DNA sensors in the nucleus detect changes in DNA. They can trigger complex biological responses such as cell cycle control and gene activation.
In the experiment shown in Figure 3, DNA-PKCS (1.9 nm) was incubated with different DNA molecules to test their ability to activate the enzyme. DNA with ends consisting of single-stranded loops or hairpins fails to efficiently stimulate kinase activity.
Transcription
Central to all life is the transfer of information stored in genes into molecules that are used to make proteins. This process is known as transcription. To begin transcription, proteins called transcription factors bind to two key bits of DNA, an enhancer and a promoter. Once these proteins are close enough together, another protein called RNA polymerase can read the DNA sequence and copy it into an RNA molecule. The resulting RNA molecule, or mRNA, contains the code that tells the cell what proteins to make.
As soon as RNA polymerase is active, it opens the DNA double helix and starts to add a strand of RNA to the opposite side. This strand is complementary to the template DNA strand and contains the same sequence of bases as the DNA, but with uracil (U) in place of thymine (T). The RNA polymerase strand then moves forward along the DNA template helix, synthesizing a new strand of RNA and reading the template strand as it goes.
As the RNA is being added, other proteins, including a pair of complexes named TFIID and TFIIH, load onto RNA polymerase. TFIIH is particularly important because it phosphorylates the tail of RNA polymerase, making it easier for this protein to disengage from DNA and move on to the next gene. The TFIID and TFIIH complexes also contain a helicase, which helps to open and then close the DNA helix.
Enhancers
A little-known, yet vitally important, aspect of gene transcription occurs when “junk DNA” binds to and activates genes. Scientists now know that this process is kicked off when proteins called transcription factors bind to two key bits of genetic sequence, an enhancer and a promoter. But until recently, it was unclear how these two elements, separated by a million or more base pairs when DNA is stretched out, come together to kick off gene expression within cells.
To answer this question, researchers turned to genome-wide functional assays and looked at thousands of different versions of enhancers side by side. They found that small changes in an enhancer’s DNA sequence can dramatically affect how well it works. For example, when a specific type of transcription factor binding site was altered on an enhancer, it stopped other types of transcription factors from binding to it.
In addition, these research findings suggest that enhancers are bound in a complex manner, with multiple transcription factors simultaneously binding to them and competing against histone octamers to stabilize the enhancer-promoter interaction. This helps explain how an enhancer can work even though it may be distant from a gene and function on a non-nucleosomal chromatin template.
Another challenge for this field is that, in contrast to protein-coding genes, the general sequence code governing the functions of enhancers is not as well understood. Despite this, many enhancers have been shown to act in ways that are specific to a cell type, tissue, time point or to particular physiological or pathological conditions.
Promoter
The promoter is the region of DNA that lies upstream of the coding sequence for a gene. The promoter contains the binding sites for proteins that initiate transcription. Genes are organized to make this initiation process as efficient as possible. The promoter may be very short (only a few nucleotides long) or quite long (hundreds of nucleotides long). The short ones are called core promoters, and they contain the binding site for the basal transcription factor. The longer ones are called proximal promoters and they contain a series of regulatory sequences that increase the likelihood that proteins will bind to them. The proximal promoters also often have regions of DNA with high C:G content (CpG islands).
Scientists have developed an entire field of study to understand how gene expression is kicked off when genes are activated. They know that this is done when protein complexes bind to two key bits of DNA, an enhancer and a promoter. But they didn’t realize how close to each other these sequences have to be for gene activation to occur.
To better understand this, researchers at Princeton University used video imaging to capture the snippets of DNA that are flipped in and out of position as they search for and connect with the gene they are meant to activate. They labeled the enhancer blue and the promoter green, and watched as they found each other and connected in real time to initiate mRNA production.
RNA Polymerase
A key step in gene regulation is RNA Polymerase recognition of promoter sequences and initiation of transcription. Several proteins enhance or inhibit this process. Once RNA Polymerase recognizes the start site of a gene, it binds to the corresponding DNA and begins transcribing a strand of RNA that matches the template. RNA Polymerase is a multisubunit enzyme that catalyzes the formation of phosphodiester bonds between nucleoside triphosphates (ATP, CTP, and UTP) to produce a growing linear chain of RNA. The chain is extended one nucleotide at a time in the 5′ to 3′ direction using a DNA template and a set of specialized RNA transcripts as primers.
RNA Polymerase can be controlled by a variety of factors including small molecules, protein factors, and signals encoded in the nascent RNA produced during transcription. The latter control the speed and pocessivity of transcription by regulating pausing and termination. The most important pause signal is a RNA hairpin that forms 10-12 nucleotides from the RNA 3′ end to halt elongation. Pausing is also induced by signals in the nascent RNA that vary in structure and sequence.
Although RNA Polymerase normally transcribes precise RNA transcripts that match the DNA template, errors can occur during transcription. These errors are called transcriptional slippage. RNA Polymerase can slip during both the initiation and elongation phases of transcription. Initiation-associated RNAP slippage seems to require a pause and homopolymeric runs of consecutive dA or dT nucleotides, while elongation-associated RNAP slippage requires longer runs of nucleotides.dna activation