Bryce Nickels Assistant Professor Rutgers University Department of Genetics Waksman Institute. Room 335 Piscataway. NJ 08854-0759 (732) 445-6852 FAX - 5735 bnickels@waksman.rutgers.edu

Mechanism of transcription, regulation of bacterial gene expression

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.
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.  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.  

Listed below are areas of particular interest:

• Factors that regulate the post-initiation activities of RNA polymerase

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 elongation and transcription termination we study regulatory factors that affect these steps in the transcription process.

• 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.  These findings have raised many questions that we are currently addressing including: 1) What are the functional roles of the σ subunit during transcription elongation? and 2) How can the association of σ with the elongation complex be influenced by regulatory factors?  

• 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 are using our knowledge of what surfaces of RNAP are likely targets of regulation to search for previously unknown regulatory factors that contact these surfaces.  To do this, we are employing a bacterial two-hybrid assay, 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.

• 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.

Selected Publications

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

Deaconescu AM, Chambers AL, Smith AJ, Nickels BE, Hochschild A, Savery NJ and Darst SA.  (2006) Structural basis for bacterial transcription-coupled DNA repair.  Cell 124:507-520.

Nickels BE, Garrity SJ, Mekler V, Minakhin L, Severinov K, Ebright RH, and Hochschild A. (2005) 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. 102:4488-93.

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

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

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

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

Jain D, Nickels BE, Sun L, Hochschild A, and Darst SA. (2004)  Structure of a ternary transcription activation complex.  Mol. Cell 13:45-53.

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

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

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

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