A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq.

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´.

Interactions between RNA polymerase and the "core recognition element" counteract pausing.

Transcription elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native elongating transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G₋10Y₋1G(+1) (where -1 corresponds to the position of the RNA 3' end). We demonstrate that sequence-specific interactions between RNAP core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a posttranslocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.

An RNA-seq method for defining endoribonuclease cleavage specificity identifies dual rRNA substrates for toxin MazF-mt3.

Toxin-antitoxin (TA) systems are widespread in prokaryotes. Among these, the mazEF TA system encodes an endoribonucleolytic toxin, MazF, that inhibits growth by sequence-specific cleavage of single-stranded RNA. Defining the physiological targets of a MazF toxin first requires the identification of its cleavage specificity, yet the current toolkit is antiquated and limited. We describe a rapid genome-scale approach, MORE (mapping by overexpression of an RNase in Escherichia coli) RNA-seq, for defining the cleavage specificity of endoribonucleolytic toxins. Application of MORE RNA-seq to MazF-mt3 from Mycobacterium tuberculosis reveals two critical ribosomal targets-the essential, evolutionarily conserved helix/loop 70 of 23S rRNA and the anti-Shine-Dalgarno (aSD) sequence of 16S rRNA. Our findings support an emerging model where both ribosomal and messenger RNAs are principal targets of MazF toxins and suggest that, as in E. coli, removal of the aSD sequence by a MazF toxin modifies ribosomes to selectively translate leaderless mRNAs in M. tuberculosis.