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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.

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.

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").

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.