Sergei Nechaev

RNA polymerase is poised for activation across the genome

Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter, followed by initiation of RNA synthesis and the transition to productive elongation. In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of genes. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region. To determine the importance of polymerase stalling for transcription regulation, we carried out a genome-wide search for Drosophila melanogaster genes with Pol II stalled within the promoter-proximal region. Our data show that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals. This finding indicates a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues.

RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo

It is widely assumed that the key rate-limiting step in gene activation is the recruitment of RNA polymerase II (Pol II) to the core promoter. Although there are well-documented examples in which Pol II is recruited to a gene but stalls, a general role for Pol II stalling in development has not been established. We have carried out comprehensive Pol II chromatin immunoprecipitation microarray (ChIP-chip) assays in Drosophila embryos and identified three distinct Pol II binding behaviors: active (uniform binding across the entire transcription unit), no binding, and stalled (binding at the transcription start site). The notable feature of the ∼10% genes that are stalled is that they are highly enriched for developmental control genes, which are either repressed or poised for activation during later stages of embryogenesis. We propose that Pol II stalling facilitates rapid temporal and spatial changes in gene activity during development.

Global Analysis of Short RNAs Reveals Widespread Promoter-Proximal Stalling and Arrest of Pol II in Drosophila

To Stall or Not to Stall Recent studies in mammals and Drosophila have shown that RNA polymerase II frequently stalls shortly after initiating messenger RNA synthesis and that this stalling is important for proper expression of genes. Although several protein factors that affect polymerase stalling are known, the role of DNA sequence in this process has remained unclear. Now Nechaev et al. (p. 335, published online 10 December) report that the initially transcribed sequences of many genes contain a signal that works like a stop sign for the elongating polymerase. Expression of genes may thus be regulated by a combination of a DNA signal that induces promoter-proximal stalling and protein factors that alter its duration. The initially transcribed sequence plays a key role in inducing polymerase stalling. Emerging evidence indicates that gene expression in higher organisms is regulated by RNA polymerase II stalling during early transcription elongation. To probe the mechanisms responsible for this regulation, we developed methods to isolate and characterize short RNAs derived from stalled RNA polymerase II in Drosophila cells. Significant levels of these short RNAs were generated from more than one-third of all genes, indicating that promoter-proximal stalling is a general feature of early polymerase elongation. Nucleotide composition of the initially transcribed sequence played an important role in promoting transcriptional stalling by rendering polymerase elongation complexes highly susceptible to backtracking and arrest. These results indicate that the intrinsic efficiency of early elongation can greatly affect gene expression.

NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly

The Negative Elongation Factor (NELF) is a transcription regulatory complex that induces stalling of RNA polymerase II (Pol II) during early transcription elongation and represses expression of several genes studied to date, including Drosophila Hsp70, mammalian proto-oncogene junB, and HIV RNA. To determine the full spectrum of NELF target genes in Drosophila, we performed a microarray analysis of S2 cells depleted of NELF and discovered that NELF RNAi affects many rapidly inducible genes involved in cellular responses to stimuli. Surprisingly, only one-third of NELF target genes were, like Hsp70, up-regulated by NELF-depletion, whereas the majority of target genes showed decreased expression levels upon NELF RNAi. Our data reveal that the presence of stalled Pol II at this latter group of genes enhances gene expression by maintaining a permissive chromatin architecture around the promoter-proximal region, and that loss of Pol II stalling at these promoters is accompanied by a significant increase in nucleosome occupancy and a decrease in histone H3 Lys 4 trimethylation. These findings identify a novel, positive role for stalled Pol II in regulating gene expression and suggest that there is a dynamic interplay between stalled Pol II and chromatin structure.

Pol II waiting in the starting gates: Regulating the transition from transcription initiation into productive elongation.

Proper regulation of gene expression is essential for the differentiation, development and survival of all cells and organisms. Recent work demonstrates that transcription of many genes, including key developmental and stimulus-responsive genes, is regulated after the initiation step, by pausing of RNA polymerase II during elongation through the promoter-proximal region. Thus, there is great interest in better understanding the events that follow transcription initiation and the ways in which the efficiency of early elongation can be modulated to impact expression of these highly regulated genes. Here we describe our current understanding of the steps involved in the transition from an unstable initially transcribing complex into a highly stable and processive elongation complex. We also discuss the interplay between factors that affect early transcript elongation and the potential physiological consequences for genes that are regulated through transcriptional pausing.

Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling

The kinetics and magnitude of cytokine gene expression are tightly regulated to elicit a balanced response to pathogens and result from integrated changes in transcription and mRNA stability. Yet, how a single microbial stimulus induces peak transcription of some genes (TNFα) within minutes whereas others (IP-10) require hours remains unclear. Here, we dissect activation of several lipopolysaccharide (LPS)-inducible genes in macrophages, an essential cell type mediating inflammatory response in mammals. We show that a key difference between the genes is the step of the transcription cycle at which they are regulated. Specifically, at TNFα, RNA Polymerase II initiates transcription in resting macrophages, but stalls near the promoter until LPS triggers rapid and transient release of the negative elongation factor (NELF) complex and productive elongation. In contrast, no NELF or polymerase is detectible near the IP-10 promoter before induction, and LPS-dependent polymerase recruitment is rate limiting for transcription. We further demonstrate that this strategy is shared by other immune mediators and is independent of the inducer and signaling pathway responsible for gene activation. Finally, as a striking example of evolutionary conservation, the Drosophila homolog of the TNFα gene, eiger, displayed all of the hallmarks of NELF-dependent polymerase stalling. We propose that polymerase stalling ensures the coordinated, timely activation the inflammatory gene expression program from Drosophila to mammals.

Stable Pausing by RNA Polymerase II Provides an Opportunity to Target and Integrate Regulatory Signals

Metazoan gene expression is often regulated after the recruitment of RNA polymerase II (Pol II) to promoters, through the controlled release of promoter-proximally paused Pol II into productive RNA synthesis. Despite the prevalence of paused Pol II, very little is known about the dynamics of these early elongation complexes or the fate of the short transcription start site-associated (tss) RNAs they produce. Here, we demonstrate that paused elongation complexes can be remarkably stable, with half-lives exceeding 15 min at genes with inefficient pause release. Promoter-proximal termination by Pol II is infrequent, and released tssRNAs are targeted for rapid degradation. Further, we provide evidence that the predominant tssRNA species observed are nascent RNAs held within early elongation complexes. We propose that stable pausing of polymerase provides a temporal window of opportunity for recruitment of factors to modulate gene expression and that the nascent tssRNA represents an appealing target for these interactions.

Promoter-proximal Pol II: When stalling speeds things up

Expression of genes was long thought to be regulated primarily at the level of RNA polymerase II (Pol II) recruitment to a gene promoter, and the dozen genes that did not fit this paradigm were regarded as exceptions. However, recent analyses of genome-wide Pol II distribution in Drosophila and mammalian systems have indicated that a large number of genes might be regulated at a step subsequent to Pol II recruitment, during early transcription elongation. At these genes, Pol II begins transcription but stalls after synthesizing a short RNA, and it is the release of this engaged Pol II from the promoter-proximal region that is rate limiting for transcription. Notably, promoter-proximal Pol II stalling is prevalent at genes involved in development and response to stimuli, suggesting that Pol II stalling during early elongation plays important roles in rapid and precise control of gene expression. Here we briefly summarize the current data on promoter-proximal Pol II stalling and discuss implications of this newly appreciated regulatory strategy.

Recombinant Thermus aquaticus RNA Polymerase, a New Tool for Structure-Based Analysis of Transcription

The three-dimensional structure of DNA-dependent RNA polymerase (RNAP) from thermophilic Thermus aquaticus has recently been determined at 3.3 A resolution. Currently, very little is known about T. aquaticus transcription and no genetic system to study T. aquaticus RNAP genes is available. To overcome these limitations, we cloned and overexpressed T. aquaticus RNAP genes in Escherichia coli. Overproduced T. aquaticus RNAP subunits assembled into functional RNAP in vitro and in vivo when coexpressed in E. coli. We used the recombinant T. aquaticus enzyme to demonstrate that transcription initiation, transcription termination, and transcription cleavage assays developed for E. coli RNAP can be adapted to study T. aquaticus transcription. However, T. aquaticus RNAP differs from the prototypical E. coli enzyme in several important ways: it terminates transcription less efficiently, has exceptionally high rate of intrinsic transcript cleavage, and is highly resistant to rifampin. Our results, together with the high-resolution structural information, should now allow a rational analysis of transcription mechanism by mutation.

The Elusive Object of Desire - Interactions of Bacteriophages and Their Hosts

Bacteria and their viruses (phages) are locked in an evolutionary contest, with each side producing constantly changing mechanisms of attack and defense that are aimed to increase the odds of survival. As a result, phages play central roles in a great variety of genetic processes and increase the rate of evolutionary change of the bacterial host, which could ultimately work to the benefit of the host in a long run.