Proposed models for regulation of differential decay rates within the same transcript.

<p>(A), A model for stabilization of the upstream gene. The mature polycistronic transcript is protected from 3’-5’ exonucleases by the 3’ terminator hairpin structure. The relatively higher ribosome density over <i>gene A</i> provides protection from endonucleases, leading the RNase E to cleave the <i>gene B</i> segment, which results in the removal of the protective terminator structure, exposing <i>gene B</i> to rapid digestion by 3’-5’ exonucleases. <i>Gene A</i> remains protected by a 3’ RNA structure that blocks processive exonuclease activity. (B) A model for stabilization of the downstream gene. The mature operon is protected by the terminator structure. The relatively higher ribosome densities over <i>gene B</i> guide the initial cleavage to <i>gene A</i>. 3’-5’ exonucleases degrade <i>gene A</i> while <i>gene B</i> maintains its ribosome densities and protective terminator structure. Endonuclease cleavage occurs upstream of a protective 5’ structure that further protects the stable transcript from RNase E.</p

Protective RNA 3’ structures at the boundaries of differentially decaying transcript regions.

<p>(A), A model for differential decay in operons in which the 5’-most gene is preferentially stabilized, as described for the maltose operon [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007354#pgen.1007354.ref008" target="_blank">8</a>]. Scissors and Pac-Man represent endonucleases and 3’-5’ exonucleases, respectively. (B-D), Differential decay in three representative <i>E</i>. <i>coli</i> operons, depicted by normalized RNA-seq coverage in steady state (black, t = 0) or at two time points (green and red) following rifampicin treatment. Bar graphs show average half-life calculations with error bars representing standard deviation. RNA-seq coverage was normalized by the number of uniquely mapped reads in each library. RNA 3’ ends detected by term-seq are shown as black arrows with the height of the arrow representing the total number of supporting reads. The sequences present immediately upstream to the recorded 3’ ends were folded using RNAfold [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007354#pgen.1007354.ref035" target="_blank">35</a>] and are shown to the right of each coverage plot, with the estimated structure stability, measured in kcal/mol.</p

RNase E cleavage and protective 5’-end structures correlate with differential decay.

<p>(A), A model for differential decay in operons in which the middle or 3’-most gene is preferentially stabilized, as described for <i>papBA</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007354#pgen.1007354.ref012" target="_blank">12</a>]. Scissors and Pac-Man represent endonucleases and 3’-5’ exonucleases, respectively. (B-D), Differential decay in three representative <i>E</i>. <i>coli</i> operons, depicted by normalized RNA-seq coverage in steady state (black, t = 0) or an additional time points (green) following rifampicin treatment. RNA-seq coverage was normalized by the number of uniquely mapped reads in each library. The position and number of reads supporting RNase E cleavage sites in the WT strain or in the RNase E mutant are shown as dark and light orange arrows, respectively. The height of the arrows represents the total number of supporting 5’ end reads across all three replicates, normalized by the number of uniquely mapped reads in each experiment. The predicted structure and stability of the RNA sequence present immediately downstream of the RNase E cleavage site end is shown next to each gene, with blue rectangles specifying the position of the structure in the genome. The 5’ cleavage position is marked by a scissors cartoon.</p

Identification of differentially decaying operons in <i>E</i>. <i>coli</i>.

<p>(A-C), Differential decay in three representative <i>E</i>. <i>coli</i> operons, depicted by normalized RNA-seq coverage in steady state (black, t = 0) or at two time points (green and red) following rifampicin treatment. RNA-seq coverage was normalized by the number of uniquely mapped reads in each library. Bar graphs show average half-life calculations from three replicates with error bars representing standard deviation. (D), Ratio of steady-state mRNA abundance (blue) and mRNA half-lives (red) shown for a subset of regulated gene-pairs in which mRNA abundances and decay rates closely matched. Gene names are marked below the x-axis. Shown is the average ratio between the genes with error bars denoting standard deviation.</p

Ribosome density can guide differential operon decay.

<p>(A), Comparison of the relative ribosome densities across uniformly (n = 533) and differentially decaying operonic gene-pairs for which ribosome densities were previously measured (n = 39) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007354#pgen.1007354.ref003" target="_blank">3</a>] is shown as grey and green box-plots, respectively. Outliers are marked as red dots and the median is marked as a horizontal line within the box. The distributions were compared using a two-sided Wilcoxon rank-sum test (<i>p</i> < 10⁻⁸). On the left, a drawing illustrates the growing differences in ribosome densities between the genes along the boxplot’s y-axis. (B), Illustration of the effect of the translation-initiation inhibitor kasugamycin and its hypothesized effect on ribosome densities in polycistronic transcripts. (C), A scatter plot showing the change in relative decay rate of regulated gene-pairs calculated using recently published decay rates [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007354#pgen.1007354.ref022" target="_blank">22</a>] for control (x-axis) and kasugamycin treated (y-axis) bacteria (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007354#pgen.1007354.s008" target="_blank">S8 Table</a>). The y = x function (denoting 1:1 ratio) is shown as a dashed line.</p

The sequence similarity space of CRISPR repeats visualized with the BioLayout (Java) program 26

<p><b>Copyright information:</b></p><p>Taken from "Evolutionary conservation of sequence and secondary structures in CRISPR repeats"</p><p>Genome Biology 2007;8(4):R61-R61.</p><p>Published online 18 Apr 2007</p><p>PMCID:PMC1896005.</p><p></p> Dots denote individual repeat sequences; connecting lines represent Smith-Waterman similarities, such that closer dots represent more similar sequences. Dot colors denote cluster association as derived from MCL clustering. The 12 largest clusters are indicated by circles together with their sequence logos, coarse phylogenetic composition, and sample secondary structures where applicable

A Global Transcriptional Switch between the Attack and Growth Forms of <i>Bdellovibrio bacteriovorus</i>

<div><p><i>Bdellovibrio bacteriovorus</i> is an obligate predator of bacteria ubiquitously found in the environment. Its life cycle is composed of two essential phases: a free-living, non-replicative, fast swimming attack phase (AP) wherein the predator searches for prey; and a non-motile, actively dividing growth phase (GP) in which it consumes the prey. The molecular regulatory mechanisms governing the switch between AP and GP are largely unknown. We used RNA-seq to generate a single-base-resolution map of the <i>Bdellovibrio</i> transcriptome in AP and GP, revealing a specific "AP" transcriptional program, which is largely mutually exclusive of the GP program. Based on the expression map, most genes in the <i>Bdellovibrio</i> genome are classified as "AP only" or "GP only". We experimentally generated a genome-wide map of 140 AP promoters, controlling the majority of AP-specific genes. This revealed a common sigma-like DNA binding site highly similar to the <i>E. coli</i> flagellar genes regulator sigma28 (FliA). Further analyses suggest that FliA has evolved to become a global AP regulator in <i>Bdellovibrio</i>. Our results also reveal a non-coding RNA that is massively expressed in AP. This ncRNA contains a c-di-GMP riboswitch. We suggest it functions as an intracellular reservoir for c-di-GMP, playing a role in the rapid switch from AP to GP.</p></div

AP-specific small RNAs.

<p>AP-specific small RNAs.</p

RNA-seq data used in this study.

<p>RNA-seq data used in this study.</p

RT-PCR verification of mutually exclusive expression.

<p>Total RNA retrieved from AP or GP <i>B. bacteriovorus</i> HD100 during a synchronous predation of <i>E. coli</i> ML35 (0.5, 1 and 3 hrs post inoculation) was subjected to RT-PCR. Sixteen representative genes predicted by RNA-seq analysis to be AP-specific (left) or GP-specific (right) were amplified. Coli, control genomic DNA of <i>E. coli</i> ML35; AP, cDNA from AP cells; GP0.5, cDNA of GP cells 0.5 hr post inoculation; GP1, cDNA of GP cells 1 hr post inoculation; GP3, cDNA of GP cells 3 hrs post inoculation. DNA, <i>B. bacteriovorus</i> genomic DNA.</p