Projects

Mechanism and regulation of 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, such as the Q antiterminator protein, that affect these steps in the transcription process. 

We have also employed high-throughput methods for the analysis of transcription elongation, native-elongating-transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), to analyze transcription pausing genome-wide in Escherichia coli. Using these approaches we have identified key sequence determinants of transcriptional pausing and established that sequence-specific interactions between RNA polymerase core enzyme and a core recognition element (CRE) modulate pausing.

 

 

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? 

 

Primer-dependent transcription initiation

The first step in transcription, initiation, consists of a number of discrete steps that culminate in the RNAP-mediated catalysis of the first phosphodiester bond formed within the nascent RNA. It had long been presumed that, in living cells, the first phosphodiester bond within the nascent RNA is formed between two nucleoside triphosphate (NTP) substrates: “de novo initiation”. However, our studies have established that a significant fraction of transcription initiation in bacteria can occur via a mechanistically distinct pathway where the first phosphodiester bond within the nascent RNA is formed between a small 2- to ~4-nt RNA primer and an NTP: “primer-dependent initiation”. Although it has long been known that primer-dependent initiation can occur when the appropriate short RNA primers are provided to in vitro transcription reactions, our experiments were the first to demonstrate its occurrence in living cells.  Current work is focused on determining the full scope of primer-dependent initiation in vivo, the mechanisms allowing or restricting primer-dependent initiation in vivo, the specificity with which primer-dependent initiation is targeted to genes in vivo, and the mechanisms by which primer-dependent initiation alters the expression of those genes.

 

‘Epitranscriptomic’ modifications of RNA 5’ ends: NAD and CoA-capping.

The chemical nature of the 5' end of RNA is a key determinant of RNA stability, processing, localization, and translation efficiency and has been proposed to provide a layer of 'epitranscriptomic' gene regulation. Recently it has been shown that some bacterial RNA species carry a 5'-end structure reminiscent of the 5' 7-methylguanylate "cap" in eukaryotic RNA. In particular, RNA species containing a 5'-end nicotinamide adenine dinucleotide (NAD+) or 3'-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH), and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps. We have shown instead that NAD+, NADH, and dpCoA are incorporated into RNA during transcription initiation, by serving as non‑canonical initiating nucleotides (NCINs) for de novo transcription initiation by bacterial RNA polymerase (RNAP). In addition, we have identified key promoter sequence determinants for NCIN-mediated initiation, shown that NCIN-mediated initiation occurs in vivo, and shown that NCIN-mediated initiation has functional consequences by increasing RNA stability in vivo. We have further shown that NCIN-mediated initiation can occur with eukaryotic RNAP II, suggesting that NCIN-mediated "ab initio capping" may occur in all organisms.

Together with our work on primer-dependent initiation, our studies of NCIN-mediated initiation add to an emerging picture that NTPs are not the only substrates for transcription initiation in vivo. In current work, we are determining the full extent to which NCIN-mediated initiation impacts gene expression in bacterial cells and investigating the possibility that NCIN-mediated initiation provides a direct regulatory connection between metabolism and gene expression.

 

Development and application of high-throughput sequencing-based approaches for analysis of transcription.

During each phase of transcription, RNAP makes extensive interactions with nucleic acids and is responsive to sequence context. In addition, as each phase of transcription is a multi-step process, different steps during initiation, elongation, and termination can be rate limiting for different transcripts, and thereby serve as potential targets for regulation. Thus, predicting how a given transcription unit (i.e. promoter and transcribed region) will respond to alterations in conditions, and identifying the sequence determinants that dictate the response, represents an immense challenge. While structural studies have revealed some RNAP-nucleic acid interactions that modulate transcription, a full understanding of the relationship between nucleic acid sequence and functional output remains a fundamental gap in our knowledge. Thus, my lab seeks to leverage the capabilities of high-throughput sequencing technologies to address this knowledge gap. In this regard, we have developed experimental platforms for massively multiplexed transcriptomics, massively multiplexed protein-DNA crosslinking, and massively multiplexed DNA footprinting (termed "MASTER," "MASTER-XL," and "MASTER-FP," where "MASTER" denotes massively systematic transcript end readout, "XL" denotes crosslinking, and "FP" denotes footprinting).

In published work, we have used MASTER and MASTER-XL to define the sequence determinants and mechanism of transcription start site selection for E. coli RNAP. In current work, we are using MASTER, MASTER-XL, and MASTER-FP to analyze transcription elongation and termination for bacterial RNAP and to define the sequence determinants and mechanisms of transcription start site selection in eukaryotes. In principle, these approaches can be readily adapted to perform a comprehensive mechanistic dissection of any process involving nucleic acid interactions. Thus, although our current studies are focused on transcription, the technical innovations derived from our studies are likely to have wide-ranging applications across many areas of biology.