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Organized by unstructured motifs The high degree of conservation in protein sequences thought to be unstructured has hinted that these regions may have important biological functions. Although unstructured regions are widely viewed to be crucial for protein signaling, localization, and stability, their roles in many other settings have remained mysterious. Cermakova et al . discovered that prominent members of the transcription elongation machinery are linked through a network of interactions involving transcription elongation factor TFIIS N-terminal domains (TNDs) and conserved unstructured sequences called “TND-interacting motifs” (TIMs). The researchers found that mutation of a single TIM in a central organizing protein of this network abolished key protein interactions and induced widespread defects in transcription elongation dynamics. —DJ

International audience; Negative elongation factor (NELF) is a critical transcriptional regulator that stabilizes paused RNA polymerase to permit rapid gene expression changes in response to environmental cues. Although NELF is essential for embryonic development, its role in adult stem cells remains unclear. In this study, through a muscle-stem-cell-specific deletion, we showed that NELF is required for efficient muscle regeneration and stem cell pool replenishment. In mechanistic studies using PRO-seq, single-cell trajectory analyses and myofiber cultures revealed that NELF works at a specific stage of regeneration whereby it modulates p53 signaling to permit massive expansion of muscle progenitors. Strikingly, transplantation experiments indicated that these progenitors are also necessary for stem cell pool repopulation, implying that they are able to return to quiescence. Thus, we identified a critical role for NELF in the expansion of muscle progenitors in response to injury and revealed that progenitors returning to quiescence are major contributors to the stem cell pool repopulation.

Negative Elongation Factor (NELF) is a critical transcriptional regulator that works through stabilizing paused RNA Polymerase to permit rapid gene expression changes in response to environmental cues. Whilst NELF is essential for embryonic development, its role in adult stem cells remains unclear. Here, through a muscle stem cell-specific deletion, we show that NELF is required for efficient muscle regeneration and replenishing the stem cell pool. Mechanistic studies using PRO-seq, single cell trajectory analyses and myofiber cultures revealed that NELF works at a specific stage of muscle regeneration whereby it mediates massive expansion of muscle progenitors. Strikingly, transplantation experiments indicate that these progenitors are also necessary for stem cell pool repopulation implying they are able to return to quiescence. Thus, we identified a critical role for NELF in expansion of muscle progenitors in response to injury and revealed that progenitors returning to quiescence are major contributors to stem cell pool repopulation.

A systems-level view of cellular gene expression requires understanding the mechanistic principles governing each step of transcription. In this chapter, we describe a massively multiplexed method for the analysis of the relationship between nucleic acid sequence and transcription termed “MASTER,” for massively systematic transcript end readout. MASTER enables parallel measurements of transcription output from at least 410 (~1,000,000) individual template sequences in vitro and in vivo. MASTER involves constructing a DNA template library of barcoded sequences, generating RNA transcripts from the library during transcription in vitro or in vivo, and analyzing the relative abundance and 5′-end sequences of the RNA transcripts by high-throughput sequencing. MASTER provides a powerful, rapid, and versatile method to identify sequence determinants of each step of transcription and to define the mechanistic basis by which these sequence determinants dictate transcription output.

Pausing for control of gene expression Pausing during gene transcription can play a critical role in gene regulation. Vvedenskaya et al. mapped pause sites across the whole genome in actively growing Escherichia coli (see the Perspective by Roberts). Thousands of undocumented pause sites were identified across well-transcribed genes, allowing the definition of a consensus pause sequence that is dependent on specific interactions of RNA polymerase with the DNA template and nascent RNA transcript. Science , this issue p. 1285 ; see also p. 1226

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.

It is often presumed that, in vivo, the initiation of RNA synthesis by DNA-dependent RNA polymerases occurs using NTPs alone. Here, using the model Gram-negative bacterium Pseudomonas aeruginosa, we demonstrate that depletion of the small-RNA-specific exonuclease, Oligoribonuclease, causes the accumulation of oligoribonucleotides 2 to ∼4 nt in length, "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. Our 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 only NTPs. Our 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.

The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ70, can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ70 can bind in trans to TECs that emanate from either a σ70-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ70 to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ70-dependent promoters. DOI: http://dx.doi.org/10.7554/eLife.10514.001, eLife digest Proteins are made following instructions that are encoded by sections of DNA called genes. In the first step of protein production, an enzyme called RNA polymerase uses the gene as a template to make molecules of messenger ribonucleic acid (mRNA). This process—known as transcription—starts when RNA polymerase binds to a site at the start of a gene. The enzyme then moves along the DNA, assembling the mRNA as it goes. This stage of transcription is known as elongation and continues until the RNA polymerase reaches the end of the gene. In bacteria, RNA polymerase needs a family of proteins called sigma factors to help it identify and bind to the start sites associated with the genes that will be transcribed. In the well studied bacterium known as E. coli, the primary sigma factor that is required for transcription initiation on most genes is called sigma 70. Recent research has shown that sigma 70 also influences the activity of RNA polymerase during elongation. During this stage, the RNA polymerase and several other proteins interact to form a complex called the transcription elongation complex (or TEC for short). However, it is not clear how sigma 70 gains access to this complex: does it simply remain with RNA polymerase after transcription starts, or is it freshly incorporated into the TEC during elongation? Goldman, Nair et al. found that sigma 70 is able to incorporate into TECs during elongation and causes them to pause at specific sites in the gene. Sigma 70 can even incorporate into TECs on genes where transcription was initiated by a different sigma factor. These findings indicate that sigma 70 can directly influence the transcription of all genes, not just the genes with start sites that are recognized by this sigma factor. Goldman et al. also observed that in cells that were growing and dividing rapidly, the pauses that occurred due to sigma 70 associating with TECs were of shorter duration than those in cells that were growing slowly. This implies that the growth status of the cells modulates the pausing of RNA polymerase during transcription. In the future, it will be important to understand how much influence the primary sigma factor has on RNA polymerase during elongation in E. coli and other bacteria. DOI: http://dx.doi.org/10.7554/eLife.10514.002

Transcription initiation that involves the use of a 2- to ~4-nt oligoribonucleotide primer, “primer-dependent initiation,” (PDI) has been shown to be widely prevalent at promoters of genes expressed during the stationary phase of growth in Escherichia coli. However, the extent to which PDI impacts E. coli physiology, and the extent to which PDI occurs in other bacteria is not known. Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation. We further demonstrate using high-throughput sequencing of RNA 5′ ends (5′ RNA-seq) that PDI occurs in the pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V. cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the two organisms. In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T−1A+1 or G−1G+1 (where position +1 corresponds to the position of de novo initiation). Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent process that leads to the preferential generation of the linear dinucleotides 5´-UA-3´ and 5´-GG-3´., Author Summary Primer-dependent transcription initiation, PDI, refers to an alternative mechanism of transcription initiation whereby the first phosphodiester bond within the nascent RNA is formed between a 2- to ~4-nt RNA primer and an incoming nucleoside triphosphate. Although PDI has been shown to occur in E. coli, the impact of PDI on E. coli physiology, and the extent to which PDI occurs in other bacteria is unknown. Here we establish that PDI modulates the ability of E. coli to form biofilms, a surface attached community of bacteria encased in a polymeric matrix. We further describe a significantly improved RNA-seq based method for the detection of PDI in cells. Using this method we document the occurrence of PDI in the pathogenic bacterium Vibrio cholerae. We further show that the pattern of PDI in V. cholerae is identical to that observed in E. coli, suggesting that PDI in these two organisms may occur through a conserved process that produces identical populations of 2- to ~4-nt RNA primers. Our findings suggest PDI may be widespread in Gammaproteobacteria.

We provide a detailed protocol for preparing cDNA libraries suitable for high throughput sequencing that are derived specifically from the 5′ ends of RNA (5′ specific RNA-seq). The protocol describes how cDNA libraries for 5′ specific RNA-seq can be tailored to analyze specific classes of RNAs based upon the phosphorylation status of the 5′ end. Thus, the analysis of cDNA libraries generated by these methods provides information regarding both the sequence and phosphorylation status of the 5′ ends of RNAs. 5′ specific RNA-seq can be used to analyze transcription initiation and post-transcriptional processing of RNAs with single base pair resolution on a genome-wide level.

We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 4(7) (∼ 16,000) barcoded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields (massively systematic transcript end readout, "MASTER"). Using MASTER, we define full inventories of transcription start sites ("TSSomes") of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo; we define the TSS-region DNA sequence determinants for TSS selection, reiterative initiation ("slippage synthesis"), and transcript yield; and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion ("scrunching") and transcription-bubble contraction ("anti-scrunching").

Membrane transport systems play important roles in enabling the uptake of essential nutrients, ions, and metabolites, as well as the expulsion of toxic compounds, cell envelope macromolecules, secondary metabolites, and the end products of metabolism. Transporters also enable communication between cells and their environments and participate in energy generation and interconversion. Secondary active transporters use chemiosmotic energy in the form of transmembrane ion or solute electrochemical gradients to drive transport. Primary active transporters use chemical, electrical, or solar energy to drive transport. ATP hydrolysis provides the energy for the majority of chemically driven active transporters, but decarboxylation or methyltransfer can drive uptake or extrusion of solutes via other such systems. Whole-genome sequencing allows comparison of cellular processes such as membrane transport at the organismal level. B. subtilis encodes within its genome six recognized channel proteins, and these proteins belong to four distinct families. Two uncharacterized Trk family paralogues, YkrM and YubG, are present in B. subtilis. They may be K+/Na+ symporters. B. subtilis encodes a large number of efflux pumps responsible for the extrusion of drugs, metabolites, and a variety of natural products. Families of sugar-transporting permeases have been tabulated and discussed, and structure-function relationships of phosphotransferase system (PTS) permeases (also known as enzyme II complexes) have been reviewed in this chapter. Although only a small fraction of the recognized B. subtilis transporters have been functionally characterized, rational functional predictions have been made for a majority of these proteins.

Identifying Abortive Initiation During transcription initiation in vitro, the RNA polymerase enzyme typically engages in cycles of synthesis and release of short RNA transcripts (“abortive initiation”) before breaking interactions with promoter DNA and beginning transcription elongation. Using hybridization methods developed to detect microRNAs, Goldman et al. (p. 927 ) directly detected products of abortive initiation in bacterial cells in vivo. Abortive initiation increased when interactions between RNA polymerase and the promoter were strengthened or when transcription was prevented. Thus, products of abortive initiation may help to regulate gene expression.

Shigella flexneri , a gram-negative enteric pathogen, is unusual in that it contains two nonredundant paralogous genes that encode the myristoyl transferase MsbB (LpxM) that catalyzes the final step in the synthesis of the lipid A moiety of lipopolysaccharide. MsbB1 is encoded on the chromosome, and MsbB2 is encoded on the large virulence plasmid present in all pathogenic shigellae. We demonstrate that myristoyl transferase activity due to MsbB2 is detected in limited magnesium medium, but not in replete magnesium medium, whereas that due to MsbB1 is detected under both conditions. MsbB2 increases overall hexa-acylation of lipid A under limited magnesium conditions. Regulation of MsbB2 by magnesium occurs at the level of transcription and is dependent on the conserved magnesium-inducible PhoPQ two-component regulatory pathway. Direct hexanucleotide repeats within the promoter upstream of msbB2 were identified as a putative PhoP binding site, and mutations within the repeats led to diminished PhoP-dependent expression of a transcriptional fusion of lacZ to this promoter. Thus, the virulence plasmid-encoded paralog of msbB is induced under limited magnesium in a PhoPQ-dependent manner. PhoPQ regulates the response of many Enterobacteriaceae to environmental signals, which include modifications of lipid A that confer increased resistance of the organism to stressful environments and antimicrobial peptides. The findings reported here are the first example of gene duplication in which one paralog has selectively acquired the mechanism for differential regulation by PhoPQ. Our findings provide molecular insight into the mechanisms by which each of the two MsbB proteins of S. flexneri likely contributes to pathogenesis.

The Shigella actin assembly protein IcsA is removed from the bacterial surface by the protease IcsP. We show that decreased intracellular spreading of virK ::Tn 10 mutants is due in part to significant increases in IcsP and IcsP-mediated cleavage of IcsA and that IcsP expression is a critical determinant of Shigella virulence.

The Shigella outer membrane protease IcsP removes the actin assembly protein IcsA from the bacterial surface, and consequently modulates Shigella actin-based motility and cell-to-cell spread. Here, we demonstrate that IcsP expression is undetectable in mutants lacking either of two transcriptional activators, VirF and VirB. In wild-type Shigella spp., virB expression is entirely dependent on VirF; therefore, to circumvent this regulatory cascade, we independently expressed VirF or VirB in Shigella strains lacking both activators and measured both IcsP levels and transcription from the icsP promoter. Our results show that VirB significantly enhanced icsP transcription, even in the absence of VirF. In contrast, when VirF was induced in the absence of VirB, VirF had variable effects. The regulation of icsP is distinctly different from the regulation of the gene encoding its major substrate, icsA , which is activated by VirF and not VirB. We propose that the different pathways regulating icsA and icsP may be critical to the modulation of IcsA-mediated actin-based motility by IcsP.

We here describe all recognized established and putative transport proteins encoded within the genome of Bacillus subtilis. These fall into four classes of established transporter types: (1) channel proteins, (2) secondary active transporters, (3) primary active transporters, and (4) group translocators of the sugar-transporting phosphotransferase system (PTS). Additionally, some transporters are recognized that utilize an unknown mode of action or energy coupling mechanism. The secondary carriers (which represent the majority of Bacillus transporters) are subdivided according to substrate specificity and family association. Characteristics of the families as well as the individual transport systems are presented when sufficient information is available. The recognized transporters fall into 58 families including 4 channel types, 42 secondary carrier types, 3 primary carrier types, 4 PTS-types and 5 unknown types.

The crystal structure of RNA polymerase open complex (RPo) indicates that RNAP core interacts with the transcription-bubble nontemplate strand segment corresponding to positions −4 to +2 in which we designated it as core recognition element, CRE. In this study, we sought to investigate the sequence determinants, the recognition mechanism and the functional roles of CRE.To determine whether RNAP-CRE interactions are sequence-specific, we constructed all possible nucleotide substitutions at each CRE position, and assessed effects on RNAP-DNA interaction in equilibrium binding experiments and high-salt-induced-dissociation off-rate experiments. We conclude that RNAP CRE interactions are specific and that the consensus CRE is t/g-n-n-n-T-G.To identify individual RNAP amino acids that mediate specificity at CRE positions +2 and +1, we constructed single Ala substitutions of RNAP residues that contact CRE positions +2 and +1, and assessed effects on RNAP DNA interactions with promoter derivatives containing all possible nucleotide substitutions at CRE positions +2 and +1. We conclude that bR151, bD446, or bR451 mediate specificity at CRE position +2, and βW183 mediates specificity at CRE position +1.To define the structural basis of specificity at CRE positions +1 and +2, we determined crystal structures of RPo derivatives containing all possible nucleotide substitutions at CRE positions +2 and +1. We conclude that specificity at CRE positions +2 and +1 manifests itself not only in quantitative differences in binding thermodynamics and kinetics, but also in qualitative differences in structure.Our results establish that RNAP-CRE interactions are sequence specific. We propose that RNAP-CRE interactions contribute to the sequence specificity of promoter binding, promoter unwinding, promoter scrunching, and promoter escape during transcription initiation and also contribute to the sequence specificity of pausing during transcription elongation.

Figure 1 gives the official results for the Hughes Trainable Text Skimmer used for MUC3 (TTS-MUC3). TTS is a largely statistical system, using a K-Nearest Neighbor classifier with the output of a shallow parser as features. (See the System Summary section of this volume for a detailed description of TTS-MUC3). The performance, on a slot by slot basis is, therefore, what one might expect: the pure set fills such as "Incident Type" and "Category" have much better performance than the string fills such as "Human Target." In addition, we can see that "Incident Date" and "Incident Location," for which special code was written, have performance above that of the string fills.