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Journal of Bacteriology, February 2002, p . 1192-1195, Vol . 184, No . 4

Two "Wild-Type" Variants of Escherichia coli {sigma}70: Context-Dependent Effects of the Identity of Amino Acid 149

Nicole E . Baldwin, Andrea McCracken, and Alicia J . Dombroski*

Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center, Houston, Texas 77030

Received 17 August 2001/ Accepted 9 November 2001


   ABSTRACT

 
The identity of amino acid 149 of Escherichia coli {sigma}70 has been reported variably as either arginine or aspartic acid . We show that the behavior of both a region 1.2 deletion and a single-amino-acid substitution at position 122 are greatly affected by the identity of amino acid 149 .


   TEXT

 
A single RNA polymerase (RNAP) is responsible for all transcription in eubacteria . The holoenzyme consists of four invariant core subunits ({alpha}2ßß') and a fifth variable subunit, sigma ({sigma}) . Although only the core subunits are required to catalyze the polymerization of RNA, association with a {sigma} factor is required for specific promoter recognition and binding (19, 27) . Like all primary {sigma} factors of the {sigma}70 family, Escherichia coli {sigma}70 has four conserved domains (9, 11, 18, 26) (Fig . 1), with a nonconserved spacer connecting regions 1 and 2 .


FIG . 1 . Linear diagram of {sigma}70 domains . Representative schematic of the domains present in E . coli {sigma}70 as determined by homology with the primary members of the {sigma}70 family of sigma factors (10) . Diagram is not to scale.

 
Region 1.1, found only in primary {sigma} factors (9, 11, 18), prevents {sigma}70 from recognizing and binding the promoter in the absence of core subunits (7), and its absence affects the rate of promoter opening and transcription initiation (29) . Regions 2.4 and 4.2 recognize the -10 and -35 promoter consensus sequences, respectively (5, 8, 25, 28, 31) . Regions 2.1, 2.2, and 3.2 have been implicated in core binding (14, 17, 24, 30), and region 2.3 is necessary for melting the DNA to form an open complex (12, 13, 15, 21) .

Transcription initiation is a multistep process (6, 22) that begins once a {sigma} factor has associated with core subunits to form holoenzyme . Footprinting binary complexes at various temperatures has been used to describe structural intermediates in the process of initiation (6) . Initial holoenzyme-promoter binding results in the formation of a short closed complex (RPC1) extending from approximately -55 to -5 relative to the transcription start site . Extension of the RNAP-DNA contacts downstream to about +20 characterizes the second closed complex (RPC2) . The DNA at the start site then isomerizes, melting to form a transcription bubble, resulting in the open complex (RPO) . In the presence of nucleoside triphosphates (NTPs), an initiated ternary complex (RPinit) forms that can remain at the promoter and produce small (2- to 12-nucleotide) abortive transcripts . RNAP can then escape the promoter and enter elongation, or it may remain trapped at the promoter, producing abortive transcripts and eventually becoming inactive (6, 16, 23) .

A literature and database survey showed that the two sequence variants encoding asparagine 149 and aspartic acid 149 are currently referenced as E . coli rpoD strains . Although both the published sequence from E . coli K-12 (3) and the sequence used to generate the partial crystal structure of E . coli {sigma}70 (20) indicate that the identity of position 149 is asparagine, the Swiss-Prot database entry and two of three cross-referenced EMBL entries show aspartic acid at position 149 .

To investigate the importance of the identity of residue 149 of {sigma}70, we directly compared the behavior of the N149 and D149 variants both in vivo and in vitro . Additionally, we characterized single-amino-acid substitutions and an internal deletion derivative of {sigma}70 lacking region 1.2 ({Delta}1.2) in each variant background .

The N149 variation of {sigma}70 was generated by PCR with the megaprimer technique (2), and both the N149 and D149 variants of {sigma}70 were subcloned into a low-copy expression vector, pPROLarAS (Clontech) . To investigate the potential effects of the variant background on secondary mutations, single-amino-acid substitutions and a region 1.2 deletion ({Delta}1.2), previously generated in the D149 background (1), were subcloned into the N149 background .

Plasmids containing the {sigma}70 derivatives were transformed into a strain carrying the rpoD800 allele, which encodes a thermolabile {sigma}70, on the chromosome (4) and tested for complementation of the temperature-sensitive growth phenotype . The behavior of the N149 and D149 variants was indistinguishable . An M100L substitution complemented the temperature-sensitive growth phenotype to the same degree as the position 149 variants (heavy streak, 1-mm colonies), while the {Delta}1.2 derivative was unable to complement in either variant background (no growth as with vector alone) . The N149 variant with an arginine to histidine change at position 122 ([N149, R122H]) was fully able to complement (heavy streak, 1-mm colonies) . However, the D149 variant with R122H ([D149, R122H]) only partially complemented, producing individual colonies one-fourth the size of the fully complemented strain . This indicated that the effect of the R122H substitution was dependent on the position 149 variant background .

In order to gain a fuller understanding of how the identity of position 149 may affect specific steps in transcription initiation, we determined the ability of reconstituted RNAP to synthesize runoff transcripts from a linear DNA template in vitro . Each of the {sigma}70 derivatives was purified with a hexahistidine tag at the C terminus, added to the core subunits to reconstitute holoenzyme (10:1, {sigma}-core), and incubated at 37°C with linear template containing {lambda}pR for ten minutes . The final concentration of RNAP was 17.8 nM, and that of DNA was 6.7 nM . A mixture of 0.2 mM NTPs and 0.02 mM [{alpha}-32P]GTP (3,000 Ci/mmol) was added, and the reaction allowed to proceed for 40 min . Transcripts of 80 nucleotides in length were resolved on an 8% denaturing polyacrylamide gel and quantified using a Packard Instantimager .

The overall transcription activities of the N149 and D149 variants did not differ significantly (Fig . 2). The position 149 variant background also did not substantially alter the activity of either the M100L substitution or the region 1.2 deletion . Confirming the results of the in vivo complementation assay, the overall transcriptional activity of the R122H substitution was significantly affected by the position 149 variant background . While [N149, R122H] resulted in similar activity to N149 alone, [D149, R122H] produced transcription activity of only 31% of that with N149 alone .


FIG . 2 . In vitro runoff transcription activity of holoenzyme with {sigma}70 variants and their derivatives . The 80-nucleotide runoff transcripts from the {lambda}pR promoter were measured, and the results are presented as a percentage of the N149 variant activity.

 
Binding of RNAP to promoter DNA comprises one of the earliest steps of transcription initiation and can be assessed using DNase I protection experiments . Holoenzymes containing the N149 and D149 variants, the {Delta}1.2 derivative in each variant background, and [D149, R122H] were examined for promoter binding properties at the {lambda}pR promoter (Fig . 3). RNAP, reconstituted as for runoff transcription, was incubated with 32P-5'-end-labeled {lambda}pR DNA for 2 min in the absence of NTPs, followed by digestion with DNase I (29) . The final concentration of RNAP was 20 nM, and that of DNA was 2 nM .


FIG . 3 . DNase I footprinting of holoenzyme complexes on the {lambda}pR promoter . The numbered arrows represent the position relative to the transcriptional start site . The {sigma} factor derivatives as a component of holoenzyme are indicted at the top of the figure.

 
RNAP with the N149 or D149 variants, as well as [D149, R122H], completely protected the {lambda}pR promoter from -55 to +20, as expected for either RPC2 or RPO . Thus, the observed defect in transcription initiation activity of [D149, R122H] was not the result of a decrease in promoter binding or a failure to form the second closed complex . RNAP with the [N149, {Delta}1.2] derivative failed to protect the promoter, and the pattern of digestion was indistinguishable from that with DNA alone . Surprisingly, the [D149, {Delta}1.2] derivative produced a very unusual digestion pattern, consisting of multiple hypersensitive bands . This mode of binding is clearly not normal and does not produce functional transcription initiation complexes, but is useful in illustrating the possible synergistic effects that are dependent on position 149 .

The {sigma} subunit of RNAP is required not only for promoter binding but also to facilitate DNA strand separation to produce open complexes . Since DNase I footprints do not distinguish between an extended closed (RPC2) and an open complex (RPO), we used KMnO4 reactivity as a measure of the extent of open complex formation (22) . Reconstituted RNAP was incubated at 37°C with linear template containing {lambda}pR for 2 or 40 min before being subjected to KMnO4 modification and piperidine cleavage (1) . The RNAP and DNA concentrations were the same as for DNase I footprinting . Cleavage patterns were resolved on a 6% denaturing polyacrylamide gel, which was autoradiographed, and the reactive positions at -4, -3, and +2 were quantified (Fig . 4). The KMnO4 reactivity of all derivatives tested was consistent with the degree of DNase I protection observed . The N149 and D149 variants as well as the [D149, R122H] derivative formed open complexes to equivalent degrees at each time point, indicating that the transcription defect of [D149, R122] is not attributable to an inability to form open complexes .


FIG . 4 . Determination of extent of open complex formation . The fraction of KMnO4-reactive complexes was determined by dividing the amount of cleaved DNA by the total DNA loaded (cleaved plus uncleaved) . This fraction was normalized to the fraction determined for the N149 variant at 40 min . Results are presented as a fraction of the reactivity observed for the N149 variant at 40 min.

 
Finally, we examined the ability of the holoenzymes to produce abortive transcripts where the RNAP remains bound at the promoter but generates short defined oligoribonucleotides by providing a dinucleotide primer (ApU) and a third radiolabeled NTP (GTP) . Binary complexes were formed as for runoff transcription, followed by addition of a mixture of 0.2 mM ApU and GTP, as well as 0.02 mM [{alpha}-32P]GTP (3,000 Ci/mmol), and the reaction was allowed to proceed for 0.5, 1, 2, 5, or 10 min and then halted with formamide stop dye . The products were resolved on a 24% denaturing polyacrylamide gel and quantified (Fig . 5) .


FIG . 5 . Abortive transcription analysis in vitro . The production of a three-nucleotide abortive transcript was monitored as a function of time . The results are presented as a fraction of the N149 variant activity at 10 min.

 
Each of the position 149 variants and the [D149, R122H] derivative produced equivalent amounts of abortive transcripts at each time point . The [D149, R122H] holoenzyme is therefore not defective in forming initiated complexes and must have a transcriptional deficiency subsequent to the formation of the second phosphodiester bond and prior to elongation that results in the observed 70% reduction in overall transcription . This demonstrates a detrimental synergistic effect of the combination of the D149 variant and the R122H mutation that is manifested either as slower promoter escape or a higher tendency to form inactive complexes at the promoter . Finally, one trivial explanation for the decreased activity of {sigma}70 mutants is a failure to associate with the core subunits to form holoenzyme . We tested core binding in a competition assay (1) and observed no defects in core binding for any of the mutations relative to the N149 variant alone (data not shown) .

In summary, whether position 149 contains asparagine or aspartic acid, the transcriptional properties examined here were the same, and therefore these variants can be used interchangeably as controls in transcription initiation activity studies involving {sigma}70 . The location of position 149 in the nonconserved spacer suggests that it probably has structural and/or conformational importance rather than a specific function . The fact that asparagine and aspartic acid are equally well tolerated at position 149 is not surprising, given the chemical similarities of these amino acids . More significant is the effect of the identity of position 149 on the behavior of additional amino acid substitutions within {sigma}70 . Both the R122H and {Delta}1.2 mutations displayed characteristics that clearly depended on the position 149 variant background .

 


   ACKNOWLEDGMENTS

 
We thank all members of the Dombroski laboratory for advice and support during the research and preparation of this work .

This work was supported by a grant from the National Institutes of Health, GM56453 .


   FOOTNOTES

 
* Corresponding author . Present address: NIH, Center for Scientific Review, 6701 Rockledge Dr., Mailstop 7808, Bethesda, MD 20892 . Phone: (301) 435-1149 . Fax: (301) 480-4042 . E-mail: dombrosa{at}csr.nih.gov .


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