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Ann Hochschild

Publications and source records attributed to Ann Hochschild.

At least 19 recordsLinked to original sources
PubMed2006

RNA-mediated destabilization of the sigma(70) region 4/beta flap interaction facilitates engagement of RNA polymerase by the Q antiterminator.

The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. During early elongation, the stability of interactions between sigma(70) (the primary sigma factor in Escherichia coli) and core decreases due to an ordered displacement of segments of sigma(70) from core triggered by growth of the nascent RNA. Here we demonstrate that the nascent RNA-mediated destabilization of an interaction between sigma(70) region 4 and the flap domain of the beta subunit is required for the bacteriophage lambda Q antiterminator protein to contact holoenzyme during early elongation. We demonstrate further that the requirement for nascent RNA in the process by which Q engages RNAP can be bypassed if sigma(70) region 4 is removed. Our findings illustrate how a regulator can exploit the nascent RNA-mediated reconfiguration of the holoenzyme to gain access to the enzyme during early elongation.

Base Sequence↗
PubMed2006

Conformational toggle triggers a modulator of RNA polymerase activity.

Members of a recently discovered class of transcription factor, which includes the Gre factors that stimulate transcript cleavage, function by directly modulating the catalytic properties of RNA polymerase (RNAP). Now, three research groups have determined crystal structures of a Gre homolog, Gfh1, which inhibits all RNAP catalytic activities. Strikingly, these structures reveal a puzzling discrepancy between the Gfh1 and GreA conformations, but the discovery that a pH-dependent conformational toggle alters Gfh1 activity suggests an elegant solution.

Bacterial Proteins↗
PubMed2006

The structural basis of cooperative regulation at an alternate genetic switch.

Bacteriophage lambda is a paradigm for understanding the role of cooperativity in gene regulation. Comparison of the regulatory regions of lambda and the unrelated temperate bacteriophage 186 provides insight into alternate ways to assemble functional genetic switches. The structure of the C-terminal domain of the 186 repressor, determined at 2.7 A resolution, reveals an unusual heptamer of dimers, consistent with presented genetic studies. In addition, the structure of a cooperativity mutant of the full-length 186 repressor, identified by genetic screens, was solved to 1.95 A resolution. These structures provide a molecular basis for understanding lysogenic regulation in 186. Whereas the overall fold of the 186 and lambda repressor monomers is remarkably similar, the way the two repressors cooperatively assemble is quite different and explains in part the differences in their regulatory activity.

Amino Acid Substitution↗
PubMed2006

Structural basis for bacterial transcription-coupled DNA repair.

Coupling of transcription and DNA repair in bacteria is mediated by transcription-repair coupling factor (TRCF, the product of the mfd gene), which removes transcription elongation complexes stalled at DNA lesions and recruits the nucleotide excision repair machinery to the site. Here we describe the 3.2 A-resolution X-ray crystal structure of Escherichia coli TRCF. The structure consists of a compact arrangement of eight domains, including a translocation module similar to the SF2 ATPase RecG, and a region of structural similarity to UvrB. Biochemical and genetic experiments establish that another domain with structural similarity to the Tudor-like domain of the transcription elongation factor NusG plays a critical role in TRCF/RNA polymerase interactions. Comparison with the translocation module of RecG as well as other structural features indicate that TRCF function involves large-scale conformational changes. These data, along with a structural model for the interaction of TRCF with the transcription elongation complex, provide mechanistic insights into TRCF function.

Amino Acid Sequence↗
PubMed2005

An artificial activator that contacts a normally occluded surface of the RNA polymerase holoenzyme.

Many activators of transcription are sequence-specific DNA-binding proteins that stimulate transcription initiation through interaction with RNA polymerase (RNAP). Such activators can be constructed artificially by fusing a DNA-binding protein to a protein domain that can interact with an accessible surface of RNAP. In these cases, the artificial activator is directed to a target promoter bearing a recognition site for the DNA-binding protein. Here we describe an artificial activator that functions by contacting a normally occluded surface of promoter-bound RNAP holoenzyme. This artificial activator consists of a DNA-binding protein fused to the bacteriophage T4-encoded transcription regulator AsiA. On its own, AsiA inhibits transcription by Escherichia coli RNAP because it remodels the holoenzyme, disrupting an intersubunit interaction that is required for recognition of the major class of bacterial promoters. However, when tethered to the DNA via a DNA-binding protein, AsiA can exert a strong stimulatory effect on transcription by disrupting the same intersubunit interaction, contacting an otherwise occluded surface of the holoenzyme. We show that mutations that affect the intersubunit interaction targeted by AsiA modulate the stimulatory effect of this artificial activator. Our results thus demonstrate that changes in the accessibility of a normally occluded surface of the RNAP holoenzyme can modulate the activity of a gene-specific regulator of transcription.

Base Sequence↗
PubMed2005

The interaction between sigma70 and the beta-flap of Escherichia coli RNA polymerase inhibits extension of nascent RNA during early elongation.

The sigma-subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. This function depends on specific intersubunit interactions that occur when sigma associates with the RNAP core enzyme to form RNAP holoenzyme. Among these interactions, that between conserved region 4 of sigma and the flap domain of the RNAP beta-subunit (beta-flap) is critical for recognition of the major class of bacterial promoters. Here, we describe the isolation of amino acid substitutions in region 4 of Escherichia coli sigma(70) that have specific effects on the sigma(70) region 4/beta-flap interaction, either weakening or strengthening it. Using these sigma(70) mutants, we demonstrate that the sigma region 4/beta-flap interaction also can affect events occurring downstream of transcription initiation during early elongation. Specifically, our results provide support for a structure-based proposal that, when bound to the beta-flap, sigma region 4 presents a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel. Our findings support the view that the transition from initiation to elongation involves a staged disruption of sigma-core interactions.

Amino Acid Substitution↗
PubMed2005

An altered-specificity DNA-binding mutant of Escherichia coli sigma70 facilitates the analysis of sigma70 function in vivo.

The sigma subunit of bacterial RNA polymerase is strictly required for promoter recognition. The primary (housekeeping) sigma factor of Escherichia coli, sigma(70), is responsible for most of the gene expression in exponentially growing cells. The fact that sigma(70) is an essential protein has complicated efforts to genetically dissect the functions of sigma(70). To facilitate the analysis of sigma(70) function in vivo, we isolated an altered-specificity DNA-binding mutant of sigma(70), sigma(70) R584A, which preferentially recognizes a mutant promoter that is not efficiently recognized by wild-type sigma(70). Exploiting this sigma(70) mutant as a genetic tool, we establish an in vivo assay for the inhibitory effect of the bacteriophage T4-encoded anti-sigma factor AsiA on sigma(70)-dependent transcription. Our results demonstrate the utility of this altered-specificity system for genetically dissecting sigma(70) and its interactions with transcription regulators.

Amino Acid Substitution↗
PubMed2004

A family of anti-sigma70 proteins in T4-type phages and bacteria that are similar to AsiA, a Transcription inhibitor and co-activator of bacteriophage T4.

Anti-sigma70 factors interact with sigma70 proteins, the specificity subunits of prokaryotic RNA polymerase. The bacteriophage T4 anti-sigma70 protein, AsiA, binds tightly to regions 4.1 and 4.2 of the sigma70 subunit of Escherichia coli RNA polymerase and inhibits transcription from sigma70 promoters that require recognition of the canonical sigma70 -35 DNA sequence. In the presence of the T4 transcription activator MotA, AsiA also functions as a co-activator of transcription from T4 middle promoters, which retain the canonical sigma70 -10 consensus sequence but have a MotA box sequence centered at -30 rather than the sigma70 -35 sequence. The E.coli anti-sigma70 protein Rsd also interacts with region 4.2 of sigma70 and inhibits transcription from sigma70 promoters. Our sequence comparisons of T4 AsiA with Rsd, with the predicted AsiA orthologs of the T4-type phages RB69, 44RR, KVP40, and Aeh1, and with AlgQ, a regulator of alginate production in Pseudomonas aeruginosa indicate that these proteins share conserved amino acid residues at positions known to be important for the binding of T4 AsiA to sigma70 region 4. We show that, like T4 AsiA, Rsd binds to sigma70 in a native protein gel and, as with T4 AsiA, a L18S substitution in Rsd disrupts this complex. Previous work has assigned sigma70 amino acid F563, within region 4.1, as a critical determinant for AsiA binding. This residue is also involved in the binding of sigma70 to the beta-flap of core, suggesting that AsiA inhibits transcription by disrupting the interaction between sigma70 region 4.1 and the beta-flap. We find that as with T4 AsiA, the interaction of KVP40 AsiA, Rsd, or AlgQ with sigma70 region 4 is diminished by the substitution F563Y. We also demonstrate that like T4 AsiA and Rsd, KVP40 AsiA inhibits transcription from sigma70-dependent promoters. We speculate that the phage AsiA orthologs, Rsd, and AlgQ are members of a related family in T4-type phage and bacteria, which interact similarly with primary sigma factors. In addition, we show that even though a clear MotA ortholog has not been identified in the KVP40 genome and the phage genome appears to lack typical middle promoter sequences, KVP40 AsiA activates transcription from T4 middle promoters in the presence of T4 MotA. We speculate that KVP40 encodes a protein that is dissimilar in sequence, but functionally equivalent, to T4 MotA.

Amino Acid Sequence↗
PubMed2004

An RNA polymerase mutant deficient in DNA melting facilitates study of activation mechanism: application to an artificial activator of transcription.

Transcription initiation is a major target for the regulation of gene expression in all organisms. Transcription activators can stimulate different steps in the initiation process including the initial binding of RNA polymerase (RNAP) to the promoter and a subsequent promoter-melting step. Typically, kinetic assays are required to determine whether an activator exerts its effect on the initial binding of RNAP or on the promoter-melting step. Here we take advantage of a mutant Escherichia coli RNAP that is deficient in promoter melting to assess the ability of an activator to stabilize the initial binding of RNAP to the promoter. For the well-characterized activator CRP, we show that this RNAP mutant can be used to distinguish between effects on initial binding and promoter melting; these results provide an independent confirmation of the results of kinetic analysis. We then employ the melting-deficient RNAP mutant to demonstrate an effect of an artificial activator of transcription on the initial binding of RNAP. Our findings demonstrate that a melting-deficient RNAP mutant can be used to trap a normally unstable intermediate in transcription initiation, thus providing a novel tool for probing activation mechanism.

Cyclic AMP Receptor Protein↗
PubMed2004

Regulation of RNA polymerase through the secondary channel.

High-resolution crystal structures have highlighted functionally important regions in multisubunit RNA polymerases, including the secondary channel, or pore, which is postulated to allow the diffusion of small molecules both into and out of the active center of the enzyme. Recent work from several groups has illustrated how regulatory factors and small molecules can exploit the secondary channel to gain access to the active site and modify the transcription properties of RNA polymerase.

Catalytic Domain↗
PubMed2004

The sigma 70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter.

The sigma(70) subunit of RNA polymerase plays an essential role in transcription initiation. In addition, sigma(70) has a critical regulatory role during transcription elongation at the bacteriophage lambda late promoter, lambda P(R'). At this promoter, sigma(70) mediates a pause in early elongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter -10 element. Here we provide evidence that sigma(70) also mediates a pause in early elongation at the lac promoter (plac). Like that at lambda P(R'), the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter -10 element. Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interaction between sigma(70) and the remainder of the transcription elongation complex. Bioinformatic analysis suggests that promoter-proximal sigma(70)-dependent pauses may play a role in the regulation of many bacterial promoters.

Base Sequence↗
PubMed2004

Cooperativity in long-range gene regulation by the lambda CI repressor.

Effective repression of cI transcription from PRM by the bacteriophage lambda CI repressor requires binding sites (OL) located 2.4 kb from the promoter. A CI tetramer bound to OL1.OL2 interacts with a tetramer bound near PRM (OR1.OR2), looping the intervening DNA. We previously proposed that in this CI octamer:DNA complex, the distant OL3 operator and the weak OR3 operator overlapping PRM are juxtaposed so that a CI dimer at OL3 can cooperate with a CI dimer binding to OR3. Here we show that OL3 is necessary for effective repression of PRM and that the repressor at OL3 appears to interact specifically with the repressor at OR3. The OL3-CI-OR3 interaction involves the same CI interface used for short-range dimer-dimer interactions and does not occur without the other four operators. The long-range interactions were incorporated into a physicochemical model, allowing estimation of the long-range interaction energies and showing the lysogenic state to be ideally poised for CI negative autoregulation. The results establish the lambda system as a powerful tool for examining long-range gene regulatory interactions in vivo.

Binding Sites↗
PubMed2004

Structure of a ternary transcription activation complex.

The cI protein of bacteriophage lambda (lambdacI) activates transcription by binding a DNA operator just upstream of the promoter and interacting with the RNA polymerase sigma subunit domain 4 (sigma(4)). We determined the crystal structure of the lambdacI/sigma(4)/DNA ternary complex at 2.3 A resolution. There are no conformational changes in either protein, which interact through an extremely small interface involving at most 6 amino acid residues. The interactions of the two proteins stabilize the binding of each protein to the DNA. The results provide insight into how activators can operate through a simple cooperative binding mechanism but affect different steps of the transcription initiation process.

Amino Acid Sequence↗
PubMed2004

A bacterial two-hybrid system based on transcription activation.

We describe the use of a bacterial two-hybrid system for the study of protein-protein interactions in Escherichia coli. This system is based on transcription activation and involves the synthesis of two fusion proteins within the bacterial cell whose interaction stimulates transcription of a reporter gene. Specifically, one of the fusion proteins can function as a transcription activator when its interaction partner is fused to a subunit of the bacterial RNA polymerase. This bacterial two-hybrid system has been used to study a number of interacting proteins from both prokaryotes and eukaryotes, and can be used to find interacting proteins from complex protein libraries.

DNA-Directed RNA Polymerases↗
PubMed2003

Region 4 of sigma as a target for transcription regulation.

Bacterial sigma factors play a key role in promoter recognition, making direct contact with conserved promoter elements. Most sigma factors belong to the sigma70 family, named for the primary sigma factor in Escherichia coli. Members of the sigma70 family typically share four conserved regions and, here, we focus on region 4, which is directly involved in promoter recognition and serves as a target for a variety of regulators of transcription initiation. We review recent advances in the understanding of the mechanism of action of regulators that target region 4 of sigma.

Bacteriophage lambda↗
PubMed2002

Protein-protein and protein-DNA interactions of sigma70 region 4 involved in transcription activation by lambdacI.

The cI protein of bacteriophage lambda (lambdacI) activates transcription from promoter P(RM) through an acidic patch on the surface of its DNA-binding domain. Genetic evidence suggests that this acidic patch stimulates transcription from P(RM) through contact with the C-terminal domain (region 4) of the sigma(70) subunit of Escherichia coli RNA polymerase. Here, we identify two basic residues in region 4 of sigma(70) that are critical for lambdacI-mediated activation of transcription from P(RM). On the basis of structural modeling, we propose that one of these sigma(70) residues, K593, facilitates the interaction between lambdacI and region 4 of sigma(70) by inducing a bend in the DNA upstream of the -35 element, whereas the other, R588, interacts directly with a critical acidic residue within the activating patch of lambdacI. Residue R588 of sigma(70) has been shown to play an important role in promoter recognition; our findings suggest that the R588 side-chain has a dual function at P(RM), facilitating the interaction of region 4 with the promoter -35 element and participating directly in the protein-protein interaction with lambdacI.

Amino Acid Sequence↗
PubMed2002

Analysis of phosphorylation-dependent protein-protein interactions using a bacterial two-hybrid system.

Phosphorylation-dependent protein-protein interactions provide the foundation for a multitude of intracellular signal transduction pathways. One of the goals of signal transduction research is to more precisely understand the nature of these phosphorylation-dependent interactions. Here, we describe a bacterial two-hybrid assay that allows for the rapid, efficient analysis of phosphorylation-dependent protein-protein interactions. In this system, the interacting protein domains are provided as fusion proteins in Escherichia coli. cells that contain a eukaryotic kinase. Specific phosphorylation of one of the fused protein domains results in a protein-protein interaction that can be detected as a change in the expression of a reporter gene. We also describe how this system can be modified to permit the use of cDNA libraries to identify either novel binding partners for a phosphorylated substrate or novel kinases that can induce a specific protein-protein interaction.

Bacteriophage lambda↗