Our lab studies transcription, the first step in gene expression, whereby the genetic information coded in the DNA is utilized for the synthesis of RNA.  Most regulation of gene expression occurs at the level of transcription.  Transcription in all cells is carried out by multisubunit RNA polymerases (RNAPs) that are conserved in sequence, structure and function from bacteria to humans.  Thus, a fundamental understanding of the diverse mechanisms employed by the bacterial cell to regulate RNAP function is important for understanding gene regulation in all organisms.  In addition, principles that emerge from investigations of the transcription apparatus and its regulation in bacterial systems permit development of new strategies to control microbial pathogens.
   We utilize genetic and biochemical approaches to gain a detailed mechanistic understanding of the processes underlying the regulation of gene expression at the level of transcription.  To facilitate our studies of transcription and its regulation we exploit the relative simplicity of the bacterial system and the power of bacterial genetics.

Bacterial RNAP

Bacteria contain a single RNAP core enzyme (subunit composition α2ββ'ω), which contains all of the catalytic machinery required for the synthesis of RNA from nucleotides.  This core enzyme combines with a σ factor to form the holoenzyme, which is competent for promoter-specific transcription initiation.  The core enzyme adopts a structure reminiscent of a crab claw (See Figure). The large β and β' subunits compose the bulk of the ≈380-kDa total molecular mass of the core RNAP and form the enzyme's main channel.  This channel accommodates the DNA template, the RNA-DNA hybrid that forms during transcription, and houses the enzyme's active center marked by a stably bound catalytic Mg2+ ion.  Although the eukaryotic enzymes consist of a dozen or more subunits, among these are subunits corresponding to β,β', α, and ω that form a structural scaffold that is remarkably congruent with the bacterial enzyme (See Figure).  Because of its simpler composition and the highly developed genetic tools available in bacteria, the bacterial RNAP provides a powerful model for studying the function of all multisubunit RNAPs.

In bacteria, the transcription process can be divided into a number of distinct steps. First, the RNAP holoenzyme binds to duplex promoter DNA to form the closed RNAP-promoter complex. Next a series of conformational changes leads to the formation of the initiation-competent open complex in which the DNA is locally melted to expose the transcription start site. RNAP can then initiate transcription, directing the synthesis of short abortive RNA products that are repetitively released and re-synthesized before RNAP breaks its contacts with the promoter and escapes into productive elongation. Elongation itself is not a monotonous and uninterrupted process; rather the transcription elongation complex encounters various pause sites and potential arrest sites at which the nascent RNA remains stably bound to the enzyme. These pause and arrest sites must be overcome in order for the elongating RNAP to complete the synthesis of the full-length transcript. Finally, upon reaching a termination site, RNAP releases the RNA transcript and dissociates from the DNA. Transcription can be regulated during initiation, elongation, and termination by an enormous variety of regulatory factors that are either recruited to specific promoters or genes by sequences in the DNA or the RNA, or bound by RNAP in a manner that does not depend on any DNA or RNA sequence determinants.

Listed below are areas of particular interest:

1. Factors that regulate transcription termination: molecular mechanism and role in virulence gene expression

A large body of work has provided a detailed picture of the mechanistic steps that underlie transcription initiation and its regulation.  In contrast, relatively little is known about the mechanistic steps that underlie the later steps in the process.  To gain a better understanding of the fundamental processes underlying transcription termination and its regulation we study regulatory factors that affect the termination properties of RNAP.

One regulator of particular interest to our lab is the bacteriophage λ Q antiterminator protein (λQ).  λQ associates with RNAP in the context of an elongation complex and prevents RNAP from terminating transcription.  Because λQ can function to prevent termination in vitro in the absence of accessory factors, it provides an especially tractable system for probing the mechanisms that underlie termination.  Recently we have obtained evidence for interaction between λQ and a domain of the β subunit of RNAP known as the “β flap”.  Biochemical evidence has previously implicated the β flap in modulating transcription termination.  Therefore, the interaction between λQ and the β flap likely represents a critical determinant of λQ’s ability to function as a transcription antiterminator.  Currently, we are investigating the mechanistic role of the λQ/β flap interaction in the process of λQ-dependent antitermination.

In addition to our studies with λQ we are also interested characterizing the Q proteins of several related lambdoid phages.  In particular, we are most interested in the characterization of Q proteins associated with prophages present in Shiga toxin-producing Escherichia coli (STEC).  STEC is the causative agent of severe foodborne illness in humans.  Previous work has indicated that expression of Shiga toxin, an important virulence factor, is controlled by the Q proteins residing on prophages present within the STEC genome.  Thus, characterization of these Q proteins may ultimately lead to the development of new therapeutic strategies to combat STEC. 

2. Post-initiation roles of the sigma subunit

Bacterial RNAP holoenzyme consists of a catalytic core enzyme complexed with a σ factor.  σ factors confer on the core enzyme the ability to initiate transcription at specific promoters.  Up until relatively recently, the prevailing view was that the σ subunit is released from the transcription complex during the transition from initiation to elongation.  Thus, it was believed that the functional roles of the σ subunit were limited to transcription initiation. However, several lines of evidence have challenged this notion and indicated that σ not only can remain associated with the elongating RNAP, but can also play functional roles during transcription elongation.  In addition, a new model (based on recent structural and biochemical data) that describes how σ’s interaction with the core enzyme changes during the transition from initiation to elongation is emerging.  According to this model, the extension of the nascent RNA during the earliest stages of transcription mediates a stepwise, staged displacement of discrete segments of σ from core.  This model does not require the complete release of σ from the transcription complex during elongation, but rather, requires the disruption of particular σ/core contacts, which play functional roles during transcription initiation, leading to a partial release of σ from the transcription complex during the transition from initiation to elongation.  Furthermore, it has also been demonstrated in vitro that free σ can bind to a transcription elongation complex.
These findings have raised many questions that we are currently addressing.  What are the functional roles of the σ subunit during transcription elongation?  How can the association of σ with the elongation complex be influenced by regulatory factors?  Does the partial release of σ confer distinct functional properties on the holoenzyme during elongation?  

3. Identification of RNAP interacting proteins

High-resolution structures of RNAP coupled with biochemical analysis have highlighted a number of discrete domains of RNAP that play functional roles during the various stages of the transcription cycle.  Given the large number of proteins with unknown functions in bacteria, it is likely that there are unidentified factors that regulate gene expression through contact with these functionally important domains of RNAP.  We will use our knowledge of what surfaces of RNAP are likely targets of regulation to search for previously unknown regulatory factors that contact these surfaces.  We are employing a bacterial two-hybrid assay (See Figure), using structurally, biochemically and genetically defined domains of RNAP implicated in various stages of the transcription cycle as “bait”, and screening genomic libraries for factors that contact these domains of RNAP.



This two hybrid based approach is advantageous compared to proteomic approaches (i.e. immunoprecipitation and mass spectrometry analysis) for several reasons.  First, in contrast with proteomic approaches, which can only identify those proteins that are synthesized under a particular growth condition, the use of genomic libraries allows all of the proteins in the genome to be sampled.  Second, the two-hybrid assay is sensitive enough to detect weak or transient interactions that may not survive biochemical manipulation.  Third, we will know what surface of RNAP is contacted by the putative regulatory factor and, in addition, have an assay allowing for genetic dissection of the interaction between the factor and RNAP, expediting the isolation of informative amino acid substitutions in RNAP and/or the putative regulatory factor.  We are also using this approach to identify regulatory factors in clinically relevant pathogens such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Staphylococcus aureus.

4. Role of abortive RNAs in gene expression

During transcription initiation, prior to escape into productive elongation, RNA polymerase (RNAP) repetitively synthesizes and releases abortive RNA products.  Abortive RNAs are small, ranging in length from 2 to 15 nucleotides and are produced in vitro by bacterial RNAP, archaeal RNAP, and eukaryotic RNAP I, RNAP II, and RNAP III.  However, due to the small size of abortive RNAs, it has not been directly demonstrated that abortive RNAsare produced in vivo, and, correspondingly, it has not yet been determined whether abortive RNAs play regulatory roles in vivo.  We are using methods designed to facilitate detection of small RNAs to investigate the production of abortive RNAs in vivo and are using complementary genetic approaches to examine whether abortive RNAs represent a new class of small regulatory RNAs.

Publications

1. Nickels BE, Roberts CW, Roberts JW and Hochschild A. RNA-mediated destabilization of the σ70 region 4/β flap interaction facilitates engagement of RNA polymerase holoenzyme by the Q antiterminator. Mol. Cell (2006) 24, 457-468.


2. Deaconescu AM, Chambers AL, Smith AJ, Nickels BE, Hochschild A, Savery NJ and Darst SA. Structural Basis for Bacterial Transcription-Coupled DNA Repair. Cell (2006) 124, 507-520.


3. Nickels BE, Garrity SJ, Mekler V, Minakhin L, Severinov K, Ebright RH, and Hochschild A. The interaction between σ70 and the β-flap of Escherichia coli RNA polymerase inhibits extension of nascent RNA during early elongation. Proc. Natl. Acad. Sci. U.S.A. (2005) 102, 4488-93.


4. Gregory BD, Nickels BE, Darst SA, and Hochschild A. An altered-specificity DNA-binding mutant of Escherichia coli σ70 facilitates the analysis of σ70 function in vivo. Mol. Microbiol. (2005) 56, 1208-1219.


5. Nickels BE and Hochschild A. Regulation of RNA Polymerase through the Secondary Channel. Cell (2004) 118, 281-284.


6. Nickels BE, Mukhopadhyay J, Garrity SJ, Ebright RH, and Hochschild A. The σ70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter. Nat. Struct. Mol. Biol. (2004) 11, 544-550.


7. Gregory BD, Nickels BE, Garrity SJ, Severinova E, Minakhin L, Bieber Urbauer RJ, Urbauer JL, Heyduk T, Severinov K, and Hochschild A. A regulator that inhibits transcription by targeting an inter-subunit interaction of the RNA polymerase holoenzyme. Proc. Natl. Acad. Sci. U.S.A. (2004) 101, 4554-4559.


8. Jain D, Nickels BE, Sun L, Hochschild A, and Darst SA. Structure of a Ternary Transcription Activation Complex. Mol. Cell (2004) 13, 45-53.


9. Nickels BE, Dove SL, Murakami KS, Darst SA, and Hochschild A. Protein-protein and protein-DNA interactions of σ70 region 4 involved in transcription activation by λcI. J. Mol. Biol. (2002) 324, 17-34.


10. Nickels BE, Roberts CW, Sun H, Roberts JW, and Hochschild A. The σ70 subunit of RNA polymerase is contacted by the λQ antiterminator during early elongation. Mol. Cell (2002) 10, 611-622.


11. Pande S, Makela A, Dove SL, Nickels BE, Hochschild A, and Hinton DM. The bacteriophage T4 transcription activator MotA interacts with the far C-terminal region of the σ70 subunit of Escherichia coli RNA polymerase. J. Bacteriol. (2002) 184, 3957-3964.


12. Kuznedelov K, Minakhin L, Niedziela-Majka A, Dove SL, Rogulja D, Nickels BE, Hochschild A, Heyduk T, and Severinov K. A role for interaction of the RNA polymerase flap domain with the σ subunit in promoter recognition. Science (2002) 295, 855-857.