Location and orientation of an activating region in the Escherichia coli transcription factor, FNR (original) (raw)
Related papers
2005
The global anaerobic regulator FNR is a DNA binding protein that activates transcription of genes required for anaerobic metabolism in Escherichia coli through interactions with RNA polymerase (RNAP). Alaninescanning mutagenesis of FNR amino acid residues 181 to 193 of FNR was utilized to determine which amino acid side chains are required for transcription of both class II and class I promoters. In vivo assays of FNR function demonstrated that a core of residues (F181, R184, S187, and R189) was required for efficient activation of class II promoters, while at a class I promoter, FF(؊61.5), only S187 and R189 were critical for FNR activation. Site-directed mutagenesis of positions 184, 187, and 189 revealed that the positive charge contributes to the function of the side chain at positions 184 and 189 while the serine hydroxyl is critical for the function of position 187. Subsequent analysis of the carboxy-terminal domain of the ␣ subunit (␣CTD) of RNAP, using an alanine library in single copy, revealed that in addition to previously characterized side chains (D305, R317, and L318), E286 and E288 contributed to FNR activation of both class II and class I promoters, suggesting that ␣CTD region 285 to 288 also participates in activation by FNR. In conclusion, this study demonstrates that multiple side chains within region 181 to 192 are required for FNR activation and the surface of ␣CTD required for FNR activation is more extensive than previously observed. A common mechanism of activation by transcription factors employs a series of macromolecular interactions, from binding DNA to multiple contacts with RNA polymerase (RNAP), which allows target promoters to overcome intrinsic defects in transcription initiation (21). This study focused on the Escherichia coli global anaerobic regulator FNR and the protein requirements essential for transcription activation. In particular, this study further investigated the residues of FNR that have been proposed to interact with the ␣ subunit of RNAP. FNR, which regulates transcription in response to O 2 deprivation, belongs to a family of transcriptional regulators related to the cyclic AMP receptor protein, CRP (13). In its active form, FNR is a homodimer (16) which protects an ϳ22-bp sequence in DNase I footprinting experiments (9, 14). The vast majority of FNR-activated promoters contain an FNR binding site centered approximately 41.5 bp upstream of the transcriptional start site and are termed class II promoters (Fig. 1A) (6). At this position, FNR is able to make multiple contacts with RNAP through three domains: activating region 1 (AR1), AR2, and AR3 (3, 5, 14, 15, 31, 33, 34). FNR-AR3 is active in the downstream subunit (3) and is likely to contact the 70 subunit of RNAP (23). FNR-AR2, which plays a minor role in activation, is also active in the downstream subunit (5), but its interaction partner is proposed to be the amino-terminal domain of the ␣ subunit of RNAP (5). FNR-AR1 is active in the upstream subunit (3, 33) and is proposed to interact with the carboxy-terminal domain of the ␣ subunit (␣CTD) of RNAP (32). In contrast, at a class I promoter, the FNR binding site is located further upstream, centered at 61.5 or 71.5 bp upstream
FEBS Letters, 2004
The Escherichia coli FNR protein is a global transcription regulator that activates gene expression via interactions with the RNA polymerase K K subunit C-terminal domain. Using preparations of E. coli RNA polymerase holoenzyme, speci¢cally labelled with a DNA cleavage reagent, we have determined the location and orientation of the C-terminal domain of the RNA polymerase K K subunit in transcriptionally competent complexes at a class II FNR-dependent promoter. We conclude that one K K subunit C-terminal domain binds immediately upstream of FNR, and that its position and orientation is the same as at similar promoters dependent on CRP, another E. coli transcription activator that is related to FNR. In complementary experiments, we show that the second K K subunit Cterminal domain of RNA polymerase can be repositioned by upstream-bound CRP, but not by upstream-bound FNR.
Nucleic Acids Research, 1989
Expression from the E.coli meiR promoter (pmelR) is normally totally dependent on the transcription activator protein, CRP. We describe experiments with a genetically engineered DNA fragment carrying pmelR in which the wild type CRP binding site was replaced with synthetic oligonucleotides containing either FNR or CRP binding sequences. When the synthetic oligonucleotide contains the 22 bp consensus for FNR binding sites, expression from pmelR is dependent on FNR but not CRP. Single changes at either of two symmetrically-related positions create sites that are recognised by both FNR and CRP. Changes at both positions result in a site that is not recognised by FNR but which binds CRP tightly.
FEMS Microbiology Letters, 1998
Naturally occurring promoters that are repressed by the Escherichia coli FNR protein are complex. In this work, we have constructed a simple semi-synthetic promoter that is repressed by FNR binding to a single site that overlaps the promoter 335 element. Our results show that a single site for FNR is sufficient for effective repression. This semi-synthetic promoter provides a simple tool for monitoring FNR binding to target sites in the absence of its activation function. We have exploited this to study FNR mutants that are defective in repressing the ndh promoter, a complex naturally occurring promoter that is repressed by FNR.
Journal of Molecular Biology, 2011
Initiation of RNA synthesis from DNA templates by RNA polymerase (RNAP) is a multi-step process, in which initial recognition of promoter DNA by RNAP triggers a series of conformational changes in both RNAP and promoter DNA. The bacterial RNAP functions as a molecular isomerization machine, using binding free energy to remodel the initial recognition complex, placing downstream duplex DNA in the active site cleft and then separating the nontemplate and template strands in the region surrounding the start site of RNA synthesis. In this initial unstable "open" complex the template strand appears correctly positioned in the active site. Subsequently, the nontemplate strand is repositioned and a clamp is assembled on duplex DNA downstream of the open region to form the highly stable open complex, RP o . The transcription initiation factor, σ 70 , plays critical roles in promoter recognition and RP o formation as well as in early steps of RNA synthesis.
Topography of intermediates in transcription initiation of E.coli
The EMBO journal, 1990
Three characteristic footprinting patterns resulted from probing the Escherichia coli RNA polymerase T7 A1 promoter complex by hydroxyl radicals in the temperature range between 4 degrees C and 37 degrees C. These were attributed to the closed complex, the intermediate complex and the open complex. In the closed complex, the RNA polymerase protects the DNA only at one side over five helical turns. In the intermediate complex, the range of the protected area is extended further downstream by two helical turns. This region of the DNA helix is fully protected, indicating that the RNA polymerase wraps around the DNA between base positions -13 and +20. In the open complex, a stretch between base positions -7 and +2, which was fully protected in the intermediate complex, becomes accessible towards hydroxyl radicals but only in the codogenic strand, indicating that the DNA strands are unwound. Our data suggest that only the DNA downstream of the promoter is involved in this unwinding process.
Journal of Molecular Biology, 1998
The s subunit of RNA polymerase orchestrates basal transcription by ®rst binding to core RNA polymerase and then recognizing promoters. Using a series of 16 alanine-substitution mutations, we show that residues in a narrow region of Escherichia coli s 70 (590 to 603) are involved in transcription activation by a mutationally altered CRP derivative, FNR and AraC. Homology modeling of region 4 of s 70 to the closely related NarL or 434 Cro proteins, suggests that the ®ve basic residues implicated in activation are either in the C terminus of a long recognition helix that includes residues recognizing the À35 hexamer region of the promoter, or in the subsequent loop, and are ideally positioned to permit interaction with activators. The only substitution that has a signi®cant effect on activator-independent transcription is at R603, indicating that this residue of s 70 may play a distinct role in transcription initiation.
Journal of Biological Chemistry
The rrnB P1 promoter of Escherichia coli (starting sequence C"4-A"S-C"2-C"1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Rea. 16,9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3+ " mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1+" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-ternary complexes carry u factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3+ ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.
Biochemistry, 2012
Differences in kinetics of transcription initiation by RNA polymerase (RNAP) at different promoters tailor the pattern of gene expression to cellular needs. After initial binding, large conformational changes occur in promoter DNA and RNAP to form initiation-capable complexes. To understand the mechanism and regulation of transcription initiation, the nature and sequence of these conformational changes must be determined. Escherichia coli RNAP uses binding free
Journal of Bacteriology, 2003
Transport and utilization of sugar phosphates in Escherichia coli depend on the transport protein encoded by the uhpT gene. Transmembrane induction of uhpT expression by external glucose 6-phosphate is positively regulated by the promoter-specific activator protein UhpA and the global regulator catabolite gene activator protein (CAP). Activation by UhpA requires a promoter element centered at ؊64 bp, relative to the start of transcription, and activation by CAP requires a DNA site centered at position ؊103.5. This DNA site binds the cyclic AMP-CAP complex in vitro, and its deletion from the promoter reduces transcription activity to 7 to 9% of the wild-type level. Ten uhpT promoter derivatives with altered spacing between the DNA site for CAP and the remainder of the promoter were constructed. Their transcription activities indicated that the action of CAP at this promoter is dependent on proper helical phasing of promoter elements, with CAP binding on the same face of the helix as RNA polymerase does. Five CAP mutants defective in transcription activation at class I and class II CAP-dependent promoters but not defective in DNA binding or DNA bending (positive control mutants) were tested for the ability to activate transcription. These CAP pc mutants exhibited little or no defect in transcription activation at uhpT, indicating that CAP action at uhpTp involves a different mechanism than that which is used for its action at other classes of CAP-dependent promoters.