Characterisation of holoenzyme lacking sigmaN regions I and II

Cannon, Wendy; Chaney, Matthew; Buck, Martin

doi:10.1093/nar/27.12.2478

Cited by 19 publications

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“…Region II is variable and in some species, such as Rhodobacter capsulatus, is almost completely absent. In many (but not all) bacteria, region II is acidic, and it has been implicated in DNA melting (126), in the transition from the closed to open complex (E. Southern and M. Merrick, submitted for publication), and in assisting 54 binding to homoduplex and heteroduplex DNA (8). The lack of conservation of region II suggests that none of its activities are essential.…”

Section: Domain Structure Promoter Recognition and Core Interactionsmentioning

confidence: 99%

The Bacterial Enhancer-Dependent ς⁵⁴(ς^N) Transcription Factor

et al. 2000

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2The initiation of transcription is a complex process involving many different steps. These steps are all potential control points for regulating gene expression, and many have been exploited by bacteria to give rise to sophisticated regulatory mechanisms that allow the cell to adapt to changing growth regimens. Before they can transcribe from specific DNA promoter sequences, bacterial core RNA polymerases (with subunit composition ␣ 2 ␤␤Ј) must combine with a dissociable sigma subunit () to form RNA polymerase holoenzyme (␣ 2 ␤␤Ј). Since the discovery of factors (6), it has become clear that these proteins are central to the function of the RNA polymerase holoenzyme. The reversible binding of alternative factors allows formation of different holoenzymes able to distinguish groups of promoters required for different cellular functions. In addition to double-strand DNA promoter recognition and binding, proteins are closely involved in promoter melting (e.g., references 31,36,49,51,74,76,128), inhibit nonspecific initiation, are targets for activators, and control early transcription through promoter clearance and release from RNA polymerase (48,49,53). Here we describe the functioning of the bacterial 54 -RNA polymerase that is the target for sophisticated signal transduction pathways (103) involving activation via remote enhancer elements (5, 95).Based on structural and functional criteria, the different factors identified in bacteria can be grouped in two classes, one of which has a single member, 54 . Many factors belong to the 70 class, the major factor which is involved in expression of most genes during exponential growth (72). 54 (also called N ) differs both in amino acid sequence and in transcription mechanism from the 70 class (80). Despite the lack of any significant sequence similarity, both types of bind the same core RNA polymerase. Nonetheless, they produce holoenzymes with different properties.With the recognition that the 54 protein represented an entirely new class of factor, what had once been regarded as an aspect of transcription restricted to higher organisms became a well-established feature of certain bacterial regulatory systems, particularly those associated with nitrogen metabolism. Activation of 54 -RNA polymerase employs specialized bacterial enhancer-binding proteins whose activating function requires nucleotide hydrolysis (94,96,122) (Fig. 1). In this system, initiation rates are controlled via regulation of the DNA melting step that is necessary for establishing the open promoter complex (85,94,97). Bacterial enhancer-dependent transcription can be studied with just two purified proteins (an activator and the 54 -RNA polymerase holoenzyme) and the appropriate DNA template, facilitating progress in understanding mechanistic aspects of 54 functioning. Below we review the biology and biochemistry of the 54 -RNA polymerase.

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Section: Domain Structure Promoter Recognition and Core Interactionsmentioning

confidence: 99%

The Bacterial Enhancer-Dependent ς⁵⁴(ς^N) Transcription Factor

et al. 2000

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“…Deletions within region II appear to reduce the rate of open-complex formation (29). Like the region I-deleted holoenzyme, holoenzyme containing 54 deleted for regions I and II binds promoter DNA and initiates transcription from a premelted heteroduplex template in the absence of enhancer-binding protein (2). The ⌬2-105 mutant cross-linked to DctD (⌬1-142) (Fig.…”

Section: Transcription Initiation Bymentioning

confidence: 99%

The Amino Terminus of Salmonella enterica Serovar Typhimurium ς ⁵⁴ Is Required for Interactions with an Enhancer-Binding Protein and Binding to Fork Junction DNA

Kelly

Hoover

2000

J Bacteriol

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(20,28).Interactions between 54 -holoenzyme and enhancer-binding proteins are transient, and little is known about the nature of these interactions. The C 4 -dicarboxylic acid transport protein D (DctD) is an enhancer-binding protein from Sinorhizobium meliloti that can be cross-linked to 54 and the ␤ subunit of RNA polymerase (16). Some mutant forms of DctD that fail to activate transcription also fail to cross-link to these subunits (27), suggesting that DctD contacts these subunits of 54 -holoenzyme to catalyze open-complex formation. To identify the region of 54 that interacts with DctD, we generated a series of truncated 54 proteins and assessed their abilities to cross-link with DctD.Deletion of 179 amino acid residues from the amino terminus of 54 disrupts cross-linking to DctD. The cross-linking reagent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) can cross-link Sinorhizobium meliloti DctD and Salmonella enterica serovar Typhimurium 54 (16). Sulfo-SMCC is a heterobifunctional cross-linking reagent that has a maleimide group and an N-hydroxysuccinimide ester group, which react preferentially with sulfhydryl groups and primary amines, respectively, and are linked by a relatively long and flexible spacer arm. Cys-307 of S. enterica serovar Typhimurium 54 is critical for cross-linking to DctD (16), presumably because it is surface exposed and reacts readily with the maleimide group of sulfo-SMCC. For the cross-linking experiments in this study, we used DctD (⌬1-142) , which is a truncated, constitutively active form of DctD (17). As in previous studies, these cross-linking assays were done with purified 54 proteins and DctD (⌬1-142) in the absence of core RNA polymerase and DNA (16, 27).We initially generated three amino-terminally truncated 54 proteins that had deletions of residues 2 to 56 (⌬2-56), 2 to 105 (⌬2-105), and 2 to 179 (⌬2-179) (Fig. 1). All deletions were generated by using PCR to introduce a unique NdeI site at the desired position of rpoN, the gene encoding 54 . The NdeI site was used to clone the truncated rpoN alleles into the expression vector pCyt3 (provided by Elliot Altman, University of Georgia), which placed the rpoN alleles under the control of the Escherichia coli lac promoter/operator. The rpoN alleles were also cloned into the expression vector pET-28a(ϩ) (Novagen), which resulted in attachment of a hexahistidine tag at the amino terminus of each truncated 54 protein. Hexahistidine-tagged 54 proteins were overexpressed in E. coli BL21 (DE3) [F Ϫ ompT (lon) hsdS B gal DE3::lacI lacUV5-gene 1 (T7 polymerase)] by growing the cells in Luria-Bertani broth supplemented with 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG). Cells were harvested, resuspended in 50 mM Tris-acetate (pH 8.2)-200 mM KCl-1 mM EDTA-1 mM dithiothreitol, and lysed in a French pressure cell at 12,000 lb/in 2 . Like the full-length hexahistidine-tagged 54 protein (15), the truncated hexahistidine-tagged 54 proteins were in the insoluble fraction following centrifugation of the cell ...

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“…When the amino-terminal domain (Region I) is deleted, 54 can still bind RNA polymerase and direct it to DNA (14,21,22). However, the bound holoenzyme fails to respond to activator and form a stable open complex that can initiate transcription.…”

Section: Control Of Transcription Frequently Involvesmentioning

confidence: 99%

Functions of the ς54 Region I in Trans and Implications for Transcription Activation

Gallegos

Cannon

Buck

1999

Journal of Biological Chemistry

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Control of transcription frequently involvesEscherichia coli RNA polymerase (RNAP) 1 is a multisubunit enzyme consisting of an exchangeable subunit and a core enzyme (␣ 2 ␤␤Ј) (1). There are a variety of E. coli factors that are expressed in response to different growth conditions and environmental stresses which regulate the expression of genes accordingly. The type of factor associated with the core alters the sequence specificity of DNA binding and directs the holoenzyme to different classes of promoters (2, 3). The major protein in E. coli is 70 , which produces most of the cellular mRNA.54 is a minor factor that is not a member of the 70 family of proteins (4) and has a distinct mechanism of transcription initiation. 54 -Holoenzyme binds promoters in a transcriptionally inactive state, forming stable closed complexes (5-7). Isomerization of the closed complex to a transcriptionally competent open complex requires an activator protein bound to a DNA sequence with enhancer-like properties (5, 8) and nucleotide hydrolysis by the activator protein (6, 9, 10). Activation appears to involve direct contact between the activator and the holoenzyme (11-13). Isomerization from the closed complex to the open complex is believed to involve a major conformational change in the holoenzyme to reveal single-strand DNA binding determinants as one step and DNA melting to reveal the template strand (14, 15) as a second step. The pathway to stable DNA melting within the holoenzyme appears to involve at least one unstable intermediate and to be driven by nucleotide hydrolysis by the activator (15).The functional domain organization of 54 is complex, but different activities reside in different sequences: DNA binding motifs are localized in the carboxyl-terminal region, the central domain is needed to bind polymerase, and amino-terminal sequences are required for proper regulation of activation (16 -20). When the amino-terminal domain (Region I) is deleted, 54 can still bind RNA polymerase and direct it to DNA (14,21,22). However, the bound holoenzyme fails to respond to activator and form a stable open complex that can initiate transcription. If stably or transiently melted DNA is used, the holoenzyme with Region I deleted can produce transcripts, showing that its catalytic activity is intact (14,22). This transcript is unusual in that it results from heparin-sensitive transcription, and its production is not enhanced by the addition of activator, implying that the amino terminus of 54 contains essential activator response determinants.Region I of 54 is closely implicated in polymerase isomerization and DNA melting and is a major determinant of enhancer responsiveness. Current models suggest that Region I contains determinants for nucleating DNA melting, for inhibiting polymerase isomerization, and for the interaction of holoenzyme with melted DNA (14,15,(22)(23)(24). Some properties of the holoenzyme that depend upon Region I sequences likely rely upon the interaction that Region I makes with the core RNAP subunits (17) To e...

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Characterisation of holoenzyme lacking sigmaN regions I and II

Cited by 19 publications

References 0 publications

The Bacterial Enhancer-Dependent ς⁵⁴(ς^N) Transcription Factor

The Bacterial Enhancer-Dependent ς⁵⁴(ς^N) Transcription Factor

The Amino Terminus of Salmonella enterica Serovar Typhimurium ς ⁵⁴ Is Required for Interactions with an Enhancer-Binding Protein and Binding to Fork Junction DNA

Functions of the ς54 Region I in Trans and Implications for Transcription Activation

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Characterisation of holoenzyme lacking sigmaN regions I and II

Cited by 19 publications

References 0 publications

The Bacterial Enhancer-Dependent ς54(ςN) Transcription Factor

The Bacterial Enhancer-Dependent ς54(ςN) Transcription Factor

The Amino Terminus of Salmonella enterica Serovar Typhimurium ς 54 Is Required for Interactions with an Enhancer-Binding Protein and Binding to Fork Junction DNA

Functions of the ς54 Region I in Trans and Implications for Transcription Activation

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The Bacterial Enhancer-Dependent ς⁵⁴(ς^N) Transcription Factor

The Bacterial Enhancer-Dependent ς⁵⁴(ς^N) Transcription Factor

The Amino Terminus of Salmonella enterica Serovar Typhimurium ς ⁵⁴ Is Required for Interactions with an Enhancer-Binding Protein and Binding to Fork Junction DNA