing E. coli two-hybrid assays, which have previously been used to detect interactions between 70 region 4 and other proteins or protein domains (10, 21, 32). We tested both wild-type 70 region 4 and 70 region 4 containing the substitution D581G. This substitution is needed to observe the interaction between 70 region 4 and the -flap region of core (21). We failed to detect an interaction between 70 region 1.1 and either wild-type 70 region 4 or 70 region 4 containing D581G by using this assay (data not shown).
Holoenzyme exchanges 1.1 for fl. Although earlier work found no difference in the binding of fl and 1.1 to core (40), this result was based on an assay that monitored the loss of function by E1.1 at the Ptac promoter and used a transcription buffer with chloride as the predominant anion. We reinvestigated this question by taking advantage of the fact that the absence of region 1.1 results in different levels of holoenzyme activity at different promoters. Thus, in the following transcription assay, we could simultaneously observe Pminor transcription, which is favored by E1.1 (39), and PuvsX transcription, which is favored by Efl (39). We also used a transcription buffer containing the physiologically relevant glutamate as the primary anion. We first reconstituted either Efl or E1.1 by incubation of core with the appropriate at 30°C. We then incubated Efl with a sixfold excess of 1.1 or incubated E1.1 with a sixfold excess of fl at 30°C or at 4°C before a single round of transcription using DNA that contained both PuvsX and Pminor. As seen in Fig. 5, incubation of Efl with excess 1.1 at either 30°C or 4°C did not affect the levels of PuvsX and Pminor RNA. However, incubation of E1.1 with fl at either temperature resulted in a rapid (at 30°C) or slower (at 4°C) change in the relative level of Pminor versus PuvsX RNA, consistent with the conversion of the E1.1 to Efl. These results suggest that holoenzyme which lacks region 1.1 is less stable than holoenzyme that contains region 1.1.
DISCUSSION
region 1.1 is the N-terminal, negatively charged domain that is common to all primary sigma factors. Previous work has indicated that for E. coli 70, region 1.1 affects the DNA binding properties of 70 both when it is free and when it is bound to core in RNA polymerase holoenzyme. In free 70, the presence of region 1.1 prevents the interaction of free 70 with DNA (9). In holoenzyme, its presence modulates the ability of holoenzyme to interact with particular promoters (39). Thus, region 1.1 plays an important role in controlling the interaction of DNA with the DNA binding regions of 70.
An understanding of exactly where region 1.1 is located within free 70 and within holoenzyme would greatly help to elucidate how region 1.1 affects the interactions of 70 with DNA. Unfortunately, efforts to determine the structure of this region have not yet been successful. However, information about the general location of region 1.1 has been inferred from other lines of evidence. Some work has argued that a direct interaction between regions 1.1 and 4.2 is responsible for the inability of free 70 to bind to DNA. The addition of region 1.1 in trans prevents 1.1 from recognizing the –35 element but not the –10 DNA element (8), and an I53A substitution in region 1.1, which renders holoenzyme unable to bind stably to PR, is suppressed by deletion of residues 609 to 613 in region 4 (2). However, other results are not compatible with this idea. An engineered cysteine substitution at 70 residue 59 within region 1.1 is more chemically reactive in free 70 than when 70 is bound to core (3). In addition, the NMR spectrum of segmentally labeled region 4.2 of free A, the 70 analog of T. maritima, is not affected by the presence or absence of region 1.1 (5). Thus, it has been suggested that region 1.1 prevents DNA binding by region 4.2 when 70 is free because region 1.1 interacts directly with other portions of region 4, because the presence of region 1.1 places region 4.2 in a conformation that is unavailable to the DNA, or because the negatively charged region 1.1 provides a steric block to the DNA (5).
The position of 70 region 1.1 in holoenzyme has been deduced from other analyses. Luminescence resonance energy transfer data indicated that when 70 binds to core, regions 4.2 and 1 move relative to region 2 so that the spacing between regions 2 and 4.2 is correct for the distance between the –10 and –35 elements (4). Modeling of fluorescent resonance energy transfer data, protein-DNA photocrosslink analyses, and crystallographic structures indicate that in holoenzyme, region 1.1 acts as a DNA mimic, residing in the channel that will hold the downstream DNA when holoenzyme stably binds to a promoter and that upon formation of the stable polymerase/promoter complex, region 1.1 moves from this downstream DNA channel (26). Hydroxyl radical protein footprinting supports these conclusions. It indicates that the protection of region 1.1 in holoenzyme is lost upon the binding of polymerase to DNA (28). Thus, region 1.1 appears to be a highly flexible domain that can reside in different positions depending on whether 70 is free, is in a complex with core, or is in the holoenzyme/promoter complex.
Both AsiA and MotA interact with portions of region 4 (7, 31, 32, 34), and structural analyses indicate that region 4 of 70 is radically remodeled by AsiA (22). This remodeling is consistent with accumulated evidence suggesting that the 70 region 4 DNA contacts must be reconfigured in order to allow MotA interaction with the MotA box sequence centered in the –30 region of T4 middle promoter DNA (reviewed in reference 18). Given that region 1.1 affects the DNA binding properties of 70, we supposed that the lack of region 1.1 might affect the ability of AsiA to inhibit transcription from 70-dependent promoters or of MotA/AsiA to activate transcription from T4 middle promoters. Our results indicate that region 1.1 is not required for transcription from PuvsX RNA by polymerase/MotA/AsiA. However, we find that AsiA inhibits and MotA/AsiA activates RNA polymerase holoenzyme significantly more rapidly when region 1.1 is missing.
We have previously shown that AsiA-inhibited polymerase is formed efficiently by a two-step process (19). AsiA first binds to free 70 and then this complex binds to core. AsiA inhibition of holoenzyme is a slow process that is consistent with the time needed for the dynamic equilibrium between holoenzyme and 70 plus core to make free 70 available for binding to AsiA (6, 12, 14, 24, 37, 41). Thus, two simple models could explain the ability of AsiA to inhibit E1.1 more readily than Efl. In one model, the portions of 70 region 4 that interact with AsiA might be available in free 70 but not available in holoenzyme because of the position of region 1.1 in holoenzyme. Thus, the absence of region 1.1 would allow AsiA to bind directly to 70 when it is present in holoenzyme. In an alternative model, the absence of region 1.1 would not allow AsiA to access holoenzyme directly, but rather it would affect the dynamic equilibrium between holoenzyme and 70 plus core. Thus, in the absence of region 1.1, this equilibrium would be shifted toward more free 70 and free core, increasing the concentration of free 70 at any given time. AsiA would then be able to bind to the available free 70 and the AsiA-associated polymerase would form more rapidly.
Our results are consistent with the second model. First, using a sensitive two-hybrid assay that has been used extensively to detect interactions of region 4 with other proteins and peptides (10, 21, 32), we do not detect an interaction of region 4 with region 1.1. Thus, our result supports other studies (3, 5) arguing that the effect of region 1.1 on the DNA binding capability of region 4.2 is indirect. Our evidence also argues that the increased rate of AsiA inhibition and MotA/AsiA activation when region 1.1 is missing is due to an indirect effect. We find that reconstituted Efl is immune to the presence of excess of 1.1, while E1.1 is converted to Efl in the presence of excess fl at either 4°C or 30°C. These results suggest that the presence of 70 region 1.1 substantially increases the stabilization of the 70/core interaction. Previous work has determined that the interaction of 70 with core is quite strong, with measured binding constants ranging from 2 x 10–7 to 5 x 10–10 M (12, 14, 24, 41). While the primary interaction of 70 with core involves the interaction of 70 regions 2.1 to 2.2 with the coiled-coil region of ' (1), the overall interface of 70 with core is extensive, involving several regions of 70 and /' (27, 28, 35, 38). The placement of the highly negatively charged region 1.1 into the downstream DNA channel of core, as predicted by modeling of fluorescent resonance energy transfer data (26), would provide multiple electrostatic interactions between 70 and core. Our finding that the presence of region 1.1 helps to stabilize holoenzyme is compatible with this prediction and suggests that the loss of region 1.1/core interaction upon formation of the stable polymerase/promoter complex may contribute to the eventual release of 70 from core when polymerase begins elongation.
ACKNOWLEDGMENTS
We gratefully acknowledge the gifts of 50 and 75 from C. W. Bowers and A. Dombroski and plasmids for the two-hybrid system from S. Dove and A. Hochschild. We also thank T. James, India Hook-Barnard, and N. Nossal for helpful discussions and A. Makela for the construction of pPwt.
This research was supported by the Intramural Research Program of the National Institutes of Health, NIDDK.
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ABSTRACT
The N-terminal region (region 1.1) of 70, the primary subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of 70 regions 2 and 4. Region 1.1 prevents the interaction of free 70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of 70-dependent transcription from promoters that require an interaction between 70 region 4 and the –35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with 70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that 70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter PuvsX. However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with 70 that lacks region 1.1 is less stable than polymerase with full-length 70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free 70 and then the AsiA/70 complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free 70 plus core, yielding more free 70 at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free 70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free 70 arises through an indirect effect.
INTRODUCTION
RNA synthesis in Escherichia coli is accomplished by an RNA polymerase holoenzyme consisting of a core of five subunits (two 's, , ', and ) and a specificity factor (reviewed in references 15 and 30). Core polymerase contains RNA synthesizing activity, but the specific start site for transcription initiation is determined by the DNA binding specificity of the particular factor. In E. coli, 70 is the primary factor that is responsible for initiation of transcription during exponential growth. Sequence and structural analyses of various proteins from many prokaryotes indicate that primary factors share four main regions of conservation (regions 1 to 4) (15, 23). Sequence recognition capability in 70 is provided by alpha helices within regions 2 and 4, which interact directly with a promoter's –10 and –35 DNA elements, respectively.
Although most family members share conservation in regions 2 through 4, the sequence of region 1.1, the N-terminal, highly negatively charged portion, is conserved only among primary proteins (15, 23, 30). Within 70, region 1.1 is composed of the first 100 residues. There is no evidence to suggest that region 1.1 interacts directly with promoter DNA. Nonetheless, its presence has a major impact on 70-DNA interactions. In free 70, the presence of region 1.1 prevents both specific and nonspecific binding to DNA (9). Early work suggested region 1.1 prevents this binding by directly interacting with region 4. However, subsequent work using nuclear magnetic resonance (NMR) spectroscopy of segmental, isotopically labeled A of Thermotoga maritima, a 70 analog, has failed to detect an interaction between regions 1.1 and 4.2 (5).
Besides its function in free 70, the presence of region 1.1 also modulates the ability of RNA polymerase holoenzyme to form stable, open complexes with certain promoters. Given the particular promoter, the presence of region 1.1 can either inhibit or promote the formation of the stable pretranscription complex (39, 40). Thus, region 1.1 provides important functions both when 70 is free and when it is present within RNA polymerase. However, how region 1.1 works is not yet clear.
The bacteriophage T4 AsiA protein has two functions. It inhibits 70-dependent transcription from promoters that require an interaction between 70 region 4 and the –35 DNA element and it coactivates transcription from T4 middle promoters that use 70 containing RNA polymerase and the T4 transcription activator MotA (reviewed in reference 18). The MotA protein binds to a DNA motif (a MotA box) centered at position –30 of middle promoter DNA, and, in addition, both AsiA and MotA interact with 70 region 4. A recent NMR structure of the AsiA/70 region 4 complex shows that AsiA contacts multiple 70 residues, including residues that make direct contact with base moieties in the –35 element when RNA polymerase binds to a typical 70-dependent promoter (22).
Because the presence of region 1.1 modulates the interaction of 70 with host promoter DNA and AsiA interacts with 70 residues that contact the –35 element, we wondered whether 70 region 1.1 was important for MotA/AsiA activation and AsiA inhibition. We show here that region 1.1 is not required for MotA/AsiA activation at a T4 middle promoter. However, RNA polymerase lacking region 1.1 is more rapidly inhibited by AsiA and more rapidly activated by MotA/AsiA. Our previous work indicated that AsiA binds efficiently to free 70 but poorly, if at all, to holoenzyme (19). Our evidence supports the idea that AsiA inhibits more rapidly when region 1.1 is absent, because the absence of region 1.1 destabilizes the interaction of 70 with core, resulting in more available free 70 at any given time.
MATERIALS AND METHODS
Proteins and buffers. MotA and AsiA were purified as described previously (16). The N-terminal His6-tagged fl, which contains the sequence MRGSHHHHHHGSSGLVPRGSELGTRL fused to the start of the 70 protein, and the N-terminal His-tagged 1.1, which contains the sequence MRGSHHHHHHTDPHASSVP fused to residue 100 of the 70 protein, were purified from the fl and 100 plasmids (40), respectively, as described previously (39). Transcription assays indicated that either saturated core at a ratio of 2:1 (I. Hook-Barnard and D. M. Hinton, unpublished data). Our sequencing of the genes within these plasmids has revealed that fl also contains the substitution V36L and that both fl and 1.1 contain the substitution N149D. (V36 is missing in the N-terminal deletion in 1.1.) We have found no detectable difference in activity between fl and purified wild-type 70 (13; data not shown). N-terminal His6-tagged mutants with N-terminal deletions of 50 (50), 75 (75), or 133 (133) residues were the generous gifts of A. Dombroski and C. W. Bowers. E. coli RNA polymerase core was purchased from Epicenter Technologies.
Incubation buffer I contained 23 mM Tris-Cl, pH 7.9; 37 mM Tris-acetate, pH 7.9; 37 mM NaCl; 22% glycerol; 0.56 mM EDTA; 0.18 mM dithiothreitol (DTT); 0.004% Triton X-100; 139 mM potassium glutamate; 3.7 mM magnesium acetate; 93 μg/ml bovine serum albumin (BSA); and 0.09 mM 2-mercaptoethanol. Incubation buffer II contained 34 mM Tris-Cl, pH 7.9; 26 mM Tris-acetate, pH 7.9; 42 mM NaCl; 34% glycerol; 0.17 mM DTT; 0.71 mM EDTA; 0.006% Triton X-100; 97 mM potassium glutamate; 2.6 mM magnesium acetate; and 65 μg/ml BSA. Incubation buffer III contained 29 mM Tris-Cl, pH 8; 26 mM Tris-acetate, pH 7.9; 38 mM NaCl; 28% glycerol; 0.65 mM EDTA; 0.15 mM DTT; 0.005% Triton X-100; 97 mM potassium glutamate; 2.6 mM magnesium acetate; 65 μg/ml BSA; and 0.06 mM 2-mercaptoethanol. DNA buffer I contained 7.4 mM Tris-Cl, pH 7.9; 51 mM Tris-acetate, pH 7.9; 57 mM NaCl; 0.66 mM EDTA; 0.13 mM DTT; 2.1% glycerol; 190 mM potassium glutamate; 5.1 mM magnesium acetate; 130 μg/ml BSA; 425 μM each of ATP, GTP, and CTP; and 110 μM [-32P]UTP (1 x 104 dpm/pmol). DNA buffer II was identical to DNA buffer I except that it lacked ribonucleoside triphosphates (rNTPs). DNA buffer III contained 4.2 mM Tris-Cl, pH 7.9; 63 mM Tris-acetate, pH 7.9; 0.58 mM EDTA; 0.16 mM DTT; 240 mM potassium glutamate; 6.3 mM magnesium acetate; 160 μg/ml BSA; 525 μM each of ATP, GTP, and CTP; and 13 μM [-32P]UTP (1 x 105 dpm/pmol). DNA buffer IV contained 5.4 mM Tris-Cl, pH 7.9; 52 mM Tris-acetate, pH 7.9; 16 mM NaCl; 0.6% glycerol; 0.61 mM EDTA; 0.13 mM DTT; 190 mM potassium glutamate; 5.2 mM magnesium acetate; 130 μg/ml BSA; 0.06 mM 2-mercaptoethanol; 525 μM each of ATP, GTP, and CTP; and 13 μM [-32P]UTP (1 x 105 dpm/pmol). NTP solution I contained 2 mM each of ATP, CTP, and GTP and 0.5 mM [-32P]UTP (1 x 104 dpm/pmol). Tris-borate-EDTA (TBE) (1x) contained 2.5 mM EDTA and 89 mM Tris-borate, pH 8.3.
DNA. pDKT90 (25) contains the promoters PuvsX and Pminor in the vector pTZ19U (United States Biochemical Co.). pPuvsX/sigma (19), which was derived from pDKT90, contains the canonical 70 –35 region TTGACA located 16 bp upstream of the PuvsX –10 region (TATAAT). pPwt, which was derived from pPuvsX/sigma, is identical to pPuvsX/sigma except for an extra T immediately downstream of the –35 element that results in a 17-bp spacer between the –10 and –35 regions. Plasmid DNA was isolated as described previously (17) and then purified by centrifuging to equilibrium in an ethidium bromide-cesium chloride gradient. Linear templates for in vitro transcription reactions, obtained after digestion with restriction enzymes, were purified by phenol extraction followed by ethanol precipitation.
The pBR-70 chimera plasmid (11) and its derivative pBR-70 (D581G) (21), used for the two-hybrid assays, have been described previously. The -70 chimera proteins from these plasmids are composed of the N-terminal domain of (amino acids 1 to 248) fused in frame to the C-terminal region (amino acids 528 to 613) of wild-type 70 or 70 containing the substitution D581G. The pACcI32 plasmid, used to construct pcI-1.1, has been described previously (20). The 70 region 1.1 fragment (corresponding to amino acids 1 to 100) cloned into pACcI32 was obtained as a PCR product of pLNH12 (29), which contains the wild-type 70 gene, by using PfuTurbo DNA polymerase (Stratagene) and primers that contained the necessary NotI or BglII sites to allow ligation with pACcI32 that had been previously cleaved with NotI and BglII. The PCR product was cloned into pACcI32 by use of standard techniques. DNA sequence analysis of pcI-1.1, performed by MGW Biotech, confirmed the expected sequence.
In vitro transcription assays. Transcription reactions were assembled as indicated in the figure legends. Upon addition of rNTPs, reactions were incubated at 37°C. Multiple-round transcription reactions were incubated for 7.5 min. Single-round transcription reactions were treated with 150 ng of rifampin 20 s after the addition of rNTPs and then incubated for an additional 7 min. Reactions were collected on dry ice. Gel load solution (1x TBE, 7 M urea, 0.1% bromophenol blue, 0.1% xylene cyanol FF) was added at a volume of five times that of the reaction aliquots, and the solution was heated at 95°C for 2 min before electrophoresis on 4% polyacrylamide-7 M urea denaturing gels run in 0.5x TBE. After autoradiography, films were scanned using a Desktop Plus scanner and quantified using Diversity One software from Protein Databases, Inc., or using a Powerlook 2100XL densitometer and Quantity One software from Bio-Rad, Inc.
Two-hybrid assay. -Galactosidase assays were performed as described previously (11), using the strain KS1 (11) and the desired pBR- and pcI plasmids, except that in some cases cultures were grown in M9 medium plus Casamino Acids (plus appropriate antibiotics) rather than LB media.
RESULTS
Region 1.1 of 70 is not required for MotA/AsiA-dependent activation of transcription from PuvsX.. Previously, portions of region 4 of 70 (amino acids 551 to 563 and 580 to 598) as well as the far C terminus of the protein (amino acids 604 to 613) have been shown to be important for the interaction of 70 with AsiA and MotA, respectively (reviewed in reference 18). To investigate the effect of region 1.1 of 70 on MotA/AsiA activation, we examined transcription from the T4 middle promoter PuvsX by using polymerases containing either the full-length 70 (fl) or 70 with various N-terminal deletions. Like other T4 middle promoters, PuvsX has an excellent match to a 70 –10 element, but it has a poor match to the 70 –35 element. Instead, it has a MotA box at position –30. When using polymerase with fl (Efl) in multiple-round transcription reactions, we observed a pattern of PuvsX transcription that was similar to that previously documented for wild-type holoenzyme (16). Transcription, which was seen with polymerase alone (Fig. 1, lane 1), was not significantly affected by MotA (Fig. 1, lane 2) but was greatly inhibited by AsiA (Fig. 1, lane 3). The presence of both MotA and AsiA resulted in a higher level of PuvsX transcription (Fig. 1, lane 4).
The amount of PuvsX RNA obtained with polymerase containing a 70 with an N-terminal deletion of 50 (50) (Fig. 1, lanes 5 to 8), 75 (75) (Fig. 1, lanes 9 to 12), or 99 (1.1) (Fig. 1, lanes 13 to 16) residues was less than that observed with Efl. However, all of these polymerases were activated by the addition of MotA and AsiA (Fig. 1, lanes 8, 12, and 16). The level of activated PuvsX transcription (observed in the presence of polymerase, MotA, and AsiA) relative to the level observed with polymerase alone was quite similar, ranging from 1.6 to 2.0, and the level of activated PuvsX transcription relative to that observed with polymerase plus AsiA was 10-fold or greater in each case. Thus, the presence of region 1.1 is not required for MotA/AsiA activation of PuvsX. Polymerase containing a deletion of 133 amino acids from the N terminus (E133), which removes region 1.2 as well as region 1.1, did not produce PuvsX RNA either in the absence or in the presence of MotA and AsiA (data not shown). This result is consistent with the very poor activity of E133 observed with other promoters (40).
Although removal of residues within region 1.1 did not affect the increase in the level of PuvsX RNA with MotA/AsiA relative to that seen with polymerase alone, it did significantly affect transcription from another promoter, Pminor (Fig. 1, lanes 1, 5, 9, and 13). Our previous investigations have indicated that the relative level of Pminor RNA is greater with E1.1 than with Efl because, in the absence of region 1.1, polymerase forms stable pretranscription complexes at this promoter more rapidly than it does when region 1.1 is present (39).
AsiA inhibits and MotA/AsiA activates more rapidly in the absence of 70 region 1.1. Transcription from 70-dependent promoters that require an interaction with the –35 region of promoter DNA is significantly inhibited when RNA polymerase is associated with AsiA (7, 31, 33, 36). Previous work has indicated that this association of AsiA with RNA polymerase occurs efficiently by a two-step process: first AsiA binds to 70 and then the AsiA/70 complex binds to core (19). Thus, AsiA inhibition of holoenzyme is a process that depends on the dynamic equilibrium between holoenzyme and free 70 plus core.
As seen in Fig. 2A, this association of AsiA with polymerase can be monitored by assaying the level of transcription when AsiA is incubated with free fl before the addition of core versus the level when AsiA is incubated directly with holoenzyme. In this assay, we used the PuvsX/sigma promoter, which is derived from the T4 middle promoter PuvsX but contains a canonical 70 –35 sequence rather than a MotA box (39). Thus, PuvsX/sigma models an excellent 70-dependent promoter. Incubation of AsiA with free fl at either 4°C or 30°C before the addition of core or incubation of AsiA with Efl for 15 min at 30°C completely inhibited the subsequent single round of PuvsX/sigma transcription, indicative of the formation of the AsiA-bound polymerase (Fig. 2A, lanes 3, 4, and 8). However, incubation of AsiA with Efl for 15 min at 4°C did not inhibit transcription (Fig. 2A, lane 7).
As seen with fl, incubation of AsiA with 1.1 before the addition of core also completely inhibited PuvsX/sigma transcription (Fig. 2B, lanes 3 and 4). However, in contrast to the results seen with Efl, incubation of AsiA with E1.1 at 4°C reduced the levels of PuvsX/sigma and Pminor RNAs to 29% and 11%, respectively, of those seen without AsiA (Fig. 2B, lane 7 versus lane 5). Thus, in the absence of region 1.1, the AsiA-associated polymerase forms much more readily at 4°C.
To quantify the effect of region 1.1 on the formation of AsiA-associated polymerase, we performed time course experiments in which we incubated AsiA with holoenzyme for various times at either 4°C or 30°C (Fig. 3) before a single round of transcription using PuvsX in the absence of MotA. Our results indicated that at 4°C about half of the E1.1 was inhibited after an incubation with AsiA for 10 min, whereas there was no detectable inhibition of Efl at this temperature. At 30°C, E1.1 was inhibited about two times more rapidly than was Efl. Thus, in the absence of region 1.1, the rate of AsiA inhibition of polymerase is significantly increased at either 4°C or 30°C. To determine whether the rate of MotA/AsiA activation was also increased in the absence of region 1.1, we repeated the time course experiments using both the T4 middle promoter PuvsX and the 70-dependent promoter Pwt and either Efl or E1.1 (Fig. 4). We found that the absence of region 1.1 increases the rates of inhibition and activation similarly.
Region 1.1 does not interact with 70 region 4 in a two-hybrid assay. We investigated whether 70 regions 1.1 and 4 interact directly by performing E. coli two-hybrid assays, which have previously been used to detect interactions between 70 region 4 and other proteins or protein domains (10, 21, 32). We tested both wild-type 70 region 4 and 70 region 4 containing the substitution D581G. This substitution is needed to observe the interaction between 70 region 4 and the -flap region of core (21). We failed to detect an interaction between 70 region 1.1 and either wild-type 70 region 4 or 70 region 4 containing D581G by using this assay (data not shown).
Holoenzyme exchanges 1.1 for fl. Although earlier work found no difference in the binding of fl and 1.1 to core (40), this result was based on an assay that monitored the loss of function by E1.1 at the Ptac promoter and used a transcription buffer with chloride as the predominant anion. We reinvestigated this question by taking advantage of the fact that the absence of region 1.1 results in different levels of holoenzyme activity at different promoters. Thus, in the following transcription assay, we could simultaneously observe Pminor transcription, which is favored by E1.1 (39), and PuvsX transcription, which is favored by Efl (39). We also used a transcription buffer containing the physiologically relevant glutamate as the primary anion. We first reconstituted either Efl or E1.1 by incubation of core with the appropriate at 30°C. We then incubated Efl with a sixfold excess of 1.1 or incubated E1.1 with a sixfold excess of fl at 30°C or at 4°C before a single round of transcription using DNA that contained both PuvsX and Pminor. As seen in Fig. 5, incubation of Efl with excess 1.1 at either 30°C or 4°C did not affect the levels of PuvsX and Pminor RNA. However, incubation of E1.1 with fl at either temperature resulted in a rapid (at 30°C) or slower (at 4°C) change in the relative level of Pminor versus PuvsX RNA, consistent with the conversion of the E1.1 to Efl. These results suggest that holoenzyme which lacks region 1.1 is less stable than holoenzyme that contains region 1.1.
DISCUSSION
region 1.1 is the N-terminal, negatively charged domain that is common to all primary sigma factors. Previous work has indicated that for E. coli 70, region 1.1 affects the DNA binding properties of 70 both when it is free and when it is bound to core in RNA polymerase holoenzyme. In free 70, the presence of region 1.1 prevents the interaction of free 70 with DNA (9). In holoenzyme, its presence modulates the ability of holoenzyme to interact with particular promoters (39). Thus, region 1.1 plays an important role in controlling the interaction of DNA with the DNA binding regions of 70.
An understanding of exactly where region 1.1 is located within free 70 and within holoenzyme would greatly help to elucidate how region 1.1 affects the interactions of 70 with DNA. Unfortunately, efforts to determine the structure of this region have not yet been successful. However, information about the general location of region 1.1 has been inferred from other lines of evidence. Some work has argued that a direct interaction between regions 1.1 and 4.2 is responsible for the inability of free 70 to bind to DNA. The addition of region 1.1 in trans prevents 1.1 from recognizing the –35 element but not the –10 DNA element (8), and an I53A substitution in region 1.1, which renders holoenzyme unable to bind stably to PR, is suppressed by deletion of residues 609 to 613 in region 4 (2). However, other results are not compatible with this idea. An engineered cysteine substitution at 70 residue 59 within region 1.1 is more chemically reactive in free 70 than when 70 is bound to core (3). In addition, the NMR spectrum of segmentally labeled region 4.2 of free A, the 70 analog of T. maritima, is not affected by the presence or absence of region 1.1 (5). Thus, it has been suggested that region 1.1 prevents DNA binding by region 4.2 when 70 is free because region 1.1 interacts directly with other portions of region 4, because the presence of region 1.1 places region 4.2 in a conformation that is unavailable to the DNA, or because the negatively charged region 1.1 provides a steric block to the DNA (5).
The position of 70 region 1.1 in holoenzyme has been deduced from other analyses. Luminescence resonance energy transfer data indicated that when 70 binds to core, regions 4.2 and 1 move relative to region 2 so that the spacing between regions 2 and 4.2 is correct for the distance between the –10 and –35 elements (4). Modeling of fluorescent resonance energy transfer data, protein-DNA photocrosslink analyses, and crystallographic structures indicate that in holoenzyme, region 1.1 acts as a DNA mimic, residing in the channel that will hold the downstream DNA when holoenzyme stably binds to a promoter and that upon formation of the stable polymerase/promoter complex, region 1.1 moves from this downstream DNA channel (26). Hydroxyl radical protein footprinting supports these conclusions. It indicates that the protection of region 1.1 in holoenzyme is lost upon the binding of polymerase to DNA (28). Thus, region 1.1 appears to be a highly flexible domain that can reside in different positions depending on whether 70 is free, is in a complex with core, or is in the holoenzyme/promoter complex.
Both AsiA and MotA interact with portions of region 4 (7, 31, 32, 34), and structural analyses indicate that region 4 of 70 is radically remodeled by AsiA (22). This remodeling is consistent with accumulated evidence suggesting that the 70 region 4 DNA contacts must be reconfigured in order to allow MotA interaction with the MotA box sequence centered in the –30 region of T4 middle promoter DNA (reviewed in reference 18). Given that region 1.1 affects the DNA binding properties of 70, we supposed that the lack of region 1.1 might affect the ability of AsiA to inhibit transcription from 70-dependent promoters or of MotA/AsiA to activate transcription from T4 middle promoters. Our results indicate that region 1.1 is not required for transcription from PuvsX RNA by polymerase/MotA/AsiA. However, we find that AsiA inhibits and MotA/AsiA activates RNA polymerase holoenzyme significantly more rapidly when region 1.1 is missing.
We have previously shown that AsiA-inhibited polymerase is formed efficiently by a two-step process (19). AsiA first binds to free 70 and then this complex binds to core. AsiA inhibition of holoenzyme is a slow process that is consistent with the time needed for the dynamic equilibrium between holoenzyme and 70 plus core to make free 70 available for binding to AsiA (6, 12, 14, 24, 37, 41). Thus, two simple models could explain the ability of AsiA to inhibit E1.1 more readily than Efl. In one model, the portions of 70 region 4 that interact with AsiA might be available in free 70 but not available in holoenzyme because of the position of region 1.1 in holoenzyme. Thus, the absence of region 1.1 would allow AsiA to bind directly to 70 when it is present in holoenzyme. In an alternative model, the absence of region 1.1 would not allow AsiA to access holoenzyme directly, but rather it would affect the dynamic equilibrium between holoenzyme and 70 plus core. Thus, in the absence of region 1.1, this equilibrium would be shifted toward more free 70 and free core, increasing the concentration of free 70 at any given time. AsiA would then be able to bind to the available free 70 and the AsiA-associated polymerase would form more rapidly.
Our results are consistent with the second model. First, using a sensitive two-hybrid assay that has been used extensively to detect interactions of region 4 with other proteins and peptides (10, 21, 32), we do not detect an interaction of region 4 with region 1.1. Thus, our result supports other studies (3, 5) arguing that the effect of region 1.1 on the DNA binding capability of region 4.2 is indirect. Our evidence also argues that the increased rate of AsiA inhibition and MotA/AsiA activation when region 1.1 is missing is due to an indirect effect. We find that reconstituted Efl is immune to the presence of excess of 1.1, while E1.1 is converted to Efl in the presence of excess fl at either 4°C or 30°C. These results suggest that the presence of 70 region 1.1 substantially increases the stabilization of the 70/core interaction. Previous work has determined that the interaction of 70 with core is quite strong, with measured binding constants ranging from 2 x 10–7 to 5 x 10–10 M (12, 14, 24, 41). While the primary interaction of 70 with core involves the interaction of 70 regions 2.1 to 2.2 with the coiled-coil region of ' (1), the overall interface of 70 with core is extensive, involving several regions of 70 and /' (27, 28, 35, 38). The placement of the highly negatively charged region 1.1 into the downstream DNA channel of core, as predicted by modeling of fluorescent resonance energy transfer data (26), would provide multiple electrostatic interactions between 70 and core. Our finding that the presence of region 1.1 helps to stabilize holoenzyme is compatible with this prediction and suggests that the loss of region 1.1/core interaction upon formation of the stable polymerase/promoter complex may contribute to the eventual release of 70 from core when polymerase begins elongation.
ACKNOWLEDGMENTS
We gratefully acknowledge the gifts of 50 and 75 from C. W. Bowers and A. Dombroski and plasmids for the two-hybrid system from S. Dove and A. Hochschild. We also thank T. James, India Hook-Barnard, and N. Nossal for helpful discussions and A. Makela for the construction of pPwt.
This research was supported by the Intramural Research Program of the National Institutes of Health, NIDDK.
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