Prokaryotic Transcription Factors Research Articles

Evaluation of the DNA Binding Tendencies of the Transition State Regulator AbrB

Global transition state regulator proteins represent one of the most diverse classes of prokaryotic transcription factors. One such transition state regulator, AbrB from Bacillus subtilis, is known to bind more than 60 gene targets yet displays specificity within this target set by binding each promoter with a different affinity. Microelectrospray ionization mass spectrometry (microESI-MS), circular dichroism, fluorescence, UV spectroscopy, and molecular modeling were used to elucidate differences among AbrB, DNA, and AbrB-DNA complexes. MicroESI-MS analysis of AbrB confirmed its stable macromolecular state as being tetrameric and verified the same stoichiometric state in complex with DNA targets. MicroESI-MS, circular dichroism, and fluorescence provided relative binding affinities for AbrB-DNA interactions in a qualitative manner. UV spectroscopy was used in a quantitative manner to determine solution phase dissociation constants for AbrB-DNA complexes. General DNA structural parameters for all known natural AbrB binding sequences were also studied and significant similarities in topological constraints (stretch, opening, and propeller twist) were observed. It is likely that these parameters contribute to the differential binding proclivities of AbrB. In addition to providing an improved understanding of transition state regulator-DNA binding properties and structural tendencies of target promoters, this comprehensive and corroborative spectroscopic study endorses the use of microESI-MS for rapidly ascertaining qualitative binding trends in noncovalent systems in a high-throughput manner.

The DNA Binding Protein H-NS Binds to and Alters the Stability of RNA in vitro and in vivo

H-NS is an abundant prokaryotic transcription factor that preferentially binds to intrinsically bent DNA. Although H-NS has been shown to reduce the transcription of over 100 genes, evidence suggests that H-NS can also affect the translation of some genes. One such gene, rpoS , specifies a sigma factor, RpoS. The ability of H-NS to bind to the rpoS mRNA and the non-coding RNA regulator, DsrA, was tested. Electrophoretic mobility-shift assays yielded an apparent binding affinity of H-NS binding to curved DNA of approximately 1 μM, whereas binding to rpoS mRNA or DsrA RNA was approximately 3 μM. This RNA binding was not prevented by an excess of competitor yeast RNA, suggesting that H-NS specifically bound these RNAs. Footprint analysis with a single strand-specific ribonuclease was used to identify the H-NS binding site(s) on DsrA and rpoS mRNA. Surprisingly, H-NS appeared to enhance the cleavage of DsrA and rpoS mRNA. The enhanced cleavage was at sites that were predicted to be single-stranded and did not result from contaminating nucleases in the H-NS protein preparation or non-specific effects of the nuclease. Quantitative RT-PCR of RNA isolated from wild-type and hns − strains revealed that H-NS also affects the stability of DsrA in vivo . Thus H-NS appears to modulate RNA stability in vivo and in vitro .

The DNA Binding Protein H-NS Binds to and Alters the Stability of RNA in vitro and in vivo

H-NS is an abundant prokaryotic transcription factor that preferentially binds to intrinsically bent DNA. Although H-NS has been shown to reduce the transcription of over 100 genes, evidence suggests that H-NS can also affect the translation of some genes. One such gene, rpoS, specifies a sigma factor, RpoS. The ability of H-NS to bind to the rpoS mRNA and the non-coding RNA regulator, DsrA, was tested. Electrophoretic mobility-shift assays yielded an apparent binding affinity of H-NS binding to curved DNA of approximately 1 μM, whereas binding to rpoS mRNA or DsrA RNA was approximately 3 μM. This RNA binding was not prevented by an excess of competitor yeast RNA, suggesting that H-NS specifically bound these RNAs. Footprint analysis with a single strand-specific ribonuclease was used to identify the H-NS binding site(s) on DsrA and rpoS mRNA. Surprisingly, H-NS appeared to enhance the cleavage of DsrA and rpoS mRNA. The enhanced cleavage was at sites that were predicted to be single-stranded and did not result from contaminating nucleases in the H-NS protein preparation or non-specific effects of the nuclease. Quantitative RT-PCR of RNA isolated from wild-type and hns − strains revealed that H-NS also affects the stability of DsrA in vivo. Thus H-NS appears to modulate RNA stability in vivo and in vitro.

Purification and in vitro characterization of the Serratia marcescens NucC protein, a zinc-binding transcription factor homologous to P2 Ogr.

NucC is structurally and functionally homologous to a family of prokaryotic zinc finger transcription factors required for late gene expression in P2- and P4-related bacteriophages. Characterization of these proteins in vitro has been hampered by their relative insolubility and tendency to aggregate. We report here the successful purification of soluble, active, wild-type NucC protein. Purified NucC exhibits site-specific binding to a conserved DNA sequence that is located upstream of NucC-dependent Serratia marcescens promoters and the late promoters of P2-related phages. This sequence is sufficient for binding of NucC in vitro. NucC binding to the S. marcescens nuclease promoter P(nucA) and to the sequence upstream of the P2 late promoter P(F) is accompanied by DNA bending. NucC protects about 25 nucleotides of the P(F) upstream region from DNase I digestion, and RNA polymerase protects the promoter region only in the presence of NucC. Template DNA, RNA polymerase holoenzyme, and purified NucC are the only macromolecular components required for transcription from P(F) in vitro.

The structure of full-length LysR-type transcriptional regulators. Modeling of the full-length OxyR transcription factor dimer.

The LysR-type transcriptional regulators (LTTRs) comprise the largest family of prokaryotic transcription factors. These proteins are composed of an N-terminal DNA binding domain (DBD) and a C-terminal cofactor binding domain. To date, no structure of the DBD has been solved. According to the SUPERFAMILY and MODBASE databases, a reliable homology model of LTTR DBDs may be built using the structure of the Escherichia coli ModE transcription factor, containing a winged helix- turn-helix (HTH) motif, as a template. The remote, but statistically significant, sequence similarity between ModE and LTTR DBDs and an alignment generated using SUPERFAMILY and MODBASE methods was independently confirmed by alignment of sequence profiles representing ModE and LTTR family DBDs. Using the crystal structure of the E.coli OxyR C-terminal domain and the DBD alignments we constructed a structural model of the full-length dimer of this LTTR family member and used it to investigate the mode of protein-DNA interaction. We also applied the model to interpret, in a structural context, the results of numerous biochemical studies of mutated LTTRs. A comparison of the LTTR DBD model with the structures of other HTH proteins also provides insights into the interaction of LTTRs with the C-terminal domain of the RNA polymerase alpha subunit.

The “glue” that controls transcription

by Mark Ptashne and Alexander Gann Cold Spring Harbor Laboratory Press (2002) 192 pages. ISBN 0-87969-633-8 $39 ![Figure][1] Guano cormorants flying home to the rock on which their baby bird is waiting to be fed face a daunting task. They must distinguish their own child from the

The zinc metalloregulatory protein Synechococcus PCC7942 SmtB binds a single zinc ion per monomer with high affinity in a tetrahedral coordination geometry.

The Synechococcus PCC7942 SmtB is a zinc-responsive transcriptional repressor and a member of the ArsR superfamily of prokaryotic metalloregulatory transcription factors. The mechanism of negative regulation by Zn(II) and other metals as well as the coordination chemistry (stoichiometry, affinity, and specificity) of SmtB is poorly understood. In contrast to previous results [Kar, S. R., Adams, A. C., Lebowitz, J., Taylor, K. B., and Hall, L. M. (1997) Biochemistry 36, 15343-15348], we find that fully reduced SmtB binds 1 mol equiv of Zn(II) with a very high affinity, K(Zn) in excess of 10(11) M(-1) (pH 7.4, 0.15 M KCl, 22 degrees C). Optical spectroscopic experiments reveal that SmtB binds 1 mol equiv of Co(II) in a tetrahedral or distorted tetrahedral environment with one or two cysteine thiolate ligands in the first coordination shell. Zn(II) and Co(II) EXAFS studies are consistent with the optical spectroscopic data, and further suggest the presence of a mixture of carboxylate and imidazole-containing ligands. K(Co) was determined to be 1.7 (+/-0.1) x 10(9) M(-1) in a chelator (EGTA) competition assay; 1 equiv of Zn(II) results in complete displacement of the bound Co(II). SmtB also binds 1 mol equiv of Ni(II), which, when formed at low Ni(II):SmtB molar ratios, adopts a non-native, six-coordinate complex characterized by at least two histidine and no thiolate ligands. The hierarchy of metal binding affinities is Zn(II) >> Co(II) >> Ni(II).

The solution structure of VAT-N reveals a ‘missing link’ in the evolution of complex enzymes from a simple βαββ element

Background: The VAT protein of the archaebacterium Thermoplasma acidophilum, like all other members of the Cdc48/p97 family of AAA ATPases, has two ATPase domains and a 185-residue amino-terminal substrate-recognition domain, VAT-N. VAT shows activity in protein folding and unfolding and thus shares the common function of these ATPases in disassembly and/or degradation of protein complexes.Results: Using nuclear magnetic resonance (NMR) spectroscopy, we found that VAT-N is composed of two equally sized subdomains. The amino-terminal subdomain VAT-Nn (comprising residues Met1–Thr92) forms a double-psi β-barrel whose pseudo-twofold symmetry is mirrored by an internal sequence repeat of 42 residues. The carboxy-terminal subdomain VAT-Nc (comprising residues Glu93–Gly185) forms a novel six-stranded β-clam fold. Together, VAT-Nn and VAT-Nc form a kidney-shaped structure, in close agreement with results from electron microscopy. Sequence and structure analyses showed that VAT-Nn is related to numerous proteins including prokaryotic transcription factors, metabolic enzymes, the protease cofactors UFD1 and PrlF, and aspartic proteinases. These proteins map out an evolutionary path from simple homodimeric transcription factors containing a single copy of the VAT-Nn repeat to complex enzymes containing four copies.Conclusions: Our results suggest that VAT-N is a precursor of the aspartic proteinases that has acquired peptide-binding activity while remaining proteolytically incompetent. We propose that the binding site of the protein is similar to that of aspartic proteinases, in that it lies between the psi-loops of the amino-terminal β-barrel and that it coincides with a crescent-shaped band of positive charge extending across the upper face of the molecule.

Characterisation of the enzymatic and RNA-binding properties of the Rhodobacter sphaeroides 2.4.1. Rho homologue

The Escherichia coli Rho is a transcription termination factor with complex enzymatic properties. Rho is a near-universal prokaryotic transcription factor, but very few non-enteric Rho factors have been studied. The expression and enzymatic activity of Rho from the GC-rich, Gram-negative bacterium Rhodobacter sphaeroides was characterised. Poly(C)-activated ATP hydrolysis, multimerisation and the abundance of the R. sphaeroides Rho were similar to the E. coli Rho. The R. sphaeroides Rho was a DNA:RNA helicase. The R. sphaeroides Rho was unique in Rho factors characterised to date in that it did not interact with the λtR1 terminator transcript and ATP hydrolysis was unusually weakly activated by poly(U) RNA. A chimeric Rho (RhoER), with the RNA-binding domain from the E. coli Rho and the ATPase domain of the R. sphaeroides Rho, was activated by RNA co-factors in a similar fashion to the E. coli Rho. The activity of RhoER suggests functional interactions between the N- and C-terminal domains of Rho monomers are highly conserved between Rho factors. The main differences between Rho factors from different bacteria is in the specificity of RNA binding although this does not appear to be necessarily dependent on the GC bias of target RNA as has been previously suggested.

The Repressor Protein, Bm3R1, Mediates an Adaptive Response to Toxic Fatty Acids in Bacillus megaterium

Bm3R1 is a helix-turn-helix transcriptional repressor from Bacillus megaterium whose binding to DNA is inhibited by fatty acids and a wide range of compounds that modulate lipid metabolism. The inactivation of Bm3R1/DNA binding activity results in the activation of transcription of the operon encoding a fatty acid hydroxylase, cytochrome P450 102. The metabolic role of this operon is unknown. It is possible that it is involved in the synthesis of modified fatty acids as part of normal cellular metabolism or may represent a protective mechanism by which B. megaterium detoxifies harmful foreign lipids. In this report we demonstrate that polyunsaturated fatty acids (PUFA) activate the transcription of the CYP102 operon. These PUFA are the most potent activators of the CYP102 operon observed to date, and we show that their effects are due to binding directly to Bm3R1. In addition, cultures that have been treated with the CYP102 inducer, nafenopin, are protected against PUFA toxicity. Resistance to PUFA toxicity is also seen in a Bm3R1-deficient strain that constitutively expresses CYP102. The resistant phenotype of this Bm3R1 mutant strain is reversed by specific chemical inactivation of CYP102. These data demonstrate that Bm3R1 can act as a direct sensor of toxic fatty acids and, in addition, provide the first direct evidence of fatty acids binding to a prokaryotic transcription factor.

Distinct functions of N and C-terminal domains of GreA, an Escherichia coli transcript cleavage factor

The prokaryotic transcription factors GreA and GreB are involved in the regulation of transcript elongation by RNA polymerase (RNAP). Their known activities include suppression of transcription arrest, enhancement of transcription fidelity, and facilitation of the transition from abortive initiation to productive elongation. Presumably, Gre proteins exert their functions by altering the conformation of the enzyme in ternary elongation complexes (TEC) and inducing the cleavage of nascent RNA. GreA and GreB have a similar structural organization and consist of two domains: a C-terminal globular and an extended N-terminal coiled-coil domain. To investigate the functional roles of Gre domains, we expressed separately the N and C-terminal domains of GreA (NTD and CTD, respectively) and characterized their activities with in vitro assays. We demonstrate that the NTD possesses the residual transcript cleavage activity of the wild-type GreA. The CTD does not display any nucleolytic activity; however, it substantially increases the cleavage activity of the NTD. In contrast to NTD, the CTD competes with GreA and GreB for binding to RNAP and inhibits their transcript cleavage and antiarrest activities. Both domains individually and together inhibit transcription elongation. From these results we conclude that the NTD is responsible for the GreA induction of nucleolytic activity while the CTD determines the binding of GreA to RNAP. Both domains are required for full functional activity of GreA.

DNA binding characteristics of CrtJ. A redox-responding repressor of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression in Rhodobacter capsulatus.

Previous genetic analysis indicated that the photosynthesis gene cluster from Rhodobacter capsulatus coded for the transcription factor, CrtJ, that is responsible for aerobic repression of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression. In this study, we have heterologously overexpressed and purified CrtJ to homogeneity and shown by gel mobility shift assays that CrtJ is biologically active. DNase I footprint analysis confirms molecular genetic studies by showing that CrtJ binds to conserved palindromic sequences that overlap the -10 and -35 promoter regions of the bchC operon. Graphs of the percentage of DNA bound versus protein concentration show sigmoidal curves, which is highly indicative of cooperative binding of CrtJ to the two palindromic sites. A binding constant for interaction of CrtJ with the palindrome that spans the -10 region was calculated to be 4.8 x 10(-9) M, whereas affinity for the palindrome that spans the -35 region was found to be 2.9 x 10(-9) M. Binding of CrtJ to the bchC promoter region was also found to be redox-sensitive, with CrtJ exhibiting a 4.5-fold higher binding affinity under oxidizing versus reducing conditions.

Study of Redox-Regulated Transcription Factors in Prokaryotes

Several prokaryotic regulatory proteins that respond to changes in oxygen tension or the presence of oxidative agents have now been identified. The Fnr protein governs the expression of numerous genes during anaerobic growth, both as a transcriptional activator and as a repressor. OxyR protein responds to cellular exposure to H2O2to stimulate transcription of several defense proteins. SoxR protein is triggered by superoxide or nitric oxide to activate a multigene regulon for antioxidant defense and antibiotic resistance. Each of these proteins has been purified and characterized for DNA binding and transcriptional activityin vitro.Fnr, OxyR, and SoxR all seem to respond directly to redox signals generated in the cell, and theirin vitroproperties support this view: Fnr has an oxygen-sensitive [4Fe-4S] center essential for DNA binding; OxyR may be activated via oxidation of a key cysteine residue; and SoxR activation depends on redox-sensitive [2Fe-2S] centers. Basic methods for genetic and biochemical analysis in these systems are presented, with emphasis on detailed methods for SoxR that illustrate general approaches for all the systems.

Analysis of CAG/CTG triplet repeats in the human genome: Implication in transcription factor gene regulation

Instability and polymorphism at several CAG/CTG trinucleotide repeat loci have been associated with human genetic disorders. In an attempt to identify novel sites that may be possible loci for expansion of CAG/CTG repeats, we searched all human sequences in the EMBL nucleotide sequence database for (CAG)5 and (CTG)5 repeats. We have identified 121 human DNA sequences of known and unknown functions that contain stretches of five or more CAG or CTG repeats. Many repeat stretches were interrupted by variant triplets, a significant number of which differ from the repeat triplet only by a single base, suggesting that these evolved from the parent triplet by point mutations. A large number of human transcription factor genes were found to contain CAG repeats within their coding sequences. Analysis of the EMBL transcription factors database showed that many transcription factor genes of other eukaryotes, including genes involved inDrosophila embryo development, possess these repeats. Interestingly, CAG repeats are absent from prokaryotic transcription factors. Different sequence entries for the human TATA box binding protein showed a polymorphism in the length of the CAG repeat in this gene, suggesting that loci other than those already known to be associated with genetic diseases may be possible sites for repeat instability related disorders. On the basis of our findings in this database analysis, we propose a role for CAG repeats as cisacting regulatory elements involved in fine-tuning gene expression.

Prediction of the Three Dimensional Structure of the DNA-Binding Domain of the LysR Family.

A considerable number of prokaryotic transcription factors belong to the LysR family. By comparison with the amino acid sequence of the putative DNA-binding domain (DBD) of BirA, for which the crystal structure has been determined, we predict that the LysR DBD has three α -helices and three β-strands, and that the third α-helix binds to DNA. Contacts between DNA bases and amino acid residues in the predicted recognition helix are discussed in comparison with those found in crystal structures of DNA-transcription factor complexes.

Cloning and expression of cyclosporin A- and FK506-sensitive nuclear factor of activated T-cells: NF45 and NF90.

Nuclear Factor of Activated T-cells (NF-AT) is a crucial transcription factor required for T-cell expression of interleukin 2. Purified NF-AT contains 45-kDa and 90-kDa subunits (Corthésy, B., and Kao, P. N. (1994) J. Biol. Chem. 269, 20682-20690). Partial internal amino acid sequences derived from each subunit indicate that these proteins are novel. The amino acid sequences were used to clone the cDNAs encoding each subunit. The cDNAs predict proteins of novel structures: NF45 has limited similarity to prokaryotic transcription factor sigma-54 and to human DNA topoisomerase II; NF90 has limited similarity to Drosophila Staufen in a domain predicted to bind double-stranded RNA. RNA encoding NF45 and NF90 exists in nonstimulated Jurkat T-cells and in all other cell types examined (HeLa, HepG2, K562). Immunofluorescence microscopy was used to demonstrate that both proteins are located in the nucleus of Jurkat T-cells. Clones NF45 and NF90 with a polyhistidine fusion tag were transiently expressed and processed in the native environment of Jurkat T-cells. Histidine-tagged NF45 and NF90 proteins, affinity-purified on nickel chelate columns, encode a NF-AT DNA-binding activity that is enhanced following T-cell stimulation, and this enhancement is blocked when T-cells are stimulated in the presence of cyclosporin A or FK506.

Oxygen and the control of gene expression

The respiration of oxygen, while essential to aerobic organisms for the generation of energy, leads to the formation of reactive oxygen intermediates (ROIs) as harmful byproducts. ROIs damage nucleic acids, lipids and proteins. Therefore, protective mechanisms against elevated intracellular ROI levels, referred to as oxidative stress, have evolved. These include the activation of transcription factors which elevate the expression of protective enzymes. Eukaryotic cells have also evolved the ability to specifically generate ROIs following stimulation with various agents. In these cases, ROIs are used as second messengers to activate gene expression. Here we will discuss both prokaryotic and eukaryotic transcription factors that respond to ROIs. In addition, transcription factors will be described that are activated by either exposure to antioxidants, which reduce the intracellular ROI concentration, or by hypoxia, the absence of oxygen.

Evidence for a trans-acting factor that regulates the transcription of class II major histocompatibility complex genes: genetic and functional analysis.

The study of specific trans-acting transcription factors in prokaryotes and lower eukaryotes has been greatly facilitated by genetic analysis of mutant strains deficient in such factors. We have developed such a system to study mammalian trans-acting factors that regulate the transcription of class II major histocompatibility complex genes, using the mutant cell lines RM2 and RM3. These cells, derived from the human B-cell line Raji, specifically fail to transcribe their class II major histocompatibility complex genes. Here we show that a transfected HLA-DR alpha class II major histocompatibility complex gene, like the endogenous HLA-DR alpha genes, is efficiently transcribed in Raji cells but not in RM2 or RM3 cells, demonstrating that the mutant cells are deficient in a specific trans-acting factor required for transcription of these genes. HLA-DR expression in RM2 and RM3 cells is rescued by fusion to another B-cell line but not by fusion to each other. Thus, the defects in the two cell lines are recessive and noncomplementing and define a locus whose wild-type product we designate TF-X1. We show that TF-X1 influences the activity of a 24-base-pair B-cell-specific cis-acting transcription element in the HLA-DR alpha promoter. However, in three different biochemical assays, we detect no difference between wild-type and mutant cells in the DNA-binding proteins that interact with these DNA sequences. Thus, the defective version of TF-X1 may be a DNA-binding protein that binds to the HLA-DR alpha promoter but fails to activate transcription. Alternatively, TF-X1 may not be a DNA-binding protein at all.