Redefining fundamental concepts of transcription initiation in bacteria

Abstract

Despite enormous progress in understanding the fundamentals of bacterial gene regulation, our knowledge remains limited when compared with the number of bacterial genomes and regulatory systems to be discovered. Derived from a small number of initial studies, classic definitions for concepts of gene regulation have evolved as the number of characterized promoters has increased. Together with discoveries made using new technologies, this knowledge has led to revised generalizations and principles. In this Expert Recommendation, we suggest precise, updated definitions that support a logical, consistent conceptual framework of bacterial gene regulation, focusing on transcription initiation. The resulting concepts can be formalized by ontologies for computational modelling, laying the foundation for improved bioinformatics tools, knowledge-based resources and scientific communication. Thus, this work will help researchers construct better predictive models, with different formalisms, that will be useful in engineering, synthetic biology, microbiology and genetics.

Figure 1 |. RNAP holoenzyme (Eσ) intrinsic recognition by a sequence is neither necessary nor sufficient for that sequence to be a promoter.

a | σ⁵⁴ is divided into three conserved regions. Region I (not shown) comprises a domain that inhibits polymerase isomerization and initiation in the absence of activation and stimulates initiation in response to activation. Due to this inhibiting element, σ⁵⁴ requires an ATP-dependent activator (green oval). Region II (not shown) is variable and is implicated in DNA melting. Region III (brown oval) is the primary DNA-binding region and recognizes the −12 element and −24 element, to which an inactive Eσ⁵⁴ (not shown) binds. Transcription initiates upon binding of an ATP-dependent activator to a sequence called enhancer. Binding sites of Eσ⁵⁴ from which no transcription occurs, possibly because there is not an enhancer nearby, are not promoters. Thus, binding of the Eσ is not sufficient to define a promoter. To be a promoter, Eσ must bind and initiate transcription. b | A schematic representing the PRE promoter that does not have autonomous capability of binding Eσ. This promoter shows little to no interaction with the Eσ in the absence of activator cII (green bubble). When cII protein is bound to its site, which overlaps the −35 element, it compensates the lack of consensus of this element, thereby allowing the Eσ interaction at PRE and allowing transcription of the CI gene (brown box). Because there are promoters that are not bound autonomously by the Eσ, this is not a necessary feature in the definition of promoter.

Figure 2 |

Relative numbers of TSSs generated by two or more σ factors according to the information in RegulonDB.

Figure 3 |. Number of transcription factor binding sites without functional assignment versus number of transcription factor regulatory sites in E. coli.

The sum of transcription factor binding sites (TFBSs) without functional assignment (purple) and transcription factor regulatory sites (TFRSs; yellow) represent the complete set of TFBSs. The figure was drawn using data from published chromatin immunoprecipitation (ChIP) experiments and data from classical experiments^,–. Only TFs with non-functional sites found by highthroughput methodologies were included. A logarithmic scale is used to help visualize the disparate numbers of sites known for Fur and Cra and other TFs, such as DpiA and TrpR.

Figure 4 |. cis-regulatory architecture of the promoter deoCp2.

The line represents the deoCp2 promoter along with its transcription factor regulatory sites (TFRS). –35 and –10 elements and transcription start site (TSS; +1) are labelled, and the cis-regulatory architecture is represented by transcription factors (TFs; bubbles) bound to their corresponding TFRS. a | Cooperative regulatory interaction between CRP and CytR in the deoCp2 promoter. CytR is recruited as a corepressor by pre-bound CRP. Since CytR necessarily requires pre-bound CRP to repress expression of the transcription unit downstream, these four sites form a TFRS module. b | The TFRS collection of promoter deoCp2 is the complete set of TFRS known to regulate the deoCp2 promoter. Although there might be indirect regulatory interactions among DeoR, ModE, Fis, CRP and CytR, the only direct and necessary interaction is the one between CytR and CRP^–. The other TFs act independently under different conditions on deoCp2, each with their own proximal sites.

Figure 5 |. Transcription unit and operon schematic.

a | When different transcription start sites (TSSs) from the same promoter are not differentially regulated, they form a single transcription unit that is limited by, but not including, a single promoter and a single terminator. b | Schematic of the gadAXW complex operon. Several internal promoters and terminators enable different sets of genes to be co-transcribed in different combinations: gadAX by the gadAp promoter; gadX and gadXW by the gadXp promoter, and gadW by the gadWp1 and gadWp2 (not shown) promoters. Red wavy lines represent mRNAs. These promoters are subject to regulation by different sets of TFs (not shown), so they are not all subject to the same signals. There are at least four operons in RegulonDB with no evidence of a single polycistronic transcript including all the genes of the operon.

Figure 6 |. Schematic of signal and effector.

A cell reacting to the environment is represented. The rectangle represents the cell membrane, separating cytoplasm and the environment. A signal is represented as an environmental molecule (brown external bubble) that elicits a cellular response. The environmental molecule is introduced to the cell (brown internal oval) through a membrane transporter (blue square) and transformed by an enzyme into another molecule (yellow oval) that plays the role of effector by binding a transcription factor (TF) and modifying its ability to recognize its DNA binding sites. The concept of genetic sensory response units (GENSOR units) captures all the elements from the signal via the effector and regulation to the final regulated gene products as the response.