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:
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.
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?
It is presumed that, in vivo, the initiation of RNA synthesis by DNA-dependent RNAPs occurs using NTPs alone. However, it is well established that prokaryotic and eukaryotic RNAP can utilize 2- to ~8-nt RNAs to prime transcription initiation in vitro. In collaboration with Simon Dove (Harvard Medical School) we have demonstrated that depletion of the small-RNA-specific exonuclease, Oligoribonuclease, in Pseudomonas aeruginosa, causes the accumulation of 2- to ~4-nt “nanoRNAs”, which serve as primers for transcription initiation at a significant fraction of promoters. Widespread use of nanoRNAs to prime transcription initiation is coupled with global alterations in gene expression. These results, obtained under conditions in which the concentration of nanoRNAs is artificially elevated, establish that small RNAs can be used to initiate transcription in vivo, challenging the idea that all cellular transcription occurs using NTPs alone. These findings further suggest that nanoRNAs could represent a distinct class of functional small RNAs that can affect gene expression through direct incorporation into a target RNA transcript rather than through a traditional antisense-based mechanism. Current work is focused on determining to what extent nanoRNA-mediated priming of transcription occurs in bacterial cells under physiological growth conditions.
During transcription initiation, prior to escape into productive elongation, 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 been difficult to study the production of abortive RNAs 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