Plasmids 101: The Promoter Region – Let's Go!

Once you've gotten to know what a plasmid is in general, it's time to take a look at some of its parts. Elements such as the antibiotic resistance gene and the origin of replication are necessary to replicate plasmids and make sure the cells maintains it. Here, we'll cover the promoter, which is necessary for getting the plasmid express our gene of interest. This element is responsible for initiating the transcription of your insert into RNA.

In practice, the term "promoter" describes the combination of the promoter (RNA polymerase binding site) and operators (response elements). Promoters are about 100 to 1000 base pairs long and found upstream of their target genes. The sequence of the promoter region controls the binding of the RNA polymerase and transcription factors, therefore promoters play a large role in determining where and when your gene of interest will be expressed. 

The RNA polymerase(s)

RNA is transcribed from DNA using an RNA polymerase (RNAP). In bacteria this is done by a single enzyme; however, eukaryotes have muliple polymerases which are each responsible for a specific subset of RNAs. To gain this specificity, the eukaryotic RNAP can recognize and bind to specific promoter elements. This means that the promoter present in your plasmid backbone must to be compatible with the type of RNA that needs to be made: if you want mRNA (for gene expression) you need to use an RNAP II promoter, whereas small RNAs (such as shRNA) are transcribed from the RNAP III promoters. This post features promoters for general RNAP II and RNAP III transcription; however, using viral LTRs as RNAP II promoters is commonly employed in lentiviral and retroviral constructs and we will discuss these in a future post on viral vector parts.

Promoter specificity

Aside from choosing a promoter based on type of RNA transcript, you will also need to make sure your plasmid has a promoter suited to working in your host organism. Because transcription machinery differs between cell types or organisms, promoters must be similarly variable. Bacterial promoters only work in prokaryotic cells and typically only in the same or closely related species from which they were derived. Similarly, the various eukaryotic cell types (mammalian, yeast, plants, etc) require unique promoters and there is very little crossover. Generally speaking, promoters in bacteria are less diverse and complex, having fewer parts than those in eukaryotic cells. Some promoters are constitutively active and on all the time while others are more carefully controlled. Regulated promoters might act only in certain tissues or at certain times in development or there may be ways to turn them on or off at will with a chemical, heat, or light. In the cell, promoters themselves are controlled by still other regulatory factors: enhancers, boundary elements, insulators, and silencers; however, some "leakiness" of transcription may occur. This is normally not a big issue for cells, but it may confound research results or even kill your cells if your gene of interest is toxic. To combat this, scientists have created synthetic promoters, which typically include some combination of other promoter elements, and tend to be more tightly regulated.

Common promoters for eukaryotes and prokaryotes

We have included two reference tables below listing some of the most common bacterial and mammalian promoters. These lists is by no means exhaustive, but should be a good place to start when trying to pick your perfect promoter!

Eukaryotic Promoters

Promoter	Primarily used for	RNA     transcript	Description	Expression	Additional considerations
CMV	General expression	mRNA	Strong mammalian expression promoter from the human cytomegalovirus	Constitutive	May contain an enhancer region. Can be silenced in some cell types.
EF1a	General expression	mRNA	Strong mammalian expression from human elongation factor 1 alpha	Constitutive	Tends to give consistent expression regardless of cell type or physiology.
SV40	General expression	mRNA	Mammalian expression promoter from the simian vacuolating virus 40	Constitutive	May include an enhancer.
PGK1 (human or mouse)	General expression	mRNA	Mammalian promoter from phosphoglycerate kinase gene.	Constitutive	Widespread expression, but may vary by cell type. Tends to resist promoter down regulation due to methylation or deacetylation.
Ubc	General expression	mRNA	Mammalian promoter from the human ubiquitin C gene	Constitutive	As the name implies, this promoter is ubiquitous.
human beta actin	General expression	mRNA	Mammalian promoter from beta actin gene	Constitutive	Ubiquitous. Chicken version is commonly used in promoter hybrids.
CAG	General expression	mRNA	Strong hybrid mammalian promoter	Constitutive	Contains CMV enhancer, chicken beta actin promoter, and rabbit beta-globin splice acceptor.
TRE	General expression	mRNA	Tetracycline response element promoter	Inducible with Tetracyline or its derivatives.	Typically contains a minimal promoter with low basal activity and several tetracycline operators. Transcription can be turned on or off depending on what tet transactivator is used.
UAS	General expression	mRNA	Yeast promoter containing Gal4 binding sites, commonly used in Drosophila	Specific	Requires the presence of Gal4 gene to activate promoter.
Ac5	General expression	mRNA	Strong insect promoter from Drosophila Actin 5c gene	Constitutive	Commonly used in expression systems for Drosophila.
Polyhedrin	General expression	mRNA	Strong insect promoter from baculovirus	Constitutive	Commonly used in expression systems for insect cells.
CaMKIIa	Gene expression for optogenetics	mRNA	Ca2+/calmodulin-dependent protein kinase II promoter	Specific	Used for neuronal/CNS expression. Modulated by calcium and calmodulin.
GAL1, 10	General expression	mRNA	Yeast adjacent, divergently transcribed promoters	Inducible with galactose; repressible with glucose	Can be used independently or together. Regulated by GAL4 and GAL 80.
TEF1	General expression	mRNA	Yeast transcription elongation factor promoter	Constitutive	Analogous to mammalian EF1a promoter.
GDS	General expression	mRNA	Strong yeast expression promoter from glyceraldehyde 3-phosphage dehydrogenase	Constitutive	Very strong, also called TDH3 or GAPDH.
ADH1	General expression	mRNA	Yeast promoter for alcohol dehydrogenase I	Repressed by ethanol	Full length version is strong with high expression. Truncated promoters are constitutive with lower expression.
CaMV35S	General expression	mRNA	Strong plant promoter from the Cauliflower Mosaic Virus	Constitutive	Active in dicots, less active in monocots, with some activity in animal cells.
Ubi	General expression	mRNA	Plant promoter from maize ubiquitin gene	Constitutive	Gives high expression in plants.
H1	small RNA expression	shRNA	From the human polymerase III RNA promoter	Constitutive	May have slightly lower expression than U6. May have better expression in neuronal cells.
U6	small RNA expression	shRNA	From the human U6 small nuclear promoter	Constitutive	Murine U6 is also used, but may be less efficient.

Prokaryotic promoters

Promoter	Primarily used for	Description	Expression	Additional considerations
T7	in vitro transcription/ general expression	Promoter from T7 bacteriophage	Constitutive, but requires T7 RNA polymerase.	When used for in vitro transcription, the promoter drives either the sense OR antisense transcript depending on its orientation to your gene.
T7lac	High levels of gene expression	Promoter from T7 bacteriophage plus lac operators	Negligible basal expression when not induced. Requires T7 RNA polymerase, which is also controlled by lac operator. Can be induced by IPTG.	Commonly found in pET vectors. Very tightly regulated by the lac operators. Good for modulating gene expression through varied inducer concentrations.
Sp6	in vitro transcription/ general expression	Promoter from Sp6 bacteriophage	Constitutive, but requires SP6 RNA polymerase.	SP6 polymerase has a high processivity. When used for in vitro transcription, the promoter drives either the sense OR antisense transcript depending on its orientation to your gene.
araBAD	General expression	Promoter of the arabinose metabolic operon	Inducible by arabinose and repressed catabolite repression in the presence of glucose or by competitive binding of the anti-inducer fucose	Weaker. Commonly found in pBAD vectors. Good for rapid regulation and low basal expression; however, not well-suited for modulating gene expression through varied inducer concentrations.
trp	High levels of gene expression	Promoter from E. coli tryptophan operon	Repressible	Gets turned off with high levels of cellular tryptophan.
lac	General expression	Promoter from lac operon	Constitutive in the absense of lac repressor (lacI or lacIq). Can be induced by IPTG or lactose.	Leaky promoter with somewhat weak expression. lacIq mutation increases expression of the repressor 10x, thus tightening regulation of lac promoter. Good for modulating gene expression through varied inducer concentrations.
Ptac	General expression	Hybrid promoter of lac and trp	Regulated like the lac promoter	Contains -35 region from trpB and -10 region from lac. Very tight regulation. Good for modulating gene expression through varied inducer concentrations. Generally better expression than lac alone.
pL	High levels of gene expression	Promoter from bacteriophage lambda	Can be temperature regulatable	Often paired with the temperature sensitive cI857 repressor.
T3	in vitro transcription/general expression	Promoter from T3 bacteriophage	Constitutive, but requires T3 RNA polymerase	When used for in vitro transcription, the promoter drives either the sense OR antisense transcript depending on its orientation to your gene

Although this list is a great place to start, the tables above do not delve into the tissue or development-specific promoters available to scientists. Plasmids are oftentimes put to therapeutic uses, and in those cases it's important to identify the right tissue-specific promoters as described by researchers at the NIH here.

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 Note: A. Max Juchheim contributed to the writing of this article.

Additional Resources on the Addgene Blog

• Browse All Plasmids 101 Posts
• Learn about Mammalian Vectors
• Read about Reporter Gene like Luciferase and GFP

Resources on Addgene.org

• Find Plasmids for Your Research