Nickels Laboratory

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?

nanoRNA-mediated priming of transcription initiation

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.

Role of abortive RNAs in gene expression

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

Recent Publications

Bao, X, Nickels BE, Fan H. 2012. Chlamydia trachomatis protein GrgA activates transcription by contacting the nonconserved region of sigma66. Proc Natl Acad Sci U S A. Abstractproceedings_of_the_national_academy_of_sciences_2012_bao.pdfWebsite

The bacterial RNA polymerase holoenzyme consists of a catalytic core enzyme in complex with a sigma factor that is required for promoter-specific transcription initiation. Primary, or housekeeping, sigma factors are responsible for most of the gene expression that occurs during the exponential phase of growth. Primary sigma factors share four regions of conserved sequence, regions 1-4, which have been further subdivided. Many primary sigma factors also contain a nonconserved region (NCR) located between subregions 1.2 and 2.1, which can vary widely in length. Interactions between the NCR of the primary sigma factor of Escherichia coli, sigma(70), and the beta' subunit of the E. coli core enzyme have been shown to influence gene expression, suggesting that the NCR of primary sigma factors represents a potential target for transcription regulation. Here, we report the identification and characterization of a previously undocumented Chlamydia trachomatis transcription factor, designated GrgA (general regulator of genes A). We demonstrate in vitro that GrgA is a DNA-binding protein that can stimulate transcription from a range of sigma(66)-dependent promoters. We further show that GrgA activates transcription by contacting the NCR of the primary sigma factor of C. trachomatis, sigma(66). Our findings suggest GrgA serves as an important regulator of sigma(66)-dependent transcription in C. trachomatis. Furthermore, because GrgA is present only in chlamydiae, our findings highlight how nonconserved regions of the bacterial RNA polymerase can be targets of regulatory factors that are unique to particular organisms.

Berdygulova, Z, Esyunina D, Miropolskaya N, Mukhamedyarov D, Kuznedelov K, Nickels BE, Severinov K, Kulbachinskiy A, Minakhin L. 2012. A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing.. Nucleic acids research. Abstractnucleic_acids_res_2012_berdygulova.pdf

Gp39, a small protein encoded by Thermus thermophilus phage P23-45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity.

Vvedenskaya, IO, Sharp JS, Goldman SR, Kanabar PN, Livny J, Dove SL, Nickels BE. 2012. Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs. Genes Dev. 26(13):1498-1507. Abstractgenes_dev_2012_vvedenskaya.pdf

Prokaryotic and eukaryotic RNA polymerases can use 2- to ~4-nt RNAs, ‘‘nanoRNAs,’’ to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.

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Nickels Laboratory

Dr. Bryce E. Nickels

Principal Investigator

Recent Publications