Mutational analysis of Escherichia coli GreA protein reveals new functional activity independent of antipause and lethal when overexpressed

There is a growing appreciation for the diverse regulatory consequences of the family of proteins that bind to the secondary channel of E. coli RNA polymerase (RNAP), such as GreA, GreB or DksA. Similar binding sites could suggest a competition between them. GreA is characterised to rescue stalled RNAP complexes due to its antipause activity, but also it is involved in transcription fidelity and proofreading. Here, overexpression of GreA is noted to be lethal independent of its antipause activity. A library of random GreA variants has been used to isolate lethality suppressors to assess important residues for GreA functionality and its interaction with the RNA polymerase. Some mutant defects are inferred to be associated with altered binding competition with DksA, while other variants seem to have antipause activity defects that cannot reverse a GreA-sensitive pause site in a fliC::lacZ reporter system. Surprisingly, apparent binding and cleavage defects are found scattered throughout both the coiled-coil and globular domains. Thus, the coiled-coil of GreA is not just a measuring stick ensuring placement of acidic residues precisely at the catalytic centre but also seems to have binding functions. These lethality suppressor mutants may provide valuable tools for future structural and functional studies.

Approximate Bayesian computation of transcriptional pausing mechanisms

At a transcriptional pause site, RNA polymerase (RNAP) takes significantly longer than average to transcribe the nucleotide before moving on to the next position. At the single-molecule level this process is stochastic, while at the ensemble level it plays a variety of important roles in biological systems. The pause signal is complex and invokes interplay between a range of mechanisms. Among these factors are: non-canonical transcription events – such as backtracking and hypertranslocation; the catalytically inactive intermediate state hypothesised to act as a precursor to backtracking; the energetic configuration of basepairing within the DNA/RNA hybrid and of those flanking the transcription bubble; and the structure of the nascent mRNA. There are a variety of plausible models and hypotheses but it is unclear which explanations are better.We performed a systematic comparison of 128 kinetic models of transcription using approximate Bayesian computation. Under this Bayesian framework, models and their parameters were assessed by their ability to predict the locations of pause sites in the E. coli genome.These results suggest that the structural parameters governing the transcription bubble, and the dynamics of the transcription bubble during translocation, play significant roles in pausing. This is consistent with a model where the relative Gibbs energies between the pre and posttranslocated positions, and the rate of translocation between the two, is the primary factor behind invoking transcriptional pausing. Whereas, hypertranslocation, backtracking, and the intermediate state are not required to predict the locations of transcriptional pause sites. Finally, we compared the predictive power of these kinetic models to that of a non-explanatory statistical model. The latter approach has significantly greater predictive power (AUC = 0.89 cf. 0.73), suggesting that, while current models of transcription contain a moderate degree of predictive power, a much greater quantitative understanding of transcriptional pausing is required to rival that of a sequence motif.Author summaryTranscription involves the copying of a DNA template into messenger RNA (mRNA). This reaction is implemented by RNA polymerase (RNAP) successively incorporating nucleotides onto the mRNA. At a transcriptional pause site, RNAP takes significantly longer than average to incorporate the nucleotide. A model which can not only predict the locations of pause sites in a DNA template, but also explain how or why they are pause sites, is sought after.Transcriptional pausing emerges from cooperation between several mechanisms. These mechanisms include non-canonical RNAP reactions; and the thermodynamic properties of DNA and mRNA. There are many hypotheses and kinetic models of transcription but it is unclear which hypotheses and models are required to predict and explain transcriptional pausing.We have developed a rigorous statistical framework for inferring model parameters and comparing hypotheses. By applying this framework to published pause-site data, we compared 128 kinetic models of transcription with the aim of finding the best models for predicting the locations of pause sites. This analysis offered insights into mechanisms of transcriptional pausing. However, the predictive power of these models lacks compared with non-explanatory statistical models - suggesting the data contains more information than can be satisfied by current quantitative understandings of transcriptional pausing.

Transcriptome-wide sites of collided ribosomes reveal principles of translational pausing

Translation initiation is the major regulatory step defining the rate of protein production from an mRNA. Meanwhile, the impact of non-uniform ribosomal elongation rates is largely unknown. Using a modified ribosome profiling protocol based on footprints from two closely packed ribosomes (disomes), we have mapped ribosomal collisions transcriptome-wide in mouse liver. We uncover that the stacking of an elongating onto a paused ribosome occurs frequently and scales with translation rate, trapping ∼10% of translating ribosomes in the disome state. A distinct class of pause sites, independent of translation rate, is indicative of deterministic pausing signals. Pause site association with specific amino acids, peptide motifs and nascent polypeptide structure, is suggestive of programmed pausing as a widespread mechanism associated with protein folding. Evolutionary conservation at disome sites indicates functional relevance of translational pausing. Collectively, our disome profiling approach allows unique insights into gene regulation occurring at the step of translation elongation.

Two transcription pause elements underlie a σ70-dependent pause cycle

The movement of RNA polymerase (RNAP) during transcription elongation is modulated by DNA-encoded elements that cause the elongation complex to pause. One of the best-characterized pause sequences is a binding site for the σ70 initiation factor that induces pausing at a site near lambdoid phage late-gene promoters. An essential component of this σ70-dependent pause is the elemental pause site (EPS), a sequence that itself induces transcription pausing throughout the Escherichia coli genome and underlies other complex regulatory pause elements, such as the ops and his operon pauses. Here, we identify and provide a detailed kinetic analysis of a transcription cycle analogous to abortive cycling that underlies the σ70-dependent pause. We show that, in σ70-dependent pausing, the elemental pause acts primarily to modulate the rate at which complexes attempt to disengage the σ70:DNA interaction. Our findings establish the σ70-dependent pause-encoding region as a multipartite element in which several pause-inducing components make distinct mechanistic contributions to the induction and maintenance of a regulatory transcription pause.