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W Cannon

Publications and source records attributed to W Cannon.

At least 19 recordsLinked to original sources
PubMed2006

A second paradigm for gene activation in bacteria.

Control of gene expression is key to development and adaptation. Using purified transcription components from bacteria, we employ structural and functional studies in an integrative manner to elaborate a detailed description of an obligatory step, the accessing of the DNA template, in gene expression. Our work focuses on a specialized molecular machinery that utilizes ATP hydrolysis to initiate DNA opening and permits a description of how the events triggered by ATP hydrolysis within a transcriptional activator can lead to DNA opening and transcription. The bacterial EBPs (enhancer binding proteins) that belong to the AAA(+) (ATPases associated with various cellular activities) protein family remodel the RNAP (RNA polymerase) holoenzyme containing the sigma(54) factor and convert the initial, transcriptionally silent promoter complex into a transcriptionally proficient open complex using transactions that reflect the use of ATP hydrolysis to establish different functional states of the EBP. A molecular switch within the model EBP we study [called PspF (phage shock protein F)] is evident, and functions to control the exposure of a solvent-accessible flexible loop that engages directly with the initial RNAP promoter complex. The sigma(54) factor then controls the conformational changes in the RNAP required to form the open promoter complex.

Bacteria↗
PubMed2002

Mechanochemical ATPases and transcriptional activation.

Transcriptional activator proteins that act upon the sigma54-containing form of the bacterial RNA polymerase belong to the extensive AAA+ superfamily of ATPases, members of which are found in all three kingdoms of life and function in diverse cellular processes, often via chaperone-like activities. Formation and collapse of the transition state of ATP for hydrolysis appears to engender the interaction of the activator proteins with sigma54 and leads to the protein structural transitions needed for RNA polymerase to isomerize and engage with the DNA template strand. The common oligomeric structures of AAA+ proteins and the creation of the active site for ATP hydrolysis between protomers suggest that the critical changes in protomer structure required for productive interactions with sigma54-holoenzyme occur as a consequence of sensing the state of the gamma-phosphate of ATP. Depending upon the form of nucleotide bound, different functional states of the activator are created that have distinct substrate and chaperone-like binding activities. In particular, interprotomer ATP interactions rely upon the use of an arginine finger, a situation reminiscent of GTPase-activating proteins.

Adenosine Triphosphatases↗
PubMed2001

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP-aluminum fluoride: insights into activator mechanochemical action.

Conformational changes in sigma 54 (sigma(54)) and sigma(54)-holoenzyme depend on nucleotide hydrolysis by an activator. We now show that sigma(54) and its holoenzyme bind to the central ATP-hydrolyzing domains of the transcriptional activators PspF and NifA in the presence of ADP-aluminum fluoride, an analog of ATP in the transition state for hydrolysis. Direct binding of sigma(54) Region I to activator in the presence of ADP-aluminum fluoride was shown and inferred from in vivo suppression genetics. Energy transduction appears to occur through activator contacts to sigma(54) Region I. ADP-aluminum fluoride-dependent interactions and consideration of other AAA+ proteins provide insight into activator mechanochemical action.

Adenosine Diphosphate↗
PubMed2001

DNA melting within a binary sigma(54)-promoter DNA complex.

The final sigma(54) subunit of the bacterial RNA polymerase requires the action of specialized enhancer-binding activators to initiate transcription. Here we show that final sigma(54) is able to melt promoter DNA when it is bound to a DNA structure representing the initial nucleation of DNA opening found in closed complexes. Melting occurs in response to activator in a nucleotide-hydrolyzing reaction and appears to spread downstream from the nucleation point toward the transcription start site. We show that final sigma(54) contains some weak determinants for DNA melting that are masked by the Region I sequences and some strong ones that require Region I. It seems that final sigma(54) binds to DNA in a self-inhibited state, and one function of the activator is therefore to promote a conformational change in final sigma(54) to reveal its DNA-melting activity. Results with the holoenzyme bound to early melted DNA suggest an ordered series of events in which changes in core to final sigma(54) interactions and final sigma(54)-DNA interactions occur in response to activator to allow final sigma(54) isomerization and the holoenzyme to progress from the closed complex to the open complex.

Bacterial Proteins↗
PubMed2000

Staged closure of complicated bronchopleural fistulas.

Bronchopleural fistulas remain a major complication after thoracic surgery. Despite continued advances in the treatment of this difficult problem, perioperative mortality remains as high as 15%. Multiple treatment strategies have been described with varying degrees of success. Successful treatment of chronic bronchopleural fistulas requires aggressive control of infection, adequate drainage of the chest cavity, closure of the fistula with vascularized tissue, and obliteration of the chest cavity. The authors present their experience with 3 patients who underwent a two-stage closure of their bronchopleural fistulas with pectoralis major muscle flaps followed by omental flap obliteration of the chest cavity. Each patient had previously undergone an Eloesser procedure for chest cavity drainage. The initial muscle flap operation is a small procedure that can be done rapidly with minimal morbidity in chronically ill patients. The intervening period between procedures allows patients to continue aggressive nutritional and physical rehabilitation until they are able to tolerate a second operation for chest cavity obliteration. All bronchopleural fistulas in our series healed, with one minor complication. A staged closure is a safe and effective alternative treatment for chronic and recurrent bronchopleural fistulas.

Aged↗
PubMed1999

Characterisation of holoenzyme lacking sigmaN regions I and II.

The sigma-N (sigmaN) protein associates with bacterial core RNA polymerase to form a holoenzyme that is silent for transcription in the absence of enhancer-binding activator proteins. Here we show that the acidic Region II of sigmaN from Klebsiella pneumoniae is dispensable for polymerase isomerisation and trans-cription under conditions where the inhibited state of the holoenzyme is relieved by removal of sigmaN Region I sequences. Holoenzymes lacking Region I or Regions I+II were equally susceptible to the order of addition-dependent inhibition or stabilisation of DNA binding afforded by in trans Region I sequences. Region I+II-deleted [sigma] formed a holoenzyme with a DNA-binding activity more susceptible to inhibition by non-specific DNA than that lacking Region I. Region II sequences appear more closely associated with formation of a holoenzyme and [sigma] proficient in DNA binding than with changes in holoenzyme conformation needed for unmasking a single-strand DNA-binding activity used for open complex for-mation. Region II may therefore function to optimise DNA interactions for an efficient sigma cycle.

Base Sequence↗
PubMed1999

Amino-terminal sequences of sigmaN (sigma54) inhibit RNA polymerase isomerization.

In bacteria, association of the specialized sigmaN protein with the core RNA polymerase subunits forms a holoenzyme able to bind promoter DNA, but unable to melt DNA and initiate transcription unless acted on by an activator protein. The conserved amino-terminal 50 amino acids of sigmaN (Region I) are required for the response to activators. We have used pre-melted DNA templates, in which the template strand is unpaired and accessible for transcription initiation, to mimic a naturally melted promoter and explore the function of Region I. Our results indicate that one activity of Region I sequences is to inhibit productive interaction of holoenzyme with pre-melted DNA. On pre-melted DNA targets, either activation of sigmaN-holoenzyme or removal of Region I allowed efficient formation of complexes in which melted DNA was sequestered by RNA polymerase. Like natural pre-initiation complexes formed on conventional DNA templates through the action of activator, such complexes were heparin-resistant and transcriptionally active. The inhibitory sigmaN Region I domain functioned in trans to confer heparin sensitivity to complexes between Region I-deleted holoenzyme and pre-melted promoter DNA. Evidence that Region I senses the conformation of the promoter was obtained from protein footprint experiments. We suggest that one function for Region I is to mask a single-strand DNA-binding activity of the holoenzyme. On the basis of extended DNA footprints of Region I-deleted holoenzyme, we also propose that Region I prevents RNA polymerase isomerization, a conformational change necessary for access to and the subsequent stable association of holoenzyme with melted DNA.

DNA↗
PubMed1997

Nucleoprotein complex formation by the enhancer binding protein nifA.

The nitrogen fixation protein NifA is a member of the protein family activating transcription by the alternative eubacterial sigmaN (sigma54) RNA polymerase holoenzyme. Binding sites for NifA, upstream activator sequences (UASs), are remotely located. Interaction between holoenzyme bound in a closed promoter complex and NiFA is facilitated by bending of the intervening DNA by integration host factor (IHF). We have examined NifA contact with the Klebsiella pneumoniae nifH promoter UAS in the presence and absence of holoenzyme and IHF. Footprints with UV light were made on 5-BrdU-substituted DNA and DNase I and laser UV footprints on conventional DNA templates. Results establish that the consensus thymidine residues of the UAS motif 5'-TGT are in close proximity to NifA. Reactivity suggests that each UAS thymidine is not structurally equivalent. Titration of NifA binding to the UAS in the presence or absence of the closed promoter complex indicates that the interaction of NifA with the UAS is not strongly co-operative with holoenzyme or IHF, a result supportive of an activation mechanism not reliant upon simple recruitment of factors to the promoter. Laser footprints demonstrated that holoenzyme suppressed reactivity of promoter consensus -14, -15 and -16 T residues, indicating close contact. Binding of holoenzyme resulted in a specific increase in 5-BrdU reactivity at -9 within the holoenzyme binding site, likely reflecting DNA distortion. Enhanced -9 reactivity required sigmaNN-terminal sequences that are necessary for activation. Since T-9 is melted in open complexes the closed complex appears poised for melting. Open promoter complex formation was accompanied by a distinct change in laser footprint signal at -11, consistent with the view that nucleation of strand separation occurs within or close to the -12 promoter element.

Azotobacter vinelandii↗
PubMed1996

Purification and activities of the Rhodobacter capsulatus RpoN (sigma N) protein.

The rpoN-encoded sigma factors (sigma N) are a distinct class of bacterial sigma factors, with no obvious homology to the major sigma 70 class. The sigma N-containing RNA polymerase holoenzyme functions in enhancer-dependent transcription to allow expression of positively controlled genes. We have purified the Rhodobacter capsulatus sigma N protein, which is distinctive in lacking an acidic region implicated in the melting of promoter DNA by the Escherichia coll sigma N holoenzyme, and may represent a minor subclass of sigma N proteins. Assays of promoter recognition and holoenzyme formation and function showed that the purified R. capsulatus sigma N protein is distinct in activity compared to the enteric proteins, but retains the broad functions described for these proteins. As first described for the Klebsiella pneumoniae protein, promoter recognition in the absence of core RNA polymerase was detected, but contact of certain promoter bases by the R. capsulatus sigma N protein and its response to core RNA polymerase was clearly different from that determined for the K. pneumoniae and E. coli proteins. Results are discussed in the context of a requirement to modulate the activity of the DNA-binding surfaces of sigma N to regulate sigma N function. Circular dichroism was used to evaluate the structure of the R. capsulatus protein and revealed differences in the tertiary signals as compared to the K. pneumoniae protein, some of which are attributable to the DNA-binding domain of sigma N.

Amino Acid Sequence↗
PubMed1995

Core RNA polymerase and promoter DNA interactions of purified domains of sigma N: bipartite functions.

The sigma N class of sigma factors confer upon RNA polymerase the requirement for enhancer-binding activator proteins. The sigma-N (sigma N) protein of Klebsiella pneumoniae was analysed by the assay of purified peptides comprising domains or regions of sigma N defined by proteolysis or by homology alignment, respectively. The NH2-terminal Region I is required for the correct interaction of holoenzyme with the promoter, and promoter complexes forming with a truncated sigma N lacking Region I are not activatable. The complexes lack the DNA structure believed to represent nucleated strand separation but still make close contacts with this promoter part. Determinants of specific DNA recognition by sigma N were shown to reside in a C-terminal 16 kDa peptide, and core RNA polymerase binding determinants in an adjacent peptide. The latter contacts and appears to pack against the DNA-binding domain. Thus the DNA-binding and core-binding domains are bipartite in function, consistent with core functioning as an allosteric effector of the sigma DNA-binding activity. The DNA-binding and core-binding domains together include Region III of sigma N. Although not the primary determinant of core or DNA recognition, the acidic Region II of sigma N influenced both activities. Regions I and II in combination with core RNA polymerase thus appear to control the activity of C-terminal DNA contacting surfaces to allow formation of a closed promoter complex that is susceptible to activation.

Allosteric Regulation↗
PubMed1995

Identification of close contacts between the sigma N (sigma 54) protein and promoter DNA in closed promoter complexes.

The complexes forming between the alternative sigma factor protein sigma N (sigma 54), its holoenzyme and promoter DNA were analysed using the hydroxyl radical probe and by photochemical footprinting of bromouridine-substituted DNA. Close contacts between the promoter, sigma N and its holoenzyme appear to be restricted predominantly to one face of the DNA helix, extending from -31 to -5. They all appear attributable to sigma N and no extra close contacts from the core RNA polymerase subunits in the holoenzyme-promoter DNA complex were detected. We suggest that the apparent absence of close core RNA polymerase contacts in the region of the promoter DNA to be melted during open complex formation is important for maintaining the closed complex. Results of the hydroxyl radical footprinting imply that sigma N makes multiple DNA backbone contacts across and beyond the -12, -24 consensus promoter elements, and the photochemical footprints indicate that consensus thymidine residues contribute important major groove contacts to sigma N. Formation of the open complex is shown to involve a major structural transition in the DNA contacted by sigma N, establishing a direct role for sigma N in formation of the activated promoter complex.

Base Sequence↗
PubMed1994

DNA distortion and nucleation of local DNA unwinding within sigma-54 (sigma N) holoenzyme closed promoter complexes.

The sigma N (sigma 54) RNA polymerase holoenzyme has the distinctive property of binding to promoters to form a closed promoter complex, which only isomerizes to the open complex when acted upon by an enhancer binding activator protein. We probed promoter complexes that form between sigma N and its holoenzyme with the conformationally sensitive footprinting reagents ortho-copper phenanthroline, potassium permanganate, and diethylpyrocarbonate. Results from these experiments indicate that the contacts sigma N makes at the -12 promoter element are necessary to promote a local DNA distortion immediately adjacent to this promoter element when the holoenzyme but not sigma N alone binds promoter DNA. Complexes in which this local distortion is not detected are not activatable, and the altered DNA conformation is diminished in the activated complex. We propose that a barrier to open complex formation in the sigma N holoenzyme closed complex is at some step or steps after the initial nucleation of DNA strand separation, which is detected as an altered DNA conformation stably maintained within the closed complex. Thus the activator protein may promote a conformational change in the sigma N holoenzyme to allow propagation of the altered DNA conformation, probably local unwinding, which we propose is necessary for formation of the melted DNA state, characteristic of the open promoter complex.

Bacterial Proteins↗
PubMed1994

Identification of a DNA-contacting surface in the transcription factor sigma-54.

The transcription factor sigma-54 (sigma 54) is a sequence-specific DNA-binding protein that directs RNA polymerase to a particular class of promoter. The interaction of sigma 54 with promoter DNA has been analysed by protein-DNA crosslinking and enzymatic and chemical proteolysis. Direct physical evidence for a DNA-contacting surface within the carboxy-terminal one-third of the protein has been obtained. This region of sigma 54 is likely to be close to the surface of the protein, and contacts DNA when either sigma 54 or the sigma 54-holoenzyme bind specifically to promoter DNA. The amino-terminal region of sigma 54 appears to be highly susceptible to proteolysis, and its integrity influences the accessibility towards proteolysis of a second region of sigma 54, which includes the DNA-contacting surface. Thus the amino-terminal region of sigma 54 may have a role in influencing its DNA-binding properties, the major determinants of which appear to reside in the carboxy-terminal one-third of the protein.

Bacterial Proteins↗
PubMed1994

Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL.

The prokaryotic enhancer-binding protein NifA stimulates transcription at a distance by binding to sequences upstream of nitrogen fixation (nif) promoters and catalyzing the formation of open promoter complexes by RNA polymerase containing the alternative sigma factor, sigma 54. The activity of NifA in vivo is modulated by the negative regulatory protein NifL in response to environmental oxygen and fixed nitrogen. To date, a detailed biochemical analysis of these proteins from the model diazotroph Klebsiella pneumoniae has been hindered by their insolubility. We have now purified NifA and NifL from Azotobacter vinelandii in their native form. NifA is competent in specific DNA binding, transcriptional activation, and response to negative regulation by NifL in vitro. In contrast to the conserved mechanism of phosphotransfer demonstrated by other two-component regulatory systems, our results support a model in which NifL regulates the activity of NifA via a protein-protein steric block interaction rather than a catalytic modification of NifA.

Azotobacter vinelandii↗
PubMed1993

The Klebsiella pneumoniae nifJ promoter: analysis of promoter elements regulating activation by the NifA promoter.

The nifJ and nifH promoters of Klebsiella pneumoniae are divergently transcribed sigma 54-dependent promoters that are positively activated by the NifA protein. NifA binds to upstream activator sequences (UASs), usually located 60-200 bp upstream of the start of transcription. Bound NifA is presented to the RNA polymerase-sigma 54 complex (E sigma 54) via DNA loop formation, mediated by the binding of integration host factor protein (IHF) between E sigma 54 and NifA. The nifJ promoter sequence contains three potential NifA binding sites (UAS1, 2 and 3) and two potential RNA polymerase-sigma 54-binding sites (downstream promoter elements, DPEs 1 and 2). DPE2 is located 420 bp into the coding region and DPE1 overlaps UAS1 by 5 bp. Mutational and footprinting analyses have shown efficient activation of the nifJ promoter requires that NifA is bound at UAS 2 and 3. Transcription is initiated at DPE1. Only a weak interaction of NifA with the UAS overlapping DPE1 was detected. Footprints demonstrated that E sigma 54 forms a closed complex at DPE1 but not DPE2 and that bound E sigma 54 closely approaches the -15 region of DPE1. Stimulation of nifJ promoter activity by IHF was not as great as that observed for other nif promoters. In the absence of IHF nifH promoter sequences stimulated activation of the nifJ promoter. This appeared to require NifA bound at the nifH UAS. Thus, one additional role of IHF may be to partition NifA between the two promoters by constraining the topology of the DNA.

Amino Acid Sequence↗
PubMed1993

Core RNA polymerase assists binding of the transcription factor sigma 54 to promoter DNA.

The sigma subunit of bacterial RNA polymerase is necessary for the specific binding of RNA polymerase holoenzyme to promoter DNA. Promoter complexes which form with holoenzyme containing sigma 54 remain as closed complexes unless they are activated by one class of enhancer binding protein. The sigma 54 transcription factor can bind specifically to certain promoter sites in the absence of the core RNA polymerase subunits. This property has allowed demonstration of a new role for core polymerase in transcription, namely that it assists the binding of sigma 54 to promoter DNA. An altered form of sigma 54 with a deletion within the amino-terminal region showed increased affinity for specific DNA-binding sites. Although able to complex with core RNA polymerase the mutant sigma 54 failed to respond to core polymerase in the manner characteristic of the wild-type sigma 54 by altering its footprint. This result indicates that sigma 54 has a latent DNA-binding activity which is revealed by core RNA polymerase, and possibly involves a change in sigma 54 conformation. Promoter complexes which formed with sigma 54-holoenzyme appeared to be qualitatively different, depending upon the target promoter sequence, suggesting that different activatable complexes form at different promoter sequences.

Bacterial Proteins↗
PubMed1992

Specific binding of the transcription factor sigma-54 to promoter DNA.

A central event in transcription is the assembly on DNA of specific complexes near the initiation sites for RNA synthesis. Activation of transcription by one class of enhancer-binding proteins requires an RNA polymerase holoenzyme containing the specialized transcription factor, sigma-54 (sigma 54). We report here that sigma 54 alone specifically binds to promoter DNA and is responsible for many of the close contacts between RNA polymerase holoenzyme and promoter DNA, a property proposed for the major sigma 70 protein family. Binding of sigma 54 to promoter DNA is not equivalent to that of holoenzyme suggesting that there is a constraint on sigma 54 conformation when bound with core RNA polymerase. Footprints indicate sigma 54 is at the leading edge of DNA-bound holoenzyme. Like the holoenzyme, sigma 54-binding to promoter DNA does not result in DNA strand separation. Instead the specific DNA-binding activity of sigma 54 assists assembly of a closed promoter complex. This complex can be isomerized to the open (DNA melted) complex by activator protein, but promoter-bound sigma 54 alone cannot be induced to melt DNA. The pathway leading to productive transcription is similar to that proposed for eukaryotic RNA polymerase II systems.

Base Sequence↗