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RESEARCH PAPER

Tethering ⁷⁰ to RNA polymerase reveals high in vivo activity of factors and ⁷⁰-dependent pausing at promoter-distal locations

Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA

	Abstract

Top Abstract Results Discussion Materials and methods Acknowledgments References

 
Bacterial factors compete for binding to RNA polymerase (RNAP) to control promoter selection, and in some cases interact with RNAP to regulate at least the early stages of transcript elongation. However, the effective concentration of s in vivo, and the extent to which can regulate transcript elongation generally, are unknown. We report that tethering ⁷⁰ to all RNAP molecules via genetic fusion of rpoD to rpoC (encoding ⁷⁰ and RNAP's ' subunit, respectively) yields viable Escherichia coli strains in which alternative -factor function is not impaired. '::⁷⁰ RNAP transcribed DNA normally in vitro, but allowed ⁷⁰-dependent pausing at extended -10-like sequences anywhere in a transcriptional unit. Based on measurement of the effective concentration of tethered ⁷⁰, we conclude that the effective concentration of ⁷⁰ in E. coli (i.e., its thermodynamic activity) is close to its bulk concentration. At this level, ⁷⁰ would be a bona fide elongation factor able to direct transcriptional pausing even after its release from RNAP during promoter escape.

[Keywords: RNA polymerase; factor; transcriptional regulation; E. coli; pausing]

Received August 8, 2003; revised version accepted October 1, 2003.

During development, differentiation, and changes in environment, binding, and release of initiation factors allow cells to alter patterns of gene expression by reprogramming RNAP. In bacteria, reprogramming is accomplished by switching among factors associated with RNAP (e.g., seven s in Escherichia coli, 18 in Bacillus subtilis, and at least 65 in Streptomyces coelicolor; Ishihama 2000; Kunst et al. 1997; Bentley et al. 2002). Among these, one is the major or housekeeping and is typically present at the highest level (⁷⁰ in E. coli).

Selection among s for RNAP binding is thought to be mediated by concentration-dependent competition among available molecules for available RNAP molecules (Zhou and Gross 1992; Hicks and Grossman 1996; Farewell et al. 1998; Gross et al. 1998; Kolesky et al. 1999; Ishihama 2000; Maeda et al. 2000). The concentrations of available s are in turn highly regulated by their expression levels, rates of degradation, and sequestration via binding to inhibitory proteins called anti-s (Ishihama 2000). Although RNAP outnumbers total in E. coli (1.7:1; Materials and Methods), s must compete for the smaller pool of available RNAP (0.6 per ), which includes newly synthesized RNAP and RNAP not engaged in transcription or sequestered nonspecifically (Ishihama 2000; see Discussion).

The idea that s are released from RNAP during transcript elongation has been challenged recently (Bar-Nahum and Nudler 2001; Mukhopadhyay et al. 2001). In one view, permanent association of ⁷⁰ with some RNAP is proposed to accelerate recycling of this RNAP for new rounds of transcription by circumventing the need to rebind ⁷⁰, which could be rate-limiting in the transcription cycle (Bar-Nahum and Nudler 2001).

⁷⁰ plays at least one regulatory role during transcript elongation. ⁷⁰ can remain bound to RNAP long enough to stimulate pausing at promoter-proximal sites that resemble -10 promoter elements (Ring et al. 1996). In this case, ⁷⁰-dependent pausing was lost when the pause site was moved 20 bp downstream and tested in vitro (Ring et al. 1996). This could be explained by stochastic ⁷⁰ release after promoter escape (Shimamoto et al. 1986) or by resistance of mature ECs to ⁷⁰-stimulated pausing.

To assess the effective concentration of ⁷⁰ in vivo and to gain insight into ⁷⁰'s effect on the EC, we created an rpoC::rpoD gene fusion that tethered ⁷⁰ to all RNAP in cells via a covalent polypeptide linkage. Tethering proteins by genetic fusion fixes the local concentration of interacting proteins (Raag and Whitlow 1995; Timpe and Peller 1995; Robinson and Sauer 1998). Depending on the length of the tether and the location of binding sites relative to the tether-attachment points, tethering can generate local protein concentrations of 10^-5 to 10 M (Robinson and Sauer 1998). The bulk concentration of ⁷⁰ in vivo is 15 µM (Materials and Methods). However, a variety of factors, including macromolecular crowding, can dramatically alter the effective concentration of ⁷⁰ in cells (i.e., its thermodynamic activity). Tethering ⁷⁰ to RNAP makes it possible to examine the effects in vivo of known local concentrations of tethered ⁷⁰, and thus gain insight into the effective concentration of ⁷⁰ in cells.

	Results

Top Abstract Results Discussion Materials and methods Acknowledgments References

 

E. coli is viable with ⁷⁰ fused to RNAPat the ' C terminus

We initially tested the in vivo function of ⁷⁰ tethered to the C terminus of ' or when corresponding gene fusions were conditionally expressed from plasmids (Table 1). (RNAP tolerates alterations at the ends of ' or ; ⁷⁰ tolerates alterations at its N terminus; Severinov et al. 1997; Sharp et al. 1999.) ::⁷⁰ was unable to complement for loss of ⁷⁰ or function. However, '::⁷⁰ complemented for loss of ⁷⁰ and, to a limited extent, '. We considered this result promising and concentrated further study on '::⁷⁰. As an initial control that '::⁷⁰ was incorporated into RNAP, we replaced region 4 of the tethered ⁷⁰ with the corresponding region of ³² (Kumar et al. 1995). This eliminated the ability of ' in the fusion to provide even weak ' function ('::^70/32; Table 1), suggesting that the weak complementation of rpoC^TS by ';::⁷⁰ reflected incorporation into a functional RNAP. Based on these results, we next asked if the rpoC;::rpoD fusion could replace rpoC in the chromosome (Materials and Methods).

View this table: [in this window] [in a new window]   Table 1. Plating efficiency of tethered 70 expressed from plasmids in wild-type and mutant strains

The resulting strain, in which ⁷⁰ was tethered to all RNAPs, proved viable. We transduced the rpoC;::rpoD allele into a strain in which rpoD expression can be shut off (Lonetto et al. 1998). This strain exhibited no defect in either growth or recovery from stationary phase when forced to rely on ';::⁷⁰ for both ' and ⁷⁰ function (Fig. 1A,B). The simplest interpretation of these results is that ';::⁷⁰ RNAP is a fully functional enzyme and that the weak complementation of rpoC^TS by the plasmid-encoded ';::⁷⁰ reflected partial overexpression toxicity (Table 1).

View larger version (45K): [in this window] [in a new window]   Figure 1. Strains and growth phenotypes. (A) Early log-phase cultures of strains with the rpoD and rpoC loci illustrated were serially diluted and plated onto LB plates or LB plates containing indole acrylic acid (IAA). Strains (top to bottom) are RL301, RL1374, CAG20153 [GenBank] , and RL1094 (Supplementary Table 1). The efficiencies of plating minus IAA versus plus IAA were 10-5 for the wild-type strain (CAG20153 [GenBank] ) and 1 for the ';::70 strain (RL1094). (B) Wild-type or ';::70 with TrpR-repressed rpoD strains (C600K- and RL1094) were monitored during growth in LB at 37°C using a Klett colorimeter (data are averages for two independent cultures). After residing in stationary phase for 12 h, the strains were diluted back to Klett <5. (C) Model of 70 tethering to ' based on the Thermus thermophilis holoenzyme structure (Vassylyev et al. 2002; ' NCD removed). The upstream and downstream faces of RNAP are shown above schematics of the E. coli ' and 70 subunits. RNAP subunits are shown in spacefill; , is shown as a C-trace colored by regions shown in the schematic. The N-terminal and C-terminal residues of and ' resolved in the structure are depicted as black spheres (corresponding to '1389 and 7091 in E. coli). region 1.1 (semitransparent white circle) is positioned based on the hydrated volume of an 82-amino acid globular protein attached to 7091.

The structure of bacterial RNAP holoenzyme (Vassylyev et al. 2002) is consistent with this interpretation (Fig. 1C). Although region 1.1 is not resolved in the structure, if spherical, it would occupy a hydrated volume 30 Å in diameter on the downstream face of RNAP. At least 27 amino acids may flexibly connect region 1.1 to ': three amino acids from the gene fusion, plus 19 C-terminal amino acids of E. coli ' that correspond to the disordered C-terminal segment in the RNAP structure, plus the last five visible amino acids of ', which form a random coil. These 27 amino acids (100 Å fully extended) are sufficient to span the 80 Å between the last resolved amino acids in ' and the likely positions of region 1.1, proposed to be within the DNA-entry channel in the holoenzyme and at the outside edge of RNAP's lobe domain in initiation complexes (Mekler et al. 2002).

To verify that ⁷⁰ remained tethered to RNAP in the viable ';::⁷⁰ strain, we examined the subunits present in cell extracts by immunoblotting with anti-' and anti-⁷⁰ antibodies (see Materials and Methods). Only ';::⁷⁰, not ' or ⁷⁰, was present in a whole-cell extract from the ';::⁷⁰ strain when rpoD expression was shut off (Fig. 2A, cf. lanes 1 and 2). Therefore, ';::⁷⁰ was not cleaved to separate ' and ⁷⁰ subunits in vivo, and this intact ';::⁷⁰ polypeptide could serve as the sole source of both ' and ⁷⁰ in viable E. coli cells.

View larger version (39K): [in this window] [in a new window]   Figure 2. Purified RNAP and immunoblot analysis. (A) (Left) Cellular extracts of wild-type (wt) or ';::70 (F) strains (RL301 and RL1094) were separated by 3%-8% Tris-Acetate (Novex), transferred to nitrocellulose, and probed with a mixture of ' and 70 antibodies (Materials and Methods). Equal amounts of cellular protein were present in each lane. (Center) Western blot of wild-type (wt) and ';::70 (F) RNAPs. Samples were separated by 3%-8% Tris Acetate (Novex) and probed as in the left panel. (Right) ';::70 RNAP (F) purified from the strain containing the rpoC;::rpoD fusion and Trp-repressed rpoD (RL1094) separated by 4%-15% PAGE gel (Pharmacia) shown next to wild-type (wt) RNAP for size comparison. (B) ';::70 (F) or wild-type (wt) RNAPs were mixed with PR template, allowing the formation of open complexes at 37°C for 20 min (heparin was present where indicated for the last 10 min), separated by native gel electrophoresis (4% NuSieve agarose, 0.5x TBE), and stained with EtBr. The absence of heparin masked the slightly faster mobility of ';::70 OCs (see legend to Fig. 4C) because ';::70 OCs appeared to aggregate more than wild-type OCs.

View larger version (58K): [in this window] [in a new window]   Figure 4. Effective local concentration of tethered 70 measured by competitive binding. (A) Equilibrium 70-binding assay (see Materials and Methods). Samples are (left to right) 0.5, 1, 2, and 5 µM [32P]70 with 1 µM wild-type RNAP, ';::70 RNAP, or no RNAP. The positions of holoenzyme (E 70) and free 70 are indicated; slower 70 bands are 70 dimers. (B) Quantitation of A. The fraction of RNAP binding 32P-labeled 70 is plotted against the amount of 32P-labeled 70 present in the reactions. (•) Wild-type RNAP; () '::70 RNAP. Effective concentrations of 70 were estimated by nonlinear regression (Materials and Methods) from the averages of three experiments. (C) OC mobility assay. '::70 or wild-type RNAPs were incubated with the indicated amounts of 70 and then with added promoter DNA (Materials and Methods). OCs and DNA were separated on a native agarose gel and stained with EtBr. The positions of the free DNA, the wild-type OC, the '::70 OC, and the supershifted '::70 OC formed with a second, untethered 70 are indicated. Heparin was omitted in this experiment to minimize smearing of the supershifted OC band, although this caused variable nonspecific binding of free DNA (evident as aggregates near the top of the gel). Addition of heparin gave constant amounts of free DNA in gels and approximately the same amounts of supershifted OC, allowing use of up to 30 µM 70 (Fig. 4D). The slightly increased mobility of '::70 OCs (see also Fig. 2B) and of '::70 RNAP (panel A) may reflect a slight structural change caused by tethering that is under study. ('::70 RNAP does, however, contain .) (D) Quantitation of OC supershifting as a function of 70 concentration. The fraction of RNAP binding a second 70 (the supershifted species in C) is plotted against the concentration of added, untethered 70 (0.1, 0.5, 1, 10, and 30 µM; average of three experiments that included heparin). Predicted binding curves (assuming all 70 species to be equivalent for binding to RNAP) for different effective local concentrations of the tethered 70 are shown for comparison (Materials and Methods).

The results described to this point confirm that RNAP tethered to ⁷⁰ supports cell growth and functions with the ⁷⁰ to which it is tethered. This suggests that '::⁷⁰ RNAP can use at least ³² and ^E, which are required for viability of E. coli at 37°C (Zhou et al. 1988; De Las Penas et al. 1997), and ^S, which is necessary for wild-type recovery from the stationary phase (Ishihama 2000). We next examined use of alternative s more carefully and tested for function of NusA, which also competes with ⁷⁰ for interaction with RNAP (Gill et al. 1991). In the presence or absence of chromosomally encoded, untethered ⁷⁰, '::⁷⁰ strains plated a phage that requires function of NusA (and other Nus proteins; Friedman et al. 1976) equivalently to wild type (Table 2). The same was true for survival at 45°C (an even more stringent requirement for ³² and ^E), for growth on medium requiring ^N function, for ^F-dependent swarming motility, and for ^S-dependent formation of peroxidase in the stationary phase (Table 2). Thus, alternative s and NusA appear to exhibit high in vivo activity, sufficient to allow normal function in '::⁷⁰ strains despite the presence of ⁷⁰ tethered to all RNAP molecules. This remains true even in '::⁷⁰ strains also containing chromosomally encoded, untethered ⁷⁰.

View this table: [in this window] [in a new window]   Table 2. Properties of wild-type and '::70 fusion strains in various conditions

'::⁷⁰ RNAPdisplays wild-type enzymatic properties and activities

We next sought to determine if '::⁷⁰ RNAP also behaved normally in vitro. For this purpose, we tested transcription by wild-type and '::⁷⁰ RNAPs of a linear DNA template encoding the well-characterized his pause site downstream of the T7 phage A1 promoter (Chan and Landick 1989). Upon initiation under conditions that allowed RNAP to transcribe only to A29, no difference was apparent between wild-type and '::⁷⁰ RNAPs in the rate of abortive initiation, the rate of productive initiation, or the ratio of abortive to productive products (as reflected by the AUC abortive RNA and A29 productive RNA; Fig. 3A). When transcription past A29 was allowed after 2 min, both RNAPs escaped the A29 position efficiently and exhibited indistinguishable kinetics of pausing and subsequent transcription to the end of the template. We also tested the ability of NusA protein to increase the duration of pausing at the his pause site and the efficiency of termination by '::⁷⁰ RNAP at several intrinsic terminators; no significant differences from wild-type RNAP were observed (data not shown).

View larger version (81K): [in this window] [in a new window]   Figure 3. (A) In vitro transcription assay. '::70 or wild-type RNAPs (40 nM) were allowed to initiate transcription on a linear DNA template (25 nM) with ApU, ATP, [32P]CTP, and GTP. Samples were mixed with 2x STOP buffer at 10, 20, 30, 45, 60, 75, 90, and 120 sec. UTP was then added to allow transcription past A29, and additional samples were taken 10, 20, 30, 45, 60, 120, 240, and 480 sec later (final sample 10 min after reaction started). The samples were separated by 15% denaturing PAGE. (AUC) Trinucleotide abortive product; (A29) A29 EC; (P) his pause RNA; (RO) run-off RNA. (B) Kinetics of promoter association, following the mechanism defined by Saecker et al. (2002). kf,obs and kr,obs were obtained for '::70 and wild-type RNAPs on the template shown in panel A (3 independent experiments; Materials and Methods).

To test whether ⁷⁰ tethering affects steps on the pathway of OC formation, we measured the overall rates of OC formation at the T7 A1 promoter (k_f,obs) and of preformed OC dissociation (k_r,obs; Fig. 3B; Materials and Methods). Both rates are composites of multiple steps (McClure 1980; Saecker et al. 2002) and would reveal any effects of the tether. Neither k_f,obs nor k_r,obs differed significantly between wild-type and '::⁷⁰ RNAPs (Fig. 3B), consistent with wild-type function of the tethered ⁷⁰.

Tethering ⁷⁰ to RNAPincreases ⁷⁰ local concentration to 55 µM

The lack of effect of tethering ⁷⁰ to RNAP caused us to consider whether tethering actually increased the local concentration of ⁷⁰ around RNAP. Such increased local concentrations have been measured for other cases of protein tethering (Timpe and Peller 1995; Robinson and Sauer 1998), and are well-grounded in polymer-chain theory (Flory 1969), but must be determined for each case.

To measure the local concentration of tethered ⁷⁰, we performed competition binding assays using a ³²P-labeled derivative of ⁷⁰ (Materials and Methods). We compared the ability of [³²P]⁷⁰ to displace untethered ⁷⁰ from wild-type RNAP and to displace tethered ⁷⁰ from '::⁷⁰ RNAP using conditions in which ⁷⁰ binding to RNAP equilibrates (Sharp et al. 1999). To detect [³²P]⁷⁰ bound to RNAP, we separated the reactions by nondenaturing PAGE and visualized RNAP and ⁷⁰ by protein staining and [³²P]⁷⁰ using a PhosphorImager (Fig. 4A). [³²P]⁷⁰ displaced untethered ⁷⁰ from the wild-type holoenzyme as predicted for equivalent K_ds of the prebound ⁷⁰ and [³²P]⁷⁰. However, only modest [³²P]⁷⁰ binding to '::⁷⁰ RNAP could be detected even at high concentrations of [³²P]⁷⁰ (Fig. 4A,B). Assuming that all ³²P at or above the position of '::⁷⁰ RNAP in the gels arose from [³²P]⁷⁰ binding to '::⁷⁰ RNAP (because binding of a second ⁷⁰ would slow migration), we calculated a local concentration of tethered ⁷⁰ of 55 µM (Fig. 4B; Materials and Methods). This is >50-fold higher than the concentration of RNAP in the assay (1 µM), but significantly less than measured for optimal cases of tethering (Robinson and Sauer 1998). The extensive topography of ⁷⁰-RNAP contacts relative to the tether attachment points on ⁷⁰ and RNAP, interference of the tether with some contacts, or the tether length may limit tether enhancement of local concentration.

This experiment unambiguously demonstrated weaker binding of free ⁷⁰ to RNAP in the presence of tethered ⁷⁰. The local concentration estimate for tethered ⁷⁰ of 55 µM should be a lower limit because it included ³²P that was nonspecifically retarded. However, our estimate conceivably could be inflated if the complex of [³²P]⁷⁰ and '::⁷⁰ RNAP dissociated during electrophoresis. This seemed unlikely because release of [³²P]⁷⁰ during electrophoresis would form a smear between the positions of holoenzyme and free ⁷⁰. No such smear was evident; rather, the amount of [³²P]⁷⁰ visible at the position of free ⁷⁰ was the same in the '::⁷⁰ lanes and the ⁷⁰ alone lanes, but was reduced in the wild-type RNAP lanes by the amount bound to RNAP (Fig. 4A, cf. the PhosphorImager densities at the bottom of the 1 µM and 2.5 µM lanes).

To confirm that tethered ⁷⁰ produced a local ⁷⁰ concentration of 55 µM, we sought to detect the binding of untethered ⁷⁰ to '::⁷⁰ RNAP in a manner that would prevent its release during electrophoresis. We reasoned that if the untethered ⁷⁰ were engaged in OC formation by '::⁷⁰ RNAP, two ⁷⁰s would be bound to RNAP because untethered ⁷⁰ would be trapped in the network of RNAP-⁷⁰-DNA interactions in the OC (Murakami et al. 2002), and tethered ⁷⁰ would remain covalently attached to RNAP. To favor trapping untethered ⁷⁰ bound to '::⁷⁰ RNAP, we used a promoter on which OCs were stable for many hours (P_UPFullcon; Materials and Methods). We equilibrated RNAPs with increasing concentrations of free ⁷⁰, incubated them with P_UPFullcon

DNA for 10 min, and then separated the reactions on a nondenaturing agarose gel. After visualizing the locations of DNA by ethidium staining, OCs were readily apparent as retarded bands for both wild-type and '::⁷⁰ RNAPs (Fig. 4C). A second, more slowly migrating species appeared when 5-10 µM ⁷⁰ was added to '::⁷⁰ RNAP (Fig. 4C, thick arrow), but was not present with wild-type RNAP. Nonspecific binding and smearing prevented us from using concentrations of ⁷⁰ above 30 µM. Because the supershifted species was also not observed with DNA alone or DNA plus 5-10 µM ⁷⁰ (data not shown), we attribute it to '::⁷⁰ RNAP complexed with a second, untethered ⁷⁰ that is engaged in promoter contacts. To estimate the local concentration of the tethered ⁷⁰, we plotted the amounts of the supershifted species versus concentrations of added ⁷⁰ and compared the data with predicted curves (Materials and Methods; Fig. 4D). They matched reasonably to the prediction for 50 µM local concentration of tethered ⁷⁰ and were more than the prediction for 100 µM. Thus, both assays give an estimate of tethered ⁷⁰ local concentration consistent with 55 µM.

Tethering ⁷⁰ to RNAPmodestly increases ⁷⁰ local concentration in vivo

To compare the local concentration of tethered ⁷⁰ with the effective concentration of wild-type ⁷⁰ in vivo, we tested the ability of chromosomally encoded '::⁷⁰ or increased amounts of plasmid-encoded, untethered ⁷⁰ to protect wild-type cells (also containing chromosomally encoded ⁷⁰) against the toxic effects of a plasmid-encoded mutant ⁷⁰ (RC584). ⁷⁰RC584 binds RNAP and interferes with promoter recognition (Siegele et al. 1989). The presence of tethered ⁷⁰ significantly increased resistance to ⁷⁰RC584 (Fig. 5, cf. closed and open circles; wild-type ⁷⁰ is not toxic when expressed at comparable levels, dashed line). Expression of untethered ⁷⁰ from a low-copy-number, compatible plasmid to about fivefold above the normal level conferred a level of ⁷⁰RC584 resistance similar to that conferred by tethered ⁷⁰ (Fig. 5, triangles).

View larger version (16K): [in this window] [in a new window]   Figure 5. '::70 inhibits toxicity caused by 70RC584. RL113 (wild type, •), RL113 + pBAD70 (wt + 5x 70; ), or RL1366 (wt + '::70; ) carrying pRM389 (70RC584 expressed under LacI control) were plated with or without IPTG as described in Materials and Methods (both strains were deleted for lacY). Data are mean values of three to six independent experiments. The dotted line shows the approximate effect of wild-type 70 expressed from pCL391 for comparison.

These results confirm that '::⁷⁰ RNAP generates a local ⁷⁰ concentration higher than that of untethered ⁷⁰ in wild-type cells. A straightforward interpretation would place the effective concentration of wild-type ⁷⁰ at 11 µM because approximately fivefold overexpression gave ⁷⁰RC584 resistance similar to 55 µM local concentration of '::⁷⁰. However, overexpressed wild-type and RC584 ⁷⁰s could affect each other's levels in ways not measurable in plating experiments. Furthermore, we did not test whether a lower level of ⁷⁰ overexpression also could protect against RC584 ⁷⁰ as '::⁷⁰. Thus, we conclude wild-type cells contain ⁷⁰ at effective concentrations 11 µM and significantly less than 55 µM.

Tethering ⁷⁰ to RNAPonly modestly affects heat shock and ³² function

We next wanted to examine the effect of tethered ⁷⁰ on function of the alternate factor ³², which is required for the well-defined heat-shock response in E. coli (Straus et al. 1987). When ⁷⁰ is overexpressed, ³² function appears to be compromised by competition for binding to core RNAP (Zhou et al. 1992). However, ³² is up-regulated in response, resulting in little if any inhibition of synthesis of heat-shock proteins or delay in the heat-shock response. We first confirmed that '::⁷⁰ RNAP could use ³² in vitro (transcription of groE by '::⁷⁰ RNAP depended on added ³²; data not shown). We next tested whether '::⁷⁰ RNAP would affect expression of heat-shock proteins and ³² levels similarly to overexpression of ⁷⁰.

The heat-shock response in '::⁷⁰ cells was slightly delayed relative to wild-type cells (Fig. 6A). The GroE synthesis rate was slower in '::⁷⁰ cells 1 or 2 min after shifting cells to 42°C, but reached the same level as wild-type by 11 min post-heat shock (Fig. 6A, plot). The characteristic suppression of ⁷⁰-dependent transcription was similar in '::⁷⁰ and wild-type cells (Fig. 6A, cf. ^* bands).

View larger version (78K): [in this window] [in a new window]   Figure 6. Heat-shock response. (A) Cells were grown in minimal media at 30°C and then shifted to 42°C. At the times indicated, samples were incubated with [35S]methionine for 2 min. Samples were separated by 4%-12% PAGE. The positions of DnaK, GroE, and an 89-kD protein likely to be Lon are indicated. (*) Major proteins whose expression decreases on heat shock; the top band is '::70, and the next-to-top band is , '. Relative GroE levels are averages of four experiments. (•) Wild-type (RL301); () '::70 strain with chromosomally encoded 70 (RL1374). (B) Immunoblot analysis using antibodies against 32 (Materials and Methods). (Lanes 1-3) Wild-type whole-cell lysate (MC1060). (Lanes 4-6) '::70 whole-cell lysate (RL1454). (Lanes 7-9) His6-tagged 32. Equal amounts of total cellular protein were electrophoresed.

To ask if heat shock was near normal because ³² levels were up-regulated in the '::⁷⁰ strain, we measured ³² levels by immunoblotting. The ³² level in '::⁷⁰ cells was 80% of that in wild-type cells, a statistically insignificant difference (Fig. 6B; Materials and Methods). The slight delay in GroE expression in heat-shocked '::⁷⁰ cells is consistent with a local concentration of tethered ⁷⁰ higher than the wild-type ⁷⁰ effective concentration, but the nonelevated ³² levels in '::⁷⁰ cells and the inhibition of ⁷⁰-directed transcription upon heat shock even when ⁷⁰ was tethered to RNAP were surprising (see Discussion).

Tethered ⁷⁰ causes ⁷⁰-dependent pausing independent of distance from the promoter

An additional consequence of tethering ⁷⁰ is that its high local concentration will be maintained during transcript elongation. Because untethered ⁷⁰ is able to stimulate pausing at -10-like sequences near a promoter (Ring et al. 1996), we wondered if tethering would allow ⁷⁰ to stimulate pausing at -10-like sequences generally. To test this possibility, we examined ⁷⁰-dependent pausing on templates originally studied by Roberts and colleagues (+16/17 and +37 pauses), and on an additional template encoding a consensus extended -10 sequence near +450 of an artificial transcription unit (+462 pause). '::⁷⁰ RNAP gave slightly increased pausing at the +16/17 site (ctcaAcgAT, -10-like ⁷⁰-binding sequence; Fig. 7A). This strong pausing also occurred at the +37 site (TGcTATAAT, consensus extended -10-like ⁷⁰-binding sequence; Fig. 7B), where wild-type RNAP pauses weakly if at all. This result confirms that wild-type RNAP fails to pause at +37 because its ⁷⁰ is released, rather than because the structure of the EC past +25 precludes pausing (Ring et al. 1996). Tethered ⁷⁰, in contrast, stimulates pausing because it remains attached to RNAP in a functional state after completion of the transition to an EC.

View larger version (61K): [in this window] [in a new window]   Figure 7. The effective concentration of tethered 70 is sufficient to cause promoter-distal, 70-dependent pausing. The effective concentration of tethered 70 is sufficient to cause promoter-distal, 70-dependent pausing. Synchronous in vitro transcription with '::70 or wild-type RNAPs (Materials and Methods; time of transcript elongation as indicated in min; C, incubation with additional 200 µM NTP for 8 min after the last indicated time point). (A) +16/17 pause template (Ring et al. 1996). The top panel shows the run-off (RO) product; the lower panel shows the pause. The schematic of the template indicates the 70-binding sequence and the position of the pause. (B) +37 pause template (+20 in Ring et al. 1996). Gel panels and schematic are as in A. The three pause positions on this template have not been mapped precisely and therefore are designated 37a, 37b, and 37c; the third pause band (relative to the +16/17 site) is probably caused by the stronger consensus 70-binding sequence. The time dependence of pause RNA levels (plot) is the average of three independent experiments. (C) +462 pause template. Gel panels and schematic are as in A. (1 µM) or NusA (10 µM) were added as indicated.

To ask if the tethered ⁷⁰ could act at even greater distances from a promoter, we tested the +462 pause template (TGcTATAAT ⁷⁰-binding sequence; Fig. 7C). As expected, wild-type RNAP was unable to recognize the +462 pause; however, '::⁷⁰ RNAP exhibited strong pausing. If pausing at +462 occurred simply because the tethered ⁷⁰ was present at high local concentration, then elevated concentrations of untethered ⁷⁰ should cause wild-type RNAP to pause. Consistent with this interpretation, wild-type RNAP recognized the +462 pause when additional ⁷⁰ (1 µM) was added to ECs (Fig. 7C). We concluded that this pause depended on the consensus extended -10-like sequence because a mutant TGcTgTAAg site nearly eliminated pausing (data not shown). This effect of nonconsensus substitutions may explain why Marr et al. (2001) found that additional ⁷⁰ (0.8 µM) did not direct pausing at the nonconsensus +16/17 pause. Pause escape by '::⁷⁰ RNAP was barely detectable at consensus extended -10-like sequences (e.g., plot in Fig. 7B). However, '::⁷⁰ RNAP was paused, not terminated, because addition of GreA or GreB protein reduced pausing dramatically (data not shown), as shown for the +16/17 pause by Marr and Roberts (2000).

Because NusA is thought to displace ⁷⁰ from the EC (Gill et al. 1991), we next asked how NusA affected recognition of the +462 pause. Addition of NusA to 10 µM slowed elongation overall, generated a new pause at 419 nt, and reduced, but did not eliminate, ⁷⁰-dependent pausing caused by untethered ⁷⁰ (Fig. 7C, wt RNAP + + NusA). However, NusA had little effect on +462 pausing by '::⁷⁰ RNAP (Fig. 7C) or by wild-type RNAP when untethered ⁷⁰ was added to 10 µM (1:1 stoichiometry with NusA; data not shown). These results establish that tethered ⁷⁰, and even untethered ⁷⁰ if added at higher concentration (but still well below that predicted to occur in vivo), can direct ⁷⁰-dependent pausing irrespective of pause site location in a transcriptional unit. The findings are consistent with the paradigm that NusA competes with ⁷⁰ for interaction with an EC (Gill et al. 1991), but suggest that NusA may not eliminate ⁷⁰-dependent pausing in vivo (see Discussion).

	Discussion

Top Abstract Results Discussion Materials and methods Acknowledgments References

 
Our study of tethering ⁷⁰ to RNAP leads to four main conclusions. First, a local ⁷⁰ concentration of 55 µM (the measured value for '::⁷⁰ RNAP) does not significantly perturb the physiology of E. coli, consistent with the effective concentration of untethered ⁷⁰ in wild-type cells being only slightly lower. Second, tethered ⁷⁰ at this modestly increased local concentration only slightly delays heat-shock gene expression and is still inactivated upon heat shock (like untethered ⁷⁰), even though ³² levels are not elevated. Third, tethered ⁷⁰ or even 1 µM untethered ⁷⁰ can stimulate pausing in vitro anywhere in a transcriptional unit. Fourth, the effective concentration of free ⁷⁰ present in cells appears sufficient to allow it to interact transiently with an EC even after ⁷⁰ release, and to stimulate pausing at extended -10-like sequences in vivo.

Effective concentration of ⁷⁰ in vivo

An accurate understanding of -factor binding by RNAP requires knowing the effective concentrations of s and RNAP in vivo. However, many factors can cause the effective concentrations of molecules in cells to differ from their bulk concentrations. First, the cytoplasm of E. coli is a highly concentrated mixture of macromolecules, small molecules, and water more akin to a gel than to a dilute solution. Macromolecular crowding will increase the effective concentrations of ⁷⁰ and RNAP significantly, quite likely by a factor of 10 or more (Record et al. 1998; Ellis 2001). The magnitude of crowding effects may depend on growth conditions, as the water content of cells changes significantly in response to osmolytes in the growth medium (Cayley et al. 1991).

Second, the effective concentrations of ⁷⁰ and RNAP will be reduced by interactions that sequester them from participation in the competitive binding equilibria among factors and RNAP. ECs sequester about two-thirds of RNAP (Ishihama 2000). Additional RNAP binds nonspecifically to DNA and possibly to RNA; these contributions depend on the binding constants, target sizes, and amounts of DNA or RNA available for nonspecific interaction. Most s in E. coli also specifically bind anti-s (FlgM for ^F, RseA for ^E, DnaK for ³², and possibly Rsd for ⁷⁰; Ishihama 2000 and references therein). 6S RNA specifically sequesters holoenzyme (Wassarman and Storz 2000). Some free or RNAP-bound could be sequestered nonspecifically. Promoter complexes also will sequester some s, but it is difficult to estimate how many because some may release slowly after initiation (Shimamoto et al. 1986; Bar-Nahum and Nudler 2001; Mukhopadhyay et al. 2001) and because some OCs appear unable to initiate transcription (Susa et al. 2002). The extent to which these various interactions reduce ⁷⁰ and RNAP availability depends on their avidity, but together they likely reduce substantially the effective concentrations of ⁷⁰ and RNAP molecules.

Finally, the association of s with RNAP is unlikely to be in equilibrium in vivo. RNAP constantly enters the pool available for binding both by release from DNA at terminators and by new synthesis. s bind RNAP tightly, which means that the off-rates may be slower than the time it takes RNAP to bind and initiate at a promoter.

Given this complexity, the prospects for calculating the effective concentration of ⁷⁰ in vivo are poor. However, the effects of tethering ⁷⁰ to RNAP at known local concentration provides some insight. Both enhanced competition against ⁷⁰RC584 (Fig. 5) and the slight delay in the heat-shock response (Fig. 6) of tethered ⁷⁰ suggest that the normal effective concentration of untethered, wild-type ⁷⁰ is modestly less than the local concentration of tethered ⁷⁰ (55 µM). Comparison to the effects of overproducing ⁷⁰ suggest it is 11 µM, close to ⁷⁰'s bulk concentration (15 µM). This is reminiscent of a similar conclusion reached for a cellular protein ordinarily present at very different bulk concentration, lac repressor (Law et al. 1993). For lac repressor, the balance of crowding and sequestration effects also yields an effective concentration near its bulk concentration (1 nM in wild-type cells).

Competition of ⁷⁰ and ³²

Despite the modest increase in the effective concentration of ⁷⁰ caused by tethering, ³² levels are not elevated in the '::⁷⁰ strain as expected for increased ⁷⁰ concentration (Zhou and Gross 1992), and the tethered ⁷⁰ is still inactivated upon heat shock. We cannot absolutely exclude the possibility that a small fraction of proteolytically cleaved '::⁷⁰ RNAP allows the near normal ³² function. However, we favor ³² function with intact '::⁷⁰ RNAP for three reasons. First, we did not detect cleavage of '::⁷⁰ RNAP after heat shock (data not shown). Second, even if some cleavage occurred, ³² must compete in these strains against additional, chromosomally encoded ⁷⁰ that also could bind any available core RNAP. Third, ³² functions with uncleaved '::⁷⁰ RNAP in vitro and this cannot be explained by proteolytic fragmentation of '::⁷⁰. Rather, we suggest the explanation for the lack of ³² overexpression when ⁷⁰ is tethered to RNAP lies in the difference between increasing ⁷⁰ concentration locally versus globally. Overexpression of ⁷⁰ produces inclusion bodies and becomes toxic to E. coli at high levels of ⁷⁰ (data not shown; see Fig. 5). Aggregates of ⁷⁰ (the precursor to inclusion bodies) may be bound to the chaperone DnaK and thus could induce E. coli's stress response and elevate the ³² level by releasing ³² from DnaK. Increasing ⁷⁰ concentration locally for RNAP, as occurs in the '::⁷⁰ strain, would not provoke this stress response because it would not elevate the global ⁷⁰ level. In essence, competition of ⁷⁰ and ³² may occur for both RNAP and DnaK upon global ⁷⁰ overexpression, but should be limited to RNAP for ⁷⁰ tethering.

The inactivation of tethered ⁷⁰ upon heat shock suggests something other than simple competition for ³² shuts off ⁷⁰-dependent gene expression. Indeed, even in wild-type cells, something in addition to an increase in ³² level seems necessary to explain the decrease in ⁷⁰-directed gene expression upon heat shock (Fig. 6A; Straus et al. 1987). Although ³² levels are elevated fivefold 15 min after shift of E. coli to 42°C, ⁷⁰ levels themselves increase 2.5-fold (Taylor et al. 1984; Straus et al. 1987). This is equivalent to 60 ³² and 900 ⁷⁰ per 1000 RNAP, suggesting that an unknown factor actively inhibits ⁷⁰ during heat shock. Whatever the mechanism of ⁷⁰ inhibition, it must also act on tethered ⁷⁰ and allow ³² to dominate during heat shock despite its lower level. Such a requirement for additional factors has been a general conclusion in most studies of -factor switching (Fujita and Sadaie 1998; Kolesky et al. 1999; Lord et al. 1999; Maeda et al. 2000; Jishage et al. 2002; Rollenhagen et al. 2003). The fact that this mechanism operates on tethered ⁷⁰ rules out explanations involving physical segregation of ⁷⁰ away from sites of RNAP function because ⁷⁰ is fixed to RNAP by covalent linkage.

⁷⁰ may function as an elongation factor in vivo

The textbook view of ⁷⁰ participation in the transcription cycle may need revision, although our results suggest that when or whether is released is not the relevant question. Even 1 µM ⁷⁰ can interact from solution with ECs in vitro and cause pausing anywhere in a transcriptional unit; this behavior is similar to that of ⁷⁰ permanently tethered to RNAP. Because ⁷⁰'s effective concentration in cells is significantly higher than 1 µM, continuous transient interactions must occur with ECs regardless of when or whether it is released after promoter escape (Fig. 8).

View larger version (15K): [in this window] [in a new window]   Figure 8. Model of 70-RNAP interactions during transcription in E. coli.

Although NusA competes with ⁷⁰ for binding to the EC (Gill et al. 1991) and may temper the effects of ⁷⁰-stimulated pausing, it is unlikely to eliminate this pausing in vivo. In vitro, a 10-fold excess of NusA reduced, but did not eliminate, ⁷⁰-stimulated pausing (Fig. 7C). Although the in vivo concentration of NusA has not been reported, a 10-fold excess over ⁷⁰ would correspond to >110 µM or more than 2.6% of total cellular protein. Thus, it is unlikely there is enough NusA in cells to eliminate ⁷⁰-dependent pausing at extended -10-like sequences.

The view that emerges from this study is that the effective concentration of ⁷⁰ in vivo is sufficient to allow interaction with the EC and stimulation of pausing at extended -10-like sequences, making