scholarly journals Identification of the binding site of the σ 54 hetero-oligomeric FleQ/FleT activator in the flagellar promoters of Rhodobacter sphaeroides

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1669-1679 ◽  
Author(s):  
J. Peña-Sánchez ◽  
S. Poggio ◽  
U. Flores-Pérez ◽  
A. Osorio ◽  
C. Domenzain ◽  
...  
Keyword(s):  
Binding Site ◽  
Rhodobacter Sphaeroides ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Conserved Motif ◽  
Sigma 54 ◽  
Divergent Promoters ◽  
Oligomeric Complex ◽  
Flagellar Genes

Expression of the flagellar genes in Rhodobacter sphaeroides is dependent on one of the four sigma-54 factors present in this bacterium and on the enhancer binding proteins (EBPs) FleQ and FleT. These proteins, in contrast to other well-characterized EBPs, carry out activation as a hetero-oligomeric complex. To further characterize the molecular properties of this complex we mapped the binding sites or upstream activation sequences (UASs) of six different flagellar promoters. In most cases the UASs were identified at approximately 100 bp upstream from the promoter. However, the activity of the divergent promoters flhAp-flgAp, which are separated by only 53 bp, is mainly dependent on a UAS located approximately 200 bp downstream from each promoter. Interestingly, a significant amount of activation mediated by the upstream or contralateral UAS was also detected, suggesting that the architecture of this region is important for the correct regulation of these promoters. Sequence analysis of the regions carrying the potential FleQ/FleT binding sites revealed a conserved motif. In vivo footprinting experiments with the motAp promoter allowed us to identify a protected region that overlaps with this motif. These results allow us to propose a consensus sequence that represents the binding site of the FleQ/FleT activating complex.

A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (11) ◽  
pp. 5648-5659
Author(s):  
F J McNally ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Protein A ◽  
Replication Initiation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Functional Components

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.

scholarly journals Regulation of the Rhodobacter sphaeroides 2.4.1 hemA Gene by PrrA and FnrL

2008 ◽  
Vol 190 (20) ◽  
pp. 6769-6778 ◽  
Author(s):  
Britton Ranson-Olson ◽  
Jill H. Zeilstra-Ryalls
Keyword(s):  
Binding Site ◽  
Rhodobacter Sphaeroides ◽  
Binding Sites ◽  
Aminolevulinic Acid ◽  
Acid Synthesis ◽  
P2 Promoter ◽  
Indirect Action ◽  
Hema Gene

ABSTRACT Part of the oxygen responsiveness of Rhodobacter sphaeroides 2.4.1 tetrapyrrole production involves changes in transcription of the hemA gene, which codes for one of two isoenzymes catalyzing 5-aminolevulinic acid synthesis. Regulation of hemA transcription from its two promoters is mediated by the DNA binding proteins FnrL and PrrA. The two PrrA binding sites, binding sites I and II, which are located upstream of the more-5′ hemA promoter (P1), are equally important to transcription under aerobic conditions, while binding site II is more important under anaerobic conditions. By using phosphoprotein affinity chromatography and immunoblot analyses, we showed that the phosphorylated PrrA levels in the cell increase with decreasing oxygen tensions. Then, using both in vivo and in vitro methods, we demonstrated that the relative affinities of phosphorylated and unphosphorylated PrrA for the two binding sites differ and that phosphorylated PrrA has greater affinity for site II. We also showed that PrrA regulation is directed toward the P1 promoter. We propose that the PrrA component of anaerobic induction of P1 transcription is attributable to higher affinity of phosphorylated PrrA than of unphosphorylated PrrA for binding site II. Anaerobic activation of the more-3′ hemA promoter (P2) is thought to involve FnrL binding to an FNR consensuslike sequence located upstream of the P2 promoter, but the contribution of FnrL to P1 induction may be indirect since the P1 transcription start is within the putative FnrL binding site. We present evidence suggesting that the indirect action of FnrL works through PrrA and discuss possible mechanisms.

scholarly journals A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (11) ◽  
pp. 5648-5659 ◽  
Author(s):  
F J McNally ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Protein A ◽  
Replication Initiation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Functional Components

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.

scholarly journals Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression.

1993 ◽  
Vol 13 (9) ◽  
pp. 5805-5813 ◽  
Author(s):  
M M Wang ◽  
R Y Tsai ◽  
K A Schrader ◽  
R R Reed
Keyword(s):  
Signal Transduction ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Olfactory Neuron ◽  
Promoter Regions ◽  
Transcriptional Initiation ◽  
Genes Encoding

Genes which mediate odorant signal transduction are expressed at high levels in neurons of the olfactory epithelium. The molecular mechanism governing the restricted expression of these genes likely involves tissue-specific DNA binding proteins which coordinately activate transcription through sequence-specific interactions with olfactory promoter regions. We have identified binding sites for the olfactory neuron-specific transcription factor, Olf-1, in the sequences surrounding the transcriptional initiation site of five olfactory neuron-specific genes. The Olf-1 binding sites described define the consensus sequence YTCCCYRGGGAR. In addition, we have identified a second binding site, the U site, in the olfactory cyclic nucleotide gated channel and type III cyclase promoters, which binds factors present in all tissue examined. These experiments support a model in which expression of Olf-1 in the sensory neurons coordinately activates a set of olfactory neuron-specific genes. Furthermore, expression of a subset of these genes may be modulated by additional binding factors.

DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF

1994 ◽  
Vol 14 (9) ◽  
pp. 5986-5996
Author(s):  
S P Hunger ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Lymphoblastic Leukemia ◽  
Consensus Sequence ◽  
Reporter Genes ◽  
Wild Type ◽  
Basic Leucine Zipper

The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-hepatic leukemia factor (HLF) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein HLF fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type HLF and chimeric E2A-HLF proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric HLF proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid-rich (PAR) and C/EBP subfamilies; however, E2A-HLF proteins were significantly less tolerant of certain deviations from the HLF consensus binding site. These differences were directly attributable to loss of an HLF ancillary DNA-binding domain in all E2A-HLF chimeras and were further exacerbated by a zipper mutation in one isolate. Both wild-type and chimeric HLF proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or C/EBP consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-HLF competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site-specific transcriptional properties for E2A-HLF which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.

scholarly journals Participation of Ets transcription factors in the glucocorticoid response of the rat tyrosine aminotransferase gene.

1994 ◽  
Vol 14 (6) ◽  
pp. 4116-4125 ◽  
Author(s):  
M L Espinás ◽  
J Roux ◽  
J Ghysdael ◽  
R Pictet ◽  
T Grange
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Cell Line ◽  
Binding Sites ◽  
Tyrosine Aminotransferase ◽  
Hierarchical Level ◽  
Detectable Effect ◽  
Independent Manner ◽  
Glucocorticoid Response

We have previously shown that two remote glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase (TAT) gene contain multiple binding sites for several transcription factor families, including the glucocorticoid receptor (GR). We report here the identification of two novel binding sites for members of the Ets family of transcription factors in one of these GRUs. One of these binding sites overlaps the major GR-binding site (GRBS), whereas the other is located in its vicinity. Inactivation of the latter binding site leads to a twofold reduction of the glucocorticoid response, whereas inactivation of the site overlapping the GRBS has no detectable effect. In vivo footprinting analysis reveals that the active site is occupied in a glucocorticoid-independent manner, in a TAT-expressing cell line, even though it is located at a position where there is a glucocorticoid-dependent alteration of the nucleosomal structure. This same site is not occupied in a cell line that does not express TAT but expresses Ets-related DNA-binding activities, suggesting the existence of an inhibitory effect of chromatin structure at a hierarchical level above the nucleosome. The inactive Ets-binding site that overlaps the GRBS is not occupied even in TAT-expressing cells. However, this same overlapping site can confer Ets-dependent stimulation of both basal and glucocorticoid-induced levels when it is isolated from the GRU and duplicated. Ets-1 expression in COS cells mimics the activity of the Ets-related activities present in hepatoma cells. These Ets-binding sites could participate in the integration of the glucocorticoid response of the TAT gene with signal transduction pathways triggered by other nonsteroidal extracellular stimuli.

scholarly journals DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF.

1994 ◽  
Vol 14 (9) ◽  
pp. 5986-5996 ◽  
Author(s):  
S P Hunger ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Lymphoblastic Leukemia ◽  
Consensus Sequence ◽  
Reporter Genes ◽  
Wild Type ◽  
Basic Leucine Zipper

The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-hepatic leukemia factor (HLF) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein HLF fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type HLF and chimeric E2A-HLF proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric HLF proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid-rich (PAR) and C/EBP subfamilies; however, E2A-HLF proteins were significantly less tolerant of certain deviations from the HLF consensus binding site. These differences were directly attributable to loss of an HLF ancillary DNA-binding domain in all E2A-HLF chimeras and were further exacerbated by a zipper mutation in one isolate. Both wild-type and chimeric HLF proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or C/EBP consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-HLF competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site-specific transcriptional properties for E2A-HLF which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals Joint Transcriptional Control of xpsR, the Unusual Signal Integrator of the Ralstonia solanacearum Virulence Gene Regulatory Network, by a Response Regulator and a LysR-Type Transcriptional Activator

1998 ◽  
Vol 180 (10) ◽  
pp. 2736-2743 ◽  
Author(s):  
Jianzhong Huang ◽  
Wandee Yindeeyoungyeon ◽  
Ram P. Garg ◽  
Timothy P. Denny ◽  
Mark A. Schell
Keyword(s):  
Binding Site ◽  
Transcriptional Control ◽  
Response Regulator ◽  
Consensus Sequence ◽  
Virulence Gene ◽  
Transcriptional Activator ◽  
Binding Assays ◽  
Environmental Signals

ABSTRACT Ralstonia (Pseudomonas)solanacearum is a soil-borne phytopathogen that causes a wilting disease of many important crops. It makes large amounts of the exopolysaccharide EPS I, which it requires for efficient colonization, wilting, and killing of plants. Transcription of the epsoperon, encoding biosynthetic enzymes for EPS I, is controlled by a unique and complex sensory network that responds to multiple environmental signals. This network is comprised of the novel transcriptional activator XpsR, three distinct two-component regulatory systems (VsrAD, VsrBC, and PhcSR), and the LysR-type regulator PhcA, which is under the control of PhcSR. Here we show that thexpsR promoter (P xpsR ) is simultaneously controlled by PhcA and VsrD, permitting XpsR to act like a signal integrator, simultaneously coordinating signal input into theeps promoter from both VsrAD and PhcSR. Additionally, we used in vivo expression analysis and in vitro DNA binding assays with substitution and deletion mutants of P xpsR to show the following. (i) PhcA primarily interacts with a typical 14-bp LysR-type consensus sequence around position −77, causing a sixfold activation of P xpsR ; a weaker, less-defined binding site between −183 and −239 likely enhances PhcA binding and activation via the −77 site another twofold. (ii) Full 70-fold activation of P xpsR requires the additional interaction of the VsrD response regulator (or its surrogate) with a 14-bp dyadic sequence centered around −315 where it enhances activation (and possibly binding) by PhcA; however, VsrD alone cannot activate P xpsR . (iii) Increasing the distance between the putative VsrD binding site from that of PhcA by up to 232 bp did not dramatically affect P xpsR activation or regulation.

scholarly journals PIN*POINT analysis on the endogenous MnSOD promoter: specific demonstration of Sp1 binding in vivo

2003 ◽  
Vol 284 (2) ◽  
pp. C528-C534 ◽  
Author(s):  
Shiuhyang Kuo ◽  
Ann L. Chokas ◽  
Richard J. Rogers ◽  
Harry S. Nick
Keyword(s):  
Binding Sites ◽  
Manganese Superoxide Dismutase ◽  
Consensus Sequence ◽  
Dimethyl Sulfate ◽  
Site Directed Mutagenesis ◽  
Cellular Respiration ◽  
Point Analysis ◽  
Specific Regulation

Manganese superoxide dismutase (MnSOD) is a critical antioxidant enzyme that protects against superoxide anion generated as a consequence of normal cellular respiration, as well as during the inflammatory response. By employing dimethyl sulfate in vivo footprinting, we have previously identified ten basal protein binding sites within the MnSODpromoter. On the basis of consensus sequence comparison and in vitro footprinting data, one would predict that Sp1 might occupy five of these binding sites. To address these findings in the context of the nucleoprotein environment, we first utilized chromatin immunoprecipitation (ChIP) to demonstrate the nuclear association of Sp1 with the MnSOD promoter region. To identify the precise location of Sp1 binding, we have modified the original protein position identification with nuclease tail (PIN*POINT) methodology, providing an approach to establish both the identity and binding occupancy of Sp1 in the context of the endogenous MnSOD promoter. These data, coupled with site-directed mutagenesis, demonstrate the functional importance of two of the Sp1 binding sites in the stimulus-specific regulation of MnSOD gene expression. We feel that the combination of ChIP and PIN*POINT analysis allows unequivocal identification and localization of protein/DNA interactions in vivo, specifically the demonstration of Sp1 with the MnSODpromoter.

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