Jay D. Gralla

The Bacterial Enhancer-Dependent ς54(ςN) Transcription Factor

The 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

DNA supercoiling promotes formation of a bent repression loop in lac DNA

Titration experiments on supercoiled lac DNA show that one repressor tetramer can bind simultaneously to the primary lac operator and to the very weak lac pseudo-operator, located 93 base-pairs apart. The formation of this complex is accompanied by the appearance of an extreme hypersensitive site in a five base-pair sequence located approximately midway between the operators. This remote sequence is hypersensitive to attack by two different chemical probes, dimethyl sulfate and potassium permanganate, the latter of which is a new probe for distorted DNA. We interpret these results in terms of a complex in which lac repressor holds two remote operators together in a DNA loop. The formation of this bent DNA loop requires negative DNA supercoiling. In vivo, both lac operators bind repressor even though the presence of multiple operator copies has forced the two operators to compete for a limited amount of repressor. This suggests that the operator and pseudo-operator have similar affinities for repressor in vivo. Such similar affinities were observed in vitro only when DNA supercoiling forced formation of a repression loop.

Escherichia coli promoter opening and −10 recognition: mutational analysis of σ70

The opening of specific segments of DNA is required for most types of genetic readout, including σ70‐dependent transcription. To learn how this occurs, a series of single point mutations were introduced into σ70 region 2. These were assayed for duplex DNA binding, DNA opening and DNA double strand–single strand fork junction binding. Band shift assays for closed complex formation implicated a series of arginine and aromatic residues within a minimal 26 amino acid region. Permanganate assays implicated two additional aromatic residues in DNA opening, known to form a parallel stack of the type that can accept a flipped‐out base. Substitution for either of these aromatics had no effect on duplex probe recognition. However, when a single unpaired −11 nucleotide is added to the probe, the mutants fail to bind appropriately to give heparin resistance. A model for DNA opening is presented in which duplex recognition by regions 2.3, 2.4 and 2.5 of sigma positions the pair of aromatic amino acids, which then create the fork junction required for stable opening.

Converting Escherichia coli RNA Polymerase into an Enhancer-Responsive Enzyme: Role of an NH2-Terminal Leucine Patch in σ54

The protein σ54 associates with Escherichia coli core RNA polymerase to form a holoenzyme that binds promoters but is inactive in the absence of enhancer activation. Here, mutants of σ54 enabled polymerases to transcribe without enhancer protein and adenosine triphosphate. The mutations are in leucines within the NH2-terminal glutamine-rich domain of σ54. Multiple leucine substitutions mimicked the effect of enhancer protein, which suggests that the enhancer protein functions to disrupt a leucine patch. The results indicate that σ54 acts both as an inhibitor of polymerase activity and as a receptor that interacts with enhancer protein to overcome this inhibition, and that these two activities jointly confer enhancer responsiveness.

All three elements of the lac pS promoter mediate its transcriptional response to DNA supercoiling

The supercoiling response of four closely related promoters was examined in vitro. It was found that changes in all three elements of the lac ps promoter, i.e. the -10 sequence, the -35 sequence, and the spacer length, alter the transcriptional response to DNA supercoiling. Thus, the promoter as a whole, not just the melted region, mediates the supercoiling response. It is proposed that DNA supercoiling changes the structure of the promoter DNA to a form that can be recognized by RNA polymerase and then easily melted. All four promoter variants tested show the same qualitative response to the introduction of DNA supercoiling; that is, transcription is increased compared to relaxed DNA. However, for three of the four promoters, the rate peaked at intermediate levels of supercoiling and declined at higher superhelicities. Each mutation was found to alter both the extent of stimulation that can be achieved and the amount of superhelicity associated with maximal stimulation. The trend is that the stronger promoters are stimulated less, and this maximal stimulation occurs on templates containing fewer superhelical turns. At the level of supercoiling that may pertain in vivo, changes in superhelicity would result in considerable differential changes in the strengths of these promoters.

Potassium glutamate as a transcriptional inhibitor during bacterial osmoregulation

Potassium glutamate accumulates upon hyper‐osmotic shock and serves as a temporary osmoprotectant. This salt leads to transcriptional activation of sets of genes that allow the cell to achieve long‐term adaptation to high osmolarity. The current experiments show that potassium glutamate also acts as an inhibitor of bulk cellular transcription. It can do so independent of the involvement of macromolecular repressors or activators by virtue of its ability to directly inhibit RNA polymerase binding to ribosomal promoters. Thus, potassium glutamate mediates a global transcription switch by acting differentially on RNA polymerase at sets of genomic promoters that differ in their built‐in direct response to this salt.

Osmo-Regulation of Bacterial Transcription via Poised RNA Polymerase

Adaptation to high-salt environments is critical for the survival of a wide range of cells, especially for pathogenic bacteria that colonize the animal gut and urinary tract. The adaptation strategy involves production of the salt potassium glutamate, which induces a specific gene expression program that produces electro-neutral osmolytes while inhibiting general sigma(70) transcription. These data show that in Escherichia coli potassium glutamate stimulates transcription by disengaging inhibitory polymerase interactions at a sigma(38) promoter. These occur in an upstream region that is marked by an osmotic shock promoter DNA consensus sequence. The disruption activates a poised RNA polymerase to transcribe. This transcription program leads to the production of osmolytes that are shown to have only minor effects on transcription and therefore help to restore normal cell function. An osmotic shock gene expression cycle is discussed.

Escherichia coli ribosomal RNA transcription: regulatory roles for ppGpp, NTPs, architectural proteins and a polymerase‐binding protein

Ribosomal RNA transcription can limit the rate of Escherichia coli growth and is subject to complex regulation. Somehow, the cell is able to sense the general nutritional environment and adjust rRNA transcription so that an appropriate number of ribosomes is produced. This review discusses the current state of affairs, including recent information about the involvement of two nucleotide regulators, two architectural protein regulators, one new co‐regulator and stalled ribosomes.

Transcription reinitiation rate: a special role for the TATA box.

Promoters need to specify both the timing of transcriptional induction and the amount of transcript synthesized. In order to explore each of these effects separately, in vitro assays for the level of active preinitiation complex formation and for the rate of continuous RNA production were done. The effects were found to be influenced differently by different promoter elements. A consensus TATA element had a very strong effect on the rate of continuous RNA production, whereas two types of activators were important primarily in forming active transcription preinitiation complexes. Consensus TATA promoters exhibited high rates of continuous transcription; they assembled active preinitiation transcription complexes slowly but then produced transcripts continuously at an approximately fivefold-higher rate. Initiator-containing TATA-less promoters produced continuous transcripts slowly. Point mutations in the TATA element led to lower levels of transcription by reducing the number of preinitiation complexes and amplifying this reduction by lowering the apparent reinitiation rate. The results allow understanding of the sequence diversity of promoter elements in terms of specifying separate controls over the sensitivity of gene induction and over the strength of the induced promoter.

TFIIH XPB mutants suggest a unified bacterial-like mechanism for promoter opening but not escape

DNA helicases open the duplex during DNA replication, repair and transcription. However, RNA polymerase II is the only member of its family with this requirement; RNA polymerases I and III and bacterial RNA polymerases open DNA without a helicase. In this report, characterization of XPB mutants indicates that its helicase activity is not used for RNA polymerase II promoter opening, which is instead driven by its ATPase activity. The mutants have parallels in σ54 bacterial transcription and this suggests a similar mode of opening DNA for both RNA polymerases, involving ATP-dependent enzyme conformational changes. Promoter escape is defective in these XPB mutants, suggesting that the XPB helicase acts as an ATP-driven motor to reorganize the tightly wrapped multiprotein eukaryotic preinitiation complex during the remodeling that precedes elongation and the coupling to RNA processing events.