Unprecedented High-Resolution View of Bacterial Operon Architecture Revealed by RNA Sequencing

Abstract

We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3′ transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5′ ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread.

Whole-genome mapping of 5′ RNA ends in bacteria by tagged sequencing: a comprehensive view in Enterococcus faecalis

“…in line with previous reports highlighting that the information provided by annotations of bacterial genomes on their gene content remains incomplete (Toledo-Arana et al 2009;Albrecht et al 2010;Irnov et al 2010;Sharma et al 2010;Mitschke et al 2011;Wurtzel et al 2012;Conway et al 2014;Thomason et al 2015).…”

“…This lower number compared with E. faecalis can be explained by the smaller number of reads obtained from this sequencing experiment (see Supplemental Material, Section S2) and the 40% larger genome of E. coli, out of which ∼33% (1.55 Mbp) appear to be transcribed in our experiment (coverage higher than 2×). Out of those 397 TSSs, 314 (79%) were found within 2 bp of a TSS reported in at least one of the three contributions described above (113 in Mendoza-Vargas et al [2009], 218 in Conway et al [2014], and 285 in Thomason et al [2015]). …”

“…Unlike in E. faecalis, transcription start sites in E. coli have been extensively studied and TSSs have been mapped in genome-wide manner in at least three separate contributions: Mendoza-Vargas et al (2009) reported TSSs for ∼1000 (∼23%) of the ∼4500 ORFs in E. coli MG1655, Conway et al (2014) mapped with high accuracy about 2100 promoters, and Thomason et al (2015) detected over 14,000 TSSs using dRNA-seq.…”

“…Yet, their functional and regulatory insights remain incomplete as primary and processed RNAs cannot be distinguished and hence transcriptional (RNA synthesis) and post-transcriptional processes (RNA processing and stability) cannot be separated. The use of differential RNA-seq (dRNA-seq), an astute method that enriches an RNA population for primary transcripts, partially overcomes these limitations and gives access to the primary transcriptome (Albrecht et al 2010;Bohn et al 2010;Irnov et al 2010;Sharma et al 2010;Conway et al 2014; Thomason et al 2015). Yet, a major limitation of dRNA-seq is that all transcripts cannot be detected in a single experiment as they are degraded by a 5 ′ -phosphate-dependent exonuclease, and thus information on post-transcriptional events is lost .…”

Inefficient translation of nsrR constrains behaviour of the NsrR regulon in Escherichia coli

“…Apparent control of poxB by NsrR results from read-through transcription from the upstream hcp-hcr genes and is, therefore, superimposed on mRNA synthesis from the NsrRindependent poxB promoter (Chang et al, 1994). Recent measurements show that only a small proportion of transcriptional terminators in E. coli can be considered strong (Chen et al, 2013), and there is evidence of pervasive read-through of terminators (Conway et al, 2014). The fact that we can measure changes in pyruvate oxidase activity in response to NsrR suggests that read-through from hcp-hcr into poxB may be physiologically significant.…”