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Core RNA polymerase (₂') requires the variable specificity subunit, sigma (), to direct promoter-dependent transcription (1, 3, 4, 12, 18, 22, 23, 26). Following promoter binding, holoenzyme (₂') progresses through several intermediate complexes, en route to a stable initiated open complex (2, 14).  factor has been implicated in stages of initiation beyond promoter recognition (8, 9, 13, 15, 17). Recently, we showed that the conserved amino terminal domain (region 1) of Escherichia coli ⁷⁰ is important for the process of strand melting and initiated complex formation at the  p_R promoter (24).

Region 1 is unique to the primary  factors, yet little is known of its function. Deletion of region 1.1 (amino acids 1 to 100) from ⁷⁰ has two major consequences for holoenzyme. The first is inefficient progression from the closed to the strand-separated open complex. This can be overcome by increasing the time allowed for formation of holoenzyme-promoter complexes and is lessened by addition of region 1.1 in trans. The second and more deleterious effect is impaired transition from the strand-separated open complex to a stable initiated complex (RP_init). According to this analysis, amino acids between positions 50 and 75 of ⁷⁰ are critical for proper initiation in vitro (24).

A comparison of region 1.1 among several primary  factors revealed conserved residues at positions 52 (glycine [G]), 53 (isoleucine [I]), 55 (valine [V]), and 61 (aspartic acid [D]), as well as a high degree of acidity (40%) within the segment from amino acids 50 to 75 (24). Here, we test whether alterations at these conserved positions or in the overall acidity of the region influence initiation by holoenzyme (E).

Site-directed mutagenesis (10) and the Expand high fidelity PCR system (Boehringer Mannheim) were used to create substitutions at positions 52, 53, 55, 61, 57, 58, 63, 64, and 69 (Table 1). rpoD was mutagenized in M13 phage (10) and amplified with oligonucleotides that incorporated restriction sites at the 5' and 3' ends of the fragment. The restricted fragments were ligated into pQE30-T (24). PCR mutagenesis was used to amplify a fragment corresponding to the 3' end of the rpoD gene with a 5' mutagenic oligonucleotide and a 3' oligonucleotide that incorporated a restriction site. A concurrent round of amplification included a 5' oligonucleotide complementary to the 5' end of rpoD and a 3' oligonucleotide with complementarity to an internal segment of rpoD, downstream from the genetic alteration(s). The 5' and 3' PCR fragments were mixed, and the full-length mutagenized rpoD gene was amplified, gel isolated, digested, ligated into pQE30-T, transformed into E. coli XL1 Blue (Stratagene), and sequenced to confirm the changes. The plasmids were transformed into E. coli 19284 (rpoD800, W3110 srl::Tn10 recA lacI^q) to test for function in vivo (24). Transformation mixtures were split and spread onto Luria-Bertani plates containing ampicillin (100 mg/ml), kanamycin (30 mg/ml), and 2% glucose and then incubated at 32 and 44°C, to evaluate complementation of the rpoD800 temperature-sensitive growth defect at 44°C. Plasmids were likewise transformed into strain CAG20176 to test growth in the absence of chromosomal rpoD expression at 32°C (11, 24).

Mutants were generated that replaced either acidic, conserved, or, at position 61, both conserved and acidic amino acids with alanine (A). Replacement of glutamic acid (E) at position 69 (E69) with A had no effect on ⁷⁰ function in vivo (Table 1). Double mutations replacing E at positions 57 and 58 or D at positions 63 and 64 reduced the overall acidity of region 1.1, but neither affected ⁷⁰ function in vivo (Table 1). Replacement of conserved D61 with A, however, rendered ⁷⁰ nonfunctional.

Additional substitutions at position 61 addressed the contribution of the amino acid side chain. Both E and serine (S) could functionally substitute for D, indicating that polarity rather than side chain charge at this position is more important for function (Table 1). D61 is found in a cluster of acidic residues; however, simultaneous substitution of D63 and D64 did not impair function in vivo, indicating that D61 does not require the flanking acidic residues. A quintuple mutation combining E57,58A with D61A and D63,64A was inactive in vivo. Since both of the double mutants were functional, the lethal phenotype is probably caused predominantly by D61A. Substitutions G52A, V55A, or V55I functioned at least as well as the wild type (Table 1). Because ⁷⁰V55A and ⁷⁰V55I function in vivo, the size and shape of the hydrophobic side chain at this position are not critical. ⁷⁰I53A was inactive in vivo and seriously defective in vitro, and its characterization has been reported elsewhere (25).

These results argue that the overall acidity of region 1.1 is not a major factor in its participation in transcription initiation. Conserved D61, however, appears to be important for ⁷⁰ structure and/or function in vivo. To address the basis for the inactivity of ⁷⁰D61A, the protein was overexpressed and purified (24) for characterization in vitro.

Run-off transcription analysis was performed to assess the overall effect of the D61A substitution on RNA synthesis (24). A time course at 37°C indicated that E⁷⁰D61A was impaired in transcription, while E⁷⁰G52A and E⁷⁰E57,58A exhibited transcription rates similar to E⁷⁰ (Fig. 1A). E⁷⁰D61A was also defective for abortive transcription (24) at 37°C (Fig. 1B). One explanation for the inactivity of ⁷⁰D61A is thermolability of the protein. Thus, we examined the effect of temperature on run-off transcription. At 25°C, E⁷⁰D61A activity was indistinguishable from E⁷⁰. At higher temperatures, a transcriptional defect became apparent (Fig. 1C), with loss of activity as the temperature increased. The functional mutants E⁷⁰G52A and E⁷⁰E57,58A were not affected by increasing temperature, as compared to E⁷⁰.

View larger version (211760K): [in this window] [in a new window]   FIG. 1.   Run-off transcription analysis. (A) A representative run-off transcription time course experiment is shown for E70 and 70 mutants at 37°C. Each experiment was repeated at least five times, and the error was less than 15%. (B) Abortive transcription by E70D61A at 37°C. Synthesis of the three nucleotide abortive RNA transcripts (ApUpG) from  pR is shown relative to the wild type. The holoenzyme concentration was 0.008 µM and the template concentration was 1.5 nM. (C) Run-off transcription as a function of temperature. A comparison of run-off transcription activities at 60 min at 25 and 42°C is shown. Activities are normalized to the wild type, and the error indicated was less than 15%. WT, wild type.

Because ⁷⁰D61A was functionally thermolabile in vitro, the possibility that it was structurally thermolabile was assessed by comparing susceptibility to trypsin digestion at 25, 37, and 42°C. Consistent with its transcriptional activity, ⁷⁰D61A exhibited increased sensitivity to trypsin digestion at 42°C (Fig. 2A). The failure of ⁷⁰D61A to complement rpoD mutant strains in vivo may therefore be caused by instability of the protein. To test this idea, we used immunoblotting to compare the levels of the wild type and ⁷⁰D61A after a shift from 37 to 44°C (Fig. 2B). At the time of the upshift, there was significantly more wild-type ⁷⁰ present, and it remained stable for longer than 80 min (Fig. 2B). Conversely, ⁷⁰D61A was much less stable, becoming nearly undetectable by 80 min. The D61A substitution appears to cause a structural disruption in ⁷⁰ that results in proteolytic instability both in vivo and in vitro and functional instability during transcription initiation. Other mutations in region 1.1 have also been reported to result in structural instability (5).

View larger version (69K): [in this window] [in a new window]   FIG. 2.   Stability of 70D61A in vitro and in vivo. (A) Limited trypsinolysis. 70D61A (2 µg) was subjected to trypsin digestion at three temperatures, as indicated, to assess possible structural defects. Wedges indicate increasing trypsin concentration (0.0125, 0.0625, and 0.025 µg). Digestion of wild-type (WT) 70 is shown for reference in the top panel. Fragments were resolved on a sodium dodecyl sulfate-8% polyacrylamide gel and visualized with Coomassie brilliant blue staining. (B) Immunoblot analysis of culture lysates of strain 19284 (wild-type [WT] 70 and 70D61A). Exponentially growing cells at 37°C were upshifted to 44°C. Lysates were prepared from cells harvested at the indicated time points following temperature upshift, and proteins were resolved on a sodium dodecyl sulfate-8% polyacrylamide gel. Following Western transfer, histidine-tagged 70 proteins were detected by using a six-His-tagged monoclonal antibody (Clontech).

A more thorough evaluation of the initiation properties of the mutants was conducted to determine if a particular step in the process was affected by the substitutions. The first step in initiation is promoter recognition and binding by RNA polymerase. Nitrocellulose filter retention has been used to evaluate DNA binding at  p_R, and the complexes retained are open complexes (6, 7, 16, 19, 20). Holoenzyme (1 nM) was incubated with a ³²P-5'-end-labeled DNA fragment containing  p_R (0.1 nM). The binding of E⁷⁰D61A to  p_R was indistinguishable from E⁷⁰ as well as the functional mutants E⁷⁰G52A and E⁷⁰E57,58A (data not shown).

Addition of nucleoside triphosphates (NTPs) to open complexes allows progression to RP_init, which are stable to an 0.8 M NaCl wash (16, 21). The ability of NTPs to stabilize E⁷⁰D61A- p_R open complexes was assessed. E⁷⁰100, previously shown to be defective in RP_init formation (24), was compared for reference. Under low-stringency wash conditions (0.1 M NaCl), at 2 and 30 min after adding holoenzyme to DNA, each E⁷⁰ derivative bound to  p_R as well as the wild type (Fig. 3). Under high-stringency wash conditions (0.8 M NaCl), the E⁷⁰E57,58A complexes were retained as well as the wild type. Interestingly, the E⁷⁰G52A complexes were less stable than E⁷⁰, but this mutant was still able to complement in vivo. E⁷⁰D61A- p_R complexes were unstable at 2 min, but by 30 min they were indistinguishable from E⁷⁰, indicating a slower rate of RP_init formation.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   Stability of initiated complexes. Nitrocellulose filter retention under low (0.1 M NaCl)- and high (0.8 M NaCl)-stringency wash conditions is shown. The fraction of mutant complexes retained is normalized to the fraction of wild-type (WT) complexes retained after allowing formation for 2 and 30 min following mixing of RNA polymerase with DNA. Amino acid substitutions are indicated.

Impaired run-off transcription, combined with the slow rate of RP_init formation by E⁷⁰D61A, could be caused by difficulty in open complex formation. KMnO₄ footprinting analysis was performed to assess the ability of E⁷⁰D61A to form open complexes (24). Strand melting for E⁷⁰D61A occurred as efficiently as for E⁷⁰ in the absence and presence of NTPs, even at the times when RP_init formation was impaired (data not shown). Open complex formation by E⁷⁰G52A was also examined, since the RP_init were slightly less stable than they were for E⁷⁰, but no differences relative to E⁷⁰ were detected.

In summary, the D61A mutation renders ⁷⁰ nonfunctional in vivo and functionally and structurally thermolabile in vitro, manifested in a slow rate of RP_init formation. Alanine substitution at acidic residues 57, 58, 63, 64, and 69 has no effect on ⁷⁰ function in vivo or in vitro. Therefore, acidity of region 1.1 is not a major contributing factor to the initiation properties of ⁷⁰, but amino acids, including D61, are very important for structural stability.