Bacterial promoters typically consist of two short DNA sequences that are separated by a defined number of bases. The two primary sequences that govern the expression level of a particular promoter in bacteria are called the -10 and -35 boxes. These sequences are positioned, perhaps unsurprisingly, at roughly -10 and -35 in relation to the start position of transcription that they initiate. The -10 box is sometimes referred to as the ‘Pribnow box’. The sequences at these positions are very important and the consensus sites are shown below.
The consensus sequence shown above has not been found upstream of any genes in bacteria, and was compiled by looking at the expression levels of a range of strong E.coli promoters to determine the optimal base to use at each position. The current hypothesis is that the use of this sequence in E.coli is detrimental to bacterial growth because transcription from this loci would be so efficient that the cell would suffer a growth burden if it were to use it. However, some promoters come very close. For example, the RecA promoter is a very strong promoter that under normal circumstances is suppressed by a protein called LexA. However, during times of stress and DNA damage the LexA repressor can be cleaved to allow RecA production. The similarity between the RecA promoter and the consensus means that the promoter mediates very high expression levels. The RecA promoter -35 box is TTGATA and the -10 box is TATAAT, with an inter-box gap of 16bp. This means that the promoter is only one base different from the consensus.
Bacterial promoters can be divided into housekeeping promoters and those that are responsive under certain physiological or environmental conditions. Whether a particular promoter is active or not is primarily determined by the Sigma factors. These are proteins that bind to RNA polymerase and allow it to dock at specific DNA sites. For the standard ‘house-keeping’ gene promoters described above, the Sigma Factor is called Sigma 70. E.coli has seven Sigma factors that regulate promoters and that are involved in different cell responses: for example, Sigma 32 is responsible for heat-shock responses and will allow RNA polymerase to dock at promoters upstream of genes involved in protecting the cell against heat damage. Once RNA polymerase is bound to a Sigma factor it can dock at the relevant DNA promoter region and initiate RNA synthesis and transcription.
In our product range we have a constitutive bacterial promoter range called OXB1-20. These promoters were primarily created by removing the LexA repressor binding site from the RecA promoter, followed by random mutagenesis and screening to create a library of variants. Each of these variants were then screened for activity. The strongest promoter was always the starting material (RecA promoter with the LexA site removed). We also tested some other standard promoters for expression levels including LacUV5 without the LacO site, and AraBAD without the AraC site. These are called OX19 and OXB1 respectively to denote their expression level relative to the modified RecA promoter (which is called OXB20).
Whilst the -10 and -35 boxes are normally necessary and sufficient for good expression levels in most bacteria, there are other sequences that can either repress or activate gene expression. For example, the UP DNA sequences are A/T rich sequences that can be found upstream of some very strong bacterial promoters, including promoters that drive expression of the ribosomal RNA gene transcripts. This particular sequence is normally located upstream of the -35 box at an approximate position of -47 to -57 bp and helps by allowing the C-terminal domain of the alpha subunit of RNA polymerase to bind to the DNA with greater affinity, thereby increasing transcription significantly.
As discussed briefly above, there are sites that can suppress transcription (for example, the LexA binding site in the RecA promoter). Other commonly studied systems include the AraBAD operon and the Lac operon which use repressors (AraC and LacI, respectively) to silence transcription until needed by the cell. These two systems constitute the main expression systems used by researchers to control transgene expression. In these systems, cells are grown containing a plasmid which encodes a gene of interest downstream of a promoter that contains a repressor binding site. Under normal conditions the promoter is prevented from expressing the gene of interest by the repressor. When a researcher wants to produce the protein of interest they add an inducing agent (for example, IPTG for Lac regulated promoters), and this allows the gene of interest to be expressed.