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.

The adnA transcriptional factor affects persistence and spread of Pseudomonas fluorescens under natural field conditions.

A soil plot was inoculated with a mixture of Pseudomonas fluorescens Pf0-2, the wild type, and Pf0-5a, a Tn5 insertion mutant in adnA, at 7.84 log CFU/g of soil. Over a period of 231 days, culturable populations of both strains were measured at selected times below and away from the point of inoculation. Pf0-5a did not spread as fast and attained significantly lower populations than Pf0-2. At sample depths below the inoculation site, the adnA mutant showed a significant decrease in CFU/g of soil as compared to Pf0-2. Pf0-2 was first detected at the 1.5-cm annular site at 3 days after inoculation, whereas Pf0-5a required 7 days to travel the same distance. At this distance, the wild-type strain could be detected at a 21.5- to 25-cm depth, whereas Pf0-5a could be detected only as deep as 15.5 to 18 cm. At 4.5 cm from the site of inoculation and in soil fractions corresponding to 13 to 18 cm, Pf0-2 was the only strain detected. These results suggest that the transcription factor AdnA provides a fitness advantage in P. fluorescens, allowing it to spread and survive in soil under field conditions.

Systematic analysis of sigma54 N-terminal sequences identifies regions involved in positive and negative regulation of transcription.

The conserved amino-terminal region of sigma 54 (Region I) contains sequences that allow response to activator proteins, and inhibit initiation in the absence of activator. Alanine-scanning mutagenesis has been used to systematically define Region I elements that contribute to each of these functions. Amino acid residues from 6 to 50 were substituted with alanine in groups of three consecutive residues, making a total of 15 mutants. Mutants were tested for their ability to mediate activation in vivo, and in vitro, and to support transcription in the absence of activator in vitro. Most mutations located between residues 15 and 47 altered sigma function, while mutations between residues 6 and 14, and 48-50 had little effect. The defective mutants ala 15-17, 42-44, and 45-47 define new amino acids required for normal sigma function. In general, there is an inverse correlation between the levels of activated and activator-independent transcription, suggesting that the two functions are linked. When activated, the defective sigma mutants, except for ala 24-26, formed heparin-resistant open complexes similar to wild-type sigma. Mutant ala 24-26 formed heparin-unstable open complexes, suggesting that this mutation interferes with a different step in the initiation pathway.

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.

Region I modifies DNA-binding domain conformation of sigma 54 within the holoenzyme.

Activation of transcription at sigma 54-dependent bacterial promoters proceeds via a mechanism that is independent of recruitment of RNA polymerase to the promoter, but is instead totally dependent on activator-driven conformational changes in the promoter-bound RNA polymerase. Understanding of the activation mechanism first requires a detailed description of the interactions taking place in the polymerase holoenzyme and closed complex. The interactions of sigma 54 with core RNA polymerase and promoter DNA were investigated using enzymatic and chemical (hydroxyl radical) protease footprinting of sigma. Regions of sigma were identified that are in direct contact with ligands, or whose conformation changes following ligand binding. A comparison of wild-type sigma and a mutant bearing a deletion of conserved Region I, which is required for response to activator proteins and regulated initiation, revealed differences in the protease sensitivity of free sigma indicating that Region I affects sigma conformation. Comparison of the holoenzyme and closed complex hydroxyl radical footprints revealed that residues of wild-type sigma protected by promoter DNA overlap, to a large extent, the residues of Region I-deleted sigma protected by core polymerase. Region I could thus modify DNA-binding by changing conformation of the DNA-binding domain of sigma 54 in a core polymerase-dependent manner. These differences can account for the modified promoter binding of the Region I-deleted sigma holoenzyme observed by DNA footprinting, and are likely of significance to the Region I-dependent activation of transcription.

Probing the assembly of transcription initiation complexes through changes in sigmaN protease sensitivity.

The alternative bacterial sigmaN RNA polymerase holoenzyme binds promoters as a transcriptionally inactive complex that is activated by enhancer-binding proteins. Little is known about how sigma factors respond to their ligands or how the responses lead to transcription. To examine the liganded state of sigmaN, the assembly of end-labeled Klebsiella pneumoniae sigmaN into holoenzyme, closed promoter complexes, and initiated transcription complexes was analyzed by enzymatic protein footprinting. V8 protease-sensitive sites in free sigmaN were identified in the acidic region II and bordering or within the minimal DNA binding domain. Interaction with core RNA polymerase prevented cleavage at noncontiguous sites in region II and at some DNA binding domain sites, probably resulting from conformational changes. Formation of closed complexes resulted in further protections within the DNA binding domain, suggesting close contact to promoter DNA. Interestingly, residue E36 becomes sensitive to proteolysis in initiated transcription complexes, indicating a conformational change in holoenzyme during initiation. Residue E36 is located adjacent to an element involved in nucleating strand separation and in inhibiting polymerase activity in the absence of activation. The sensitivity of E36 may reflect one or both of these functions. Changing patterns of protease sensitivity strongly indicate that sigmaN can adjust conformation upon interaction with ligands, a property likely important in the dynamics of the protein during transcription initiation.

A TEF-1-independent mechanism for activation of the simian virus 40 (SV40) late promoter by mutant SV40 large T antigens.

Simian virus 40 (SV40) large tumor antigen (T antigen) stimulates the activity of the SV40 late promoter and a number of cellular and other viral promoters. We have characterized the ability of T antigens with mutations in the DNA-binding domain and within the N-terminal 85 residues to activate the SV40 late promoter. T antigens lacking both nonspecific and sequence-specific DNA-binding activities were able to induce the late promoter. Mutations within the N-terminal 85 residues of T antigen diminished activation by less than twofold. Activation by wild-type and most of the mutant T antigens required intact binding sites for the cellular transcription factor TEF-1 in the late promoter. Curiously, two mutants altered in the N-terminal region and an additional mutant altered in the DNA-binding domain activated a late promoter derivative lacking TEF-1 binding sites, indicating the existence of a TEF-1-independent pathway for activation of the late promoter. A consensus binding site for the TATA binding protein, TBP, was created in variants of late promoters either containing or lacking TEF-1 binding sites. Basal expression was increased by the consensus TBP binding site only when TEF-1 binding sites were present, leading to a reduction in the degree of activation by T antigen. However, activation by a mutant T antigen of the promoter lacking TEF-1 sites was unchanged or slightly enhanced by the consensus TBP binding site. These results suggest that some mutant T antigens can stabilize an interaction between TBP and additional factors bound to the late promoter.

trans activation of the simian virus 40 late promoter by large T antigen requires binding sites for the cellular transcription factor TEF-1.

Simian virus 40 (SV40) T antigen stimulates the level of transcription from several RNA polymerase II promoters, including the SV40 late promoter. The mechanism of trans activation appears to be indirect since binding of T antigen to specific DNA sequences is not required. However, specific promoter elements that respond to T antigen have not previously been defined. We identified DNA sequences from the SV40 late promoter whose ability to stimulate transcription is induced by the expression of T antigen. In particular, the Sph I + II motifs of the SV40 enhancer can confer T-antigen inducibility to the normally uninducible herpes simplex virus thymidine kinase gene promoter when multiple copies of the sequence are inserted 5' of the transcription initiation site and TATA sequence. Binding sites for the cellular transcription factor TEF-1 and octamer binding proteins are contained within the Sph I + II motifs, as well as at other positions in the SV40 promoter. To study the role of individual protein-binding sites in trans activation by T antigen, mutations were constructed in various TEF-1 and octamer protein-binding sites of the SV40 late promoter. These mutations did not significantly affect basal promoter activity. However, mutation of all three TEF-1 sites prevented detectable activation by T antigen. DNase I footprinting of the mutated promoters with purified proteins demonstrated that inducibility by T antigen correlated with binding affinity of TEF-1 for the DNA and not with binding affinity of an octamer binding protein.