Jonah Patel
The activity of constitutive promoters is an important area of study in genetics, particularly in the context of gene regulation and the design of synthetic genetic circuits. In the discussion of promoter activity, the question of whether downstream promoter elements can affect upstream promoters is a complex one. Promoters are DNA sequences that initiate gene transcription by providing a binding site for RNA polymerase and transcription factors. These promoters can be found in close proximity to each other, and their interaction can influence gene expression. Enhancers, which are clusters of transcription factor binding sites, can also impact promoter activity and are often located downstream of the promoter region. Understanding the effects of downstream promoter elements on upstream promoters is crucial for advancing our knowledge of gene regulation and developing applications in plant biotechnology.
| Characteristics | Values |
|---|---|
| Definition of Promoter | A sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter |
| Promoter Length | About 100-1000 base pairs long |
| Promoter Location | Upstream on the DNA (towards the 5' region of the sense strand) |
| Promoter Function | Control the binding of RNA polymerase to DNA |
| Promoter Activity | Depends on the free RNA polymerase concentration and on two promoter-specific parameters: Vmax and Km |
| Promoter Types | Constitutive, Synthetic, Eukaryotic, Prokaryotic, Bacterial |
| Promoter Elements | CpG islands, TATA box, Initiator (Inr), Upstream and downstream TFIIB recognition elements (BREu and BREd), Downstream core promoter element (DPE) |
| Promoter Studies | In vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli |
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What You'll Learn
Interference between closely spaced promoters
Closely spaced promoters are promoters that are very close to each other in the DNA. They have been observed in the DNAs of all life forms, from prokaryotes to humans, and are highly conserved. The most important aspect of two closely spaced promoters is that they will, most likely, interfere with each other.
Pairs of promoters can be positioned in divergent, tandem, and convergent directions. They can also be regulated by transcription factors and differ in various features, such as the nucleotide distance between them, the two promoter strengths, etc. The degree of correlation between polymerases depends on the geometry, the distance between transcription start sites (TSSs), and repressors. Small changes in the distance between TSSs can cause abrupt changes in behaviour patterns, suggesting that the sequence between adjacent promoters may be subject to strong selective pressure.
Several studies have explored the interference between closely spaced promoters using both analytical and stochastic models. One study measured most genes controlled by tandem promoters in E. coli. Two main forms of interference were measured. One is when a RNA polymerase (RNAP) is on the downstream promoter, blocking the movement of RNAPs elongating from the upstream promoter. This is known as "occlusion" or "sitting duck" interference. The other is when the two promoters are so close that when an RNAP sits on one of the promoters, it blocks any other RNAP from reaching the other promoter. These events are possible because the RNAP occupies several nucleotides when bound to the DNA, including in transcription start sites. Similar events occur when the promoters are in divergent and convergent formations. The possible events also depend on the distance between them.
Another study on the effect of DNA bending on transcriptional interference in the systems of closely spaced convergent promoters found that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilisation, while DksA interferes directly with nucleotide positioning into the RNAP active site. At the iNTPs-sensitive rRNA promoters, ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter, DksA reduces the effect of ppGpp in accordance with their antagonistic role.
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RNAP blocking movement from upstream promoter
In genetics, promoters are vital components of expression vectors as they control the binding of RNA polymerase to DNA. Promoters are typically found upstream of the gene in question, and their positions are designated relative to the transcriptional start site (TSS), where DNA transcription begins. The TSS is the most 3' portion of the core promoter, which is where transcription actually begins. Promoters can be about 100-1000 base pairs long and are usually located near the TSS of genes.
The bacterial RNA polymerase (RNAP) recognizes promoters through sequence-specific contacts with two DNA sequence motifs: the upstream ('-35') promoter motif and the downstream ('-10') motif. During transcription initiation, the RNAP and the promoter form an open complex, which involves upstream DNA wrapping, downstream promoter bending, and associated large-scale protein rearrangements.
In the context of closely spaced promoters, RNAP blocking movement from the upstream promoter can occur when an RNAP is on the downstream promoter, physically obstructing the movement of RNAPs elongating from the upstream promoter. This interference is possible because the RNAP occupies several nucleotides when bound to the DNA, including transcription start sites.
In bacterial transcription, the late transcription coactivator gp33 has been found to repress transcription by preventing RNAP binding to internal sites on DNA. Gp33 binds to the upstream end of the open promoter complex and can remain with or reattach to the elongating transcription complex. This repression is due to gp33 blocking an upstream sequence-independent DNA-binding site on RNAP.
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RNAP blocking access to other promoters
In the context of gene expression, promoters are vital components of expression vectors as they control the binding of RNA polymerase to DNA. Promoters are typically located upstream on the DNA (towards the 5' region of the sense strand) and are about 100–1000 base pairs long. The process of transcription involves RNA polymerase synthesising RNA from DNA. For this to occur, the RNA polymerase must attach to the DNA near a gene.
Promoters contain specific DNA sequences, such as response elements, that provide a secure initial binding site for RNA polymerase and for proteins called transcription factors that recruit RNA polymerase. Transcription factors act at the gene-specific level and form a network to coordinate gene expression in response to environmental challenges. Their expressions and activities are tissue-specific in multicellular organisms.
When promoters are closely spaced, they will likely interfere with each other. One form of interference is when an RNAP is on the downstream promoter, blocking the movement of RNAPs elongating from the upstream promoter. This is known as promoter occlusion, a mechanism of transcriptional interference (TI). Elongating RNAPs positioned over a promoter prevent other RNAPs in solution from accessing the promoter. This can lead to a reduction in promoter activity.
RNAP pausing at a protein roadblock can enhance TI by promoter occlusion. Using the Lac repressor as a 'roadblock' to induce pausing over a target promoter, researchers found a small increase in TI, with mathematical modelling suggesting that rapid termination of the stalled RNAP limited the occlusion effect.
In addition, elongating RNAPs can interfere with transcription from downstream promoters by inhibiting DNA binding by RNAP and activators. However, it has been shown that simple RNAP elongation cannot produce the strong asymmetric interference observed between a natural face-to-face promoter pair in bacteriophage lambda.
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Promoter-specific parameters
The promoter region controls the binding of RNA polymerase and transcription factors, determining when and where the gene of interest will be expressed. The promoter region is about 100-1000 base pairs long and is usually found upstream (5') of the sense or coding strand of the transcribed gene. The sequence of the promoter region is highly dependent on the gene and product of transcription, the type or class of RNA polymerase recruited to the site, and the species of the organism.
Promoters contain specific DNA sequences such as response elements that provide a secure initial binding site for RNA polymerase and proteins called transcription factors that recruit RNA polymerase. These transcription factors have specific activator or repressor sequences of corresponding nucleotides that attach to specific promoters and regulate gene expression. The binding of transcription factors to promoters is influenced by promoter-specific parameters, including the presence of certain DNA sequences and the flexibility of transcription start sites.
The presence of specific DNA sequences within the promoter region can influence the binding of transcription factors. For example, CpG islands are present in about 70% of promoters, and a high CpG content status is a predictor of promoter variability. The TATA box, present in about 24% of promoters, is a DNA sequence within the core promoter region where general transcription factor proteins and histones can bind. The presence of the TATA box is also a predictor of promoter variability. Initiator (Inr) is present in about 49% of promoters and causes a 28% increase in expression. Other promoter-specific DNA sequences include upstream and downstream TFIIB recognition elements (BREu and BREd), present in about 22% of promoters, and the downstream core promoter element (DPE), present in about 12% of promoters.
The flexibility of transcription start sites (TSS) within a promoter is another parameter that can influence the binding of transcription factors. Promoters with a rigid TSS architecture are more prone to variable expression and are associated with genetic variants with large effect sizes. On the other hand, a flexible usage of TSS within a promoter attenuates expression variability and limits genotypic effects.
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Enhancers and UPEs
In genetics, promoters are a vital component of expression vectors as they control the binding of RNA polymerase to DNA. Promoters are about 100-1000 base pairs long and are usually upstream of the sense or coding strand of the transcribed gene. The coding strand is the DNA strand that encodes codons and whose sequence corresponds to the mRNA transcript produced.
Enhancers are regions of the genome that are major gene-regulatory elements. They are short (50-1500 base pairs) regions of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur. These proteins are usually referred to as transcription factors. Enhancers are found in both prokaryotes and eukaryotes. They can be located upstream or downstream of the gene they regulate and can be found within introns. Enhancers do not act on the promoter region itself but are bound by activator proteins. They can also be found at the exonic region of an unrelated gene and they may act on genes on another chromosome. Enhancers are scattered across the 98% of the human genome that does not encode proteins.
Enhancers can act independently of orientation, distance, and location with respect to the target gene. They can be located over as much as a million base pairs away from the gene. Multiple enhancers can exist in a cluster to form a super-enhancer. They are found mostly in the intergenic and intronic regions, while a few enhancers have been found within exons. Enhancers consist of dense clusters of transcription factor binding sites (TFBS) and are bound by cell type-specific TFs, coregulators, chromatin modifiers, architectural proteins, other enzymes, and RNAPII.
UPEs, or upstream promoter elements, are similar to enhancers in that they share the same activators. The boundary between the upstream promoter region and the enhancer region is arbitrary and determined by the investigator. UPEs are located upstream of the core promoter region, which itself is upstream of the transcription start site (TSS). The core promoter region contains a number of sequence elements located both upstream and downstream from the TSS.
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Frequently asked questions
Promoters are a vital component of expression vectors because they control the binding of RNA polymerase to DNA. They are usually about 100-1000 base pairs long and are located upstream of the sense or coding strand of the transcribed gene.
Upstream promoter elements (UP elements) are DNA sequences found upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding of RNAP to DNA.
The activity of constitutive promoters is expected to depend on the free RNA polymerase concentration and promoter-specific parameters like maximum promoter activity and the free RNA polymerase concentration at which the transcript initiation rate is half-maximal. The presence of two closely spaced promoters can also lead to interference, where an RNAP on the downstream promoter blocks the movement of RNAPs elongating from the upstream promoter.
Some examples of constitutive promoters include the spc ribosomal protein operon promoter Pspc, the β-lactamase gene promoter Pblaof, and the replication control promoters PRNAI and PRNAII.
