Instruct: Given a question, retrieve relevant passages that answer the question.
Query: What triggers transcription in DNA?
In genetics, a transcription terminator is a section of nucleic acid sequence that marks the end of a gene or operon in genomic DNA during transcription. This sequence mediates transcriptional termination by providing signals in the newly synthesized transcript RNA that trigger processes which release the transcript RNA from the transcriptional complex. These processes include the direct interaction of the mRNA secondary structure with the complex and/or the indirect activities of recruited termination factors. Release of the transcriptional complex frees RNA polymerase and related transcriptional machinery to begin transcription of new mRNAs.
In cells, DNA is housed in the nucleus and it never leaves. The DNA within the nucleus is transcribed by tRNA, and the transcriptions of the DNA become RNA. The RNA (ribonucleic acid) leaves the nucleus to float freely around the cytoplasm, and it contains the instructions of the DNA that are vital to the coding, decoding, regulation and expression of genes. The ribosomes then take the RNA messages and turn them into proteins that build cells. When a virus enters a cell and inserts its DNA code into the nucleus, it gets transcribed and released into the cell for the ribosome to make it into a protein. This is how viral infections occur. The cell then explodes with the overproduction of the virus, releasing it into the body to infect any and all other cells it finds. It is theorized that through evolution, cells developed a defense system called RNAi (RNA interference) to stop the production of the proteins that have suspicious mirror image messages of the RNA. They don’t just destroy the suspicious messages, but the correct messages as well to stop all production of that message. This is a cell’s self-destruct mechanism and every cell, plant and animal, have RNAi: a way to turn off the production of a certain gene inside the protein.
In the absence of hormone, estrogen receptors are largely located in the cytosol. Hormone binding to the receptor triggers a number of events starting with migration of the receptor from the cytosol into the nucleus, dimerization of the receptor, and subsequent binding of the receptor dimer to specific sequences of DNA known as hormone response elements. The DNA/receptor complex then recruits other proteins that are responsible for the transcription of downstream DNA into mRNA and finally protein that results in a change in cell function. Estrogen receptors also occur within the cell nucleus, and both estrogen receptor subtypes have a DNA-binding domain and can function as transcription factors to regulate the production of proteins.
One strand of the DNA, the "template strand" (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy (which elongates during the traversal). Although RNA polymerase traverses the template strand from 3' → 5', the coding (non-template) strand and newly formed RNA can also be used as reference points, so transcription can be described as occurring 5' → 3'. This produces an RNA molecule from 5' → 3', an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one fewer oxygen atom) in its sugar-phosphate backbone).
Studies with mouse HES7 have demonstrated that the gene contains 4 exons. There are three known human HES7 variants due to alterative splicing. The gene product is a transcription factor protein. The Helix-loop-helix domain is located at the N-terminal end of the protein. Both the human and the mouse protein have been shown to contain 225 amino acids and feature an orange domain as well as a conserved sequence of four amino acids at the C-terminal end. The four amino acids are Tryptophan-Arginine-Proline-Tryptophan. This conserved sequence of four amino acids binds Groucho/TLE family members. Groucho/TLE are transcriptional corepressors. Hairy-related proteins can direct these transcriptional corepressors to target genes. Orange domains are motifs of ~35 amino acids that are found on the C-terminal side of basic helix-loop-helix domains in some transcription factors in eukaryotic organisms. Proteins with Orange domains are divided into four subfamilies, three in which all proteins contain a basic helix-loop-helix domain. The subfamilies that contain Orange domains and basic helix-loop-helix domains are Hairy, Enhancer of Split, and Hey. Transcription factors with Orange domains bind DNA and regulate biological processes such as cell differentiation and embryonic patterning.
At first, regulatory RNA was thought to be a eukaryotic phenomenon, a part of the explanation for why so much more transcription in higher organisms was seen than had been predicted. But as soon as researchers began to look for possible RNA regulators in bacteria, they turned up there as well. Currently, the ubiquitous nature of systems of RNA regulation of genes has been discussed as support for the RNA World theory. Bacterial small RNAs generally act via antisense pairing with mRNA to down-regulate its translation, either by affecting stability or affecting cis-binding ability. Riboswitches have also been discovered. They are cis-acting regulatory RNA sequences acting allosterically. They change shape when they bind metabolites so that they gain or lose the ability to bind chromatin to regulate expression of genes.
Cells in the body function because of DNA. DNA sits within the nucleus of a cell. Here, parts of DNA are copied and sent to the body of the cell via RNA. The RNA is then used to create proteins which form the basis for cells, their activity, and their products. Proteins dictate cell function and gene expression, a cell is able to self-regulate by the amount of proteins produced. However, not all cells have DNA – some cells such as mature red blood cells lose their nucleus as they mature.
cDNA cloning is a major technology for analysis of the expression of genomes. The full-length first-strand can be synthesized through the commercially available reverse transcriptases. Synthesis of the second-strand was once a major limitation to cDNA cloning. Two groups of methods differing by the mechanism of initiation were developed to synthesize the second-strand. In a first group of methods, initiation of second-strand synthesis takes place within the sequence of the first strand. However, the digestion of the 3' end of the first strand is required and therefore results in the loss of the sequences corresponding to the 5'end of the mRNA. In a second group of methods, initiation of second-strand synthesis takes place outside the sequence of the first strand. This group of methods does not require digestion of the 3' end of the first strand. However, the limitation of this group of method lies upon the elongation. Cloning with T7 DNA polymerase helps overcome this limitation by allowing digestion of the poly(dT) tract during the second-strand synthesis reaction. Therefore, the size of the tract synthesized with terminal transferase is not required to be within a given size range and the resulting clones contain a tract of a limited size. Moreover, due to high 3’ exonuclease activity of T7 DNA polymerase, high yield of the full-length second-strand can be obtained.
DNA synthesis is the natural or artificial creation of deoxyribonucleic acid (DNA) molecules. The term DNA synthesis can refer to DNA replication - DNA biosynthesis ("in vivo" DNA amplification), polymerase chain reaction - enzymatic DNA synthesis ("in vitro" DNA amplification) or gene synthesis - physically creating artificial gene sequences.
In nature, DNA molecules are created by all living cells through the process of DNA replication, with replication initiator proteins splitting the cell's DNA and copying each split strand, with the copied strands then joining together with their template strand into a new DNA molecule. Various means exist to artificially stimulate the replication of naturally occurring DNA, or to create artificial gene sequences. The process of DNA replication occurs typically as part of cell division, during interphase.
Various genetic discoveries have been essential in the development of genetic engineering. Genetic inheritance was first discovered by Gregor Mendel in 1865 following experiments crossing peas. Although largely ignored for 34 years he provided the first evidence of hereditary segregation and independent assortment. In 1889 Hugo de Vries came up with the name "(pan)gene" after postulating that particles are responsible for inheritance of characteristics and the term "genetics" was coined by William Bateson in 1905. In 1928 Frederick Griffith proved the existence of a "transforming principle" involved in inheritance, which Avery, MacLeod and McCarty later (1944) identified as DNA. Edward Lawrie Tatum and George Wells Beadle developed the central dogma that genes code for proteins in 1941. The double helix structure of DNA was identified by James Watson and Francis Crick in 1953.As well as discovering how DNA works, tools had to be developed that allowed it to be manipulated. In 1970 Hamilton Smith's lab discovered restriction enzymes that allowed DNA to be cut at specific places and separated out on an electrophoresis gel. This enabled scientists to isolate genes from an organism's genome. DNA ligases, that join broken DNA together, had been discovered earlier in 1967 and by combining the two enzymes it was possible to "cut and paste" DNA sequences to create recombinant DNA. Plasmids, discovered in 1952, became important tools for transferring information between cells and replicating DNA sequences. Frederick Sanger developed a method for sequencing DNA in 1977, greatly increasing the genetic information available to researchers. Polymerase chain reaction (PCR), developed by Kary Mullis in 1983, allowed small sections of DNA to be amplified and aided identification and isolation of genetic material.
In 1961, Jacques Monod, Jean-Pierre Changeux and François Jacob discovered the lac operon in the bacterium "Escherichia coli". It was a cluster of genes, arranged in a feedback control loop so that its products would only be made when "switched on" by an environmental stimulus. One of these products was an enzyme that splits a sugar, lactose; and lactose itself was the stimulus that switched the genes on. This was a revelation, as it showed for the first time that genes, even in an organism as small as a bacterium, were subject to fine-grained control. The implication was that many other genes were also elaborately regulated.
At the time of the hypercycle theory formulation, ribozymes were not known. After the breakthrough of discovering RNA's catalytic properties in 1982, it was realized that RNA had the ability to integrate protein and nucleotide-chain properties into one entity. Ribozymes potentially serving as templates and catalysers of replication can be considered components of quasispecies that can self-organize into a hypercycle without the need to invent a translation process. In 2001, a partial RNA polymerase ribozyme was designed via directed evolution. Nevertheless, it was able to catalyse only a polymerization of a chain having the size of about 14 nucleotides, even though it was 200 nucleotides long. The most up-to-date version of this polymerase was shown in 2013. While it has an ability to catalyse polymerization of longer sequences, even of its own length, it cannot replicate itself due to a lack of sequence generality and its inability to transverse secondary structures of long RNA templates. However, it was recently shown that those limitations could in principle be overcome by the assembly of active polymerase ribozymes from several short RNA strands. In 2014, a cross-chiral RNA polymerase ribozyme was demonstrated. It was hypothesized that it offers a new mode of recognition between an enzyme and substrates, which is based on the shape of the substrate, and allows avoiding the Watson-Crick pairing and, therefore, may provide greater sequence generality. Various other experiments have shown that, besides bearing polymerase properties, ribozymes could have developed other kinds of evolutionarily useful catalytic activity such as synthase, ligase, or aminoacylase activities. Ribozymal aminoacylators and ribozymes with the ability to form peptide bonds might have been crucial to inventing translation. An RNA ligase, in turn, could link various components of quasispecies into one chain, beginning the process of a genome integration. An RNA with a synthase or a synthetase activity could be critical for building compartments and providing building blocks for growing RNA and protein chains as well as other types of molecules. Many examples of this kind of ribozyme are currently known, including a peptidyl transferase ribozyme, a ligase, and a nucleotide synthetase. A transaminoacylator described in 2013 has five nucleotides, which is sufficient for a trans-amino acylation reaction and makes it the smallest ribozyme that has been discovered. It supports a peptidyl-RNA synthesis that could be a precursor for the contemporary process of linking amino acids to tRNA molecules. An RNA ligase's catalytic domain, consisting of 93 nucleotides, proved to be sufficient to catalyse a linking reaction between two RNA chains. Similarly, an acyltransferase ribozyme 82 nucleotides long was sufficient to perform an acyltransfer reaction. Altogether, the results concerning the RNA ligase's catalytic domain and the acyltransferase ribozyme are in agreement with the estimated upper limit of 100 nucleotides set by the error threshold problem. However, it was hypothesized that even if the putative first RNA-dependent RNA-polymerases are estimated to be longer—the smallest reported up-to-date RNA-dependent polymerase ribozyme is 165 nucleotides long—they did not have to arise in one step. It is more plausible that ligation of smaller RNA chains performed by the first RNA ligases resulted in a longer chain with the desired catalytically active polymerase domain.
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and DNA are nucleic acids, and, along with lipids, proteins and carbohydrates, constitute the four major macromolecules essential for all known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it is more often found in nature as a single-strand folded onto itself, rather than a paired double-strand. Cellular organisms use messenger RNA (mRNA) to convey genetic information (using the nitrogenous bases of guanine, uracil, adenine, and cytosine, denoted by the letters G, U, A, and C) that directs synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome.
DNA, RNA and proteins are most common sequence-controlled polymers in living creatures. Inspired by them, polymerization methods, utilizing DNA or RNA as templates to control sequences of polymer, are developed. At first, taking DNA or RNA as templates, scientists developed a series of peptide nucleic acid (PNA)-based polymers, without using DNA polymerases. But this method is limited to polymerization scale and yield. After that, polymerase chain reaction (PCR) is developed, which currently is still the most extensively used sequence-regulated method. By employing enzymes, the yields and scales are greatly increased, but the specificity of enzymes towards natural peptides limits this technique to a certain degree. Nowadays, more attention is paid to utilization of ribosomes to directly mimic the transcription and translation process. This technology called protein engineering is considered as the most promising biological polymerization method for synthesis of sequence-controlled polymers.
The HIV genome presented several additional mysteries. The virus genome contained regions that might specify as yet unknown proteins. The Haseltine laboratory discovered a novel transacting protein and a second protein, both required for virus replication. They named the transactivator tat and the second protein art; the latter was renamed rev. He predicted that drugs that target these proteins will also be effective in treating the infection. A drug that inhibits the "rev" protein has been approved by the USA Food and Drug administration for treatment of HIV infection. His laboratory went on to discover additional viral genes and proteins – "vpr", "vpu", "vif", and "nef" - required for efficient virus growth in some but not all circumstances. The discoveries were summarized in an article in 1988 in "Scientific American".
Usually, a set of individually designed oligonucleotides is made on automated solid-phase synthesizers, purified and then connected by specific annealing and standard ligation or polymerase reactions. To improve specificity of oligonucleotide annealing, the synthesis step relies on a set of thermostable DNA ligase and polymerase enzymes. To date, several methods for gene synthesis have been described, such as the ligation of phosphorylated overlapping oligonucleotides, the Fok I method and a modified form of ligase chain reaction for gene synthesis. Additionally, several PCR assembly approaches have been described. They usually employ oligonucleotides of 40-50 nucleotides long that overlap each other. These oligonucleotides are designed to cover most of the sequence of both strands, and the full-length molecule is generated progressively by overlap extension (OE) PCR, thermodynamically balanced inside-out (TBIO) PCR or combined approaches. The most commonly synthesized genes range in size from 600 to 1,200 bp although much longer genes have been made by connecting previously assembled fragments of under 1,000 bp. In this size range it is necessary to test several candidate clones confirming the sequence of the cloned synthetic gene by automated sequencing methods.
Artificial gene synthesis, sometimes known as DNA printing is a method in synthetic biology that is used to create artificial genes in the laboratory. Based on solid-phase DNA synthesis, it differs from molecular cloning and polymerase chain reaction (PCR) in that it does not have to begin with preexisting DNA sequences. Therefore, it is possible to make a completely synthetic double-stranded DNA molecule with no apparent limits on either nucleotide sequence or size
Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence. To amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed, and a variety of specialised cloning vectors (small piece of DNA into which a foreign DNA fragment can be inserted) exist that allow protein production, affinity tagging, single stranded RNA or DNA production and a host of other molecular biology tools.
As noted above, Colin Blake, the author of the Gilbert-Blake theory for the origin of introns and exons, states, “Recent work by Senapathy, when applied to RNA, comprehensively explains the origin of the segregated form of RNA into coding and noncoding regions. It also suggests why a splicing mechanism was developed at the start of primordial evolution.”
Mitochondria, as a major energy factory of the cell, was previously a free-living prokaryote that invades the eukaryotic cells by the endosymbiosis theory, which gained acceptance around the 1970s. Under this theory, symbiotic organelles gradually transferred their genes to the eukaryotic genome, implying that mtDNA was gradually integrated into the nuclear genome. Despite the metabolic alterations and functional adaptations in the host eukaryotes, circular mitochondrial DNA is contained within the organelles. Containing 37 genes, mitochondrial DNA has an essential role in the production of necessary compounds, such as required enzymes for the proper function of mitochondria. Specifically, it has been suggested that certain genes (such as the genes for cytochrome oxidase subunits I and II) within the organelle are necessary to regulate redox balance throughout membrane-associated electron transport chains. These parts of the mitochondrial genome have been reported to be the most frequently employed. Mitochondria is not the only location within which the cell mtDNA, mitochondrial DNA, can be found; sometimes transfer of mitochondrial DNA from organelles to the nucleus can occur; the evidence of such translocation has been seen through the comparison of mitochondrial DNA sequence with the genome sequence of the counterparts. The integration and recombination of cytoplasmic mtDNA into the nuclear DNA is called Nuclear Mitochondrial DNA, which is abbreviated as NUMT.
The possible presence of organelle DNA inside the nuclear genome was suggested after finding of homologous structure to the mitochondrial DNA, which was shortly after the discovery of the presence of an independent DNA within the organelles in 1967. This topic stayed untouched until the 1980s. Initial evidence that DNA could move among cell compartments came when fragments of chloroplast DNA were found in the maize mitochondrial genome with the help of cross-hybridization, between chloroplast and mitochondrial DNA, and physical mapping of homologous regions. After this initial observation, Ellis coined the name "promiscuous DNA" in order to signify the transfer of DNA intracellularly from one organelle to the other and is the presence of organelle DNA in multiple cellular compartments. This is not only an important discovery on its own, but is also highly informative and helpful for understanding the evolutionary process and the time period different occurrence might take place. The searching for mtDNA in nuclear DNA continued until 1994 when the recent remarkable transposition of 7.9 kb of a typically 17.0-kb mitochondrial genome to a specific nuclear chromosomal position in the domestic cat was reported. This is the time that NUMT was coined to designate the large stretches of mitochondrial DNA in the genome.
Up to now, the whole genomes of many eukaryotes, both vertebrate, and invertebrate, have been sequenced and NUMT was observed in the nuclear genome of various organisms, including yeast, "Podospora", sea urchin, locust, honey bee, "Tribolium", rat, maize, rice, and primates. In Plasmodium, "Anopheles gambiae", and "Aedes aegypti" mosquitoes NUMT can barely be detected. In contrast, the conserved fragments of NUMT have now few were identified in genome data for "Ciona intestinalis", "Neurospora crassa", "Schizosaccharomyces pombe", "Caenorhabditis elegans", "Drosophila melanogaster", and "Rattus norvegicus". Antunes and Ramos were found the presence of NUMT in the fish genome for the first time in 2005 using of BLASTN, MAFFT, very vigorous genome mappings, and phylogenic analysis. Across the animal kingdom, "Apis mellifera", from phylum "Arthropoda", and "Hydra magnipapillata", from phylum "Cnidaria", are respectively the first and second animals with the highest ratio of NUMTs to the total size of the nuclear genome while "Monodelphis Domestica", or Gray short-tailed opossum, is the record holder for NUMT frequency among vertebrates. Similar to animals, NUMTs are abundant in the plants and the longest NUMT fragment known so far, a 620-kb partially duplicated insertion of the 367-kb mtDNA of "Arabidopsis thaliana", was reported.
However, in 2012, the Australian Safe Food Foundation alleged that the RNA trigger designed to change wheat's starch content might interfere with the gene for a human liver enzyme. Supporters countered that RNA does not appear to make it past human saliva or stomach acids. The US National Honey Bee Advisory Board told EPA that using RNAi would put natural systems at "the epitome of risk". The beekeepers cautioned that pollinators could be hurt by unintended effects and that the genomes of many insects are still unknown. Other unassessed risks include ecological (given the need for sustained presence for herbicide and other applications) and the possible for RNA drift across species boundaries.
Simply twisting DNA can expose internal bases to the outside, without the aid of any proteins. Also, transcription itself contorts DNA in living human cells, tightening some parts of the coil and loosening it in others. That stress triggers changes in shape, most notably opening up the helix to be read. Unfortunately, these interactions are very difficult to study because biological molecules morph shapes so easily. In 2008 it was noted that transcription twists DNA, leaving a trail of undercoiled (or negatively supercoiled) DNA in its wake. Moreover, they discovered that the DNA sequence itself effects how the molecule responds to supercoiling. For example, the researchers identified a specific sequence of DNA that regulates transcription speed; as the amount of supercoil rises and falls, it slows or speeds the pace at which molecular machinery reads DNA. It is hypothesized that these structural changes might trigger stress elsewhere along its length, which in turn might provide trigger points for replication or gene expression. This implies that it is a very dynamic process in which both DNA and proteins each influences how the other acts and reacts.
Activation of RNA polymerase activity to produce primary transcripts is often controlled by sequences of DNA called enhancers. Transcription factors, proteins that bind to DNA elements to either activate or repress transcription, bind to enhancers and recruit enzymes that alter nucleosome components, causing DNA to be either more or less accessible to RNA polymerase. The unique combinations of either activating or inhibiting transcription factors that bind to enhancer DNA regions determine whether or not the gene that enhancer interacts with is activated for transcription or not. Activation of transcription depends on whether or not the transcription elongation complex, itself consisting of a variety of transcription factors, can induce RNA polymerase to dissociate from the Mediator complex that connects an enhancer region to the promoter.
The activation of gene transcription is a multistep process that is triggered by factors that recognize transcriptional enhancer sites in DNA. These factors work with co-activators to direct transcriptional initiation by the RNA polymerase II apparatus. The protein encoded by this gene is a subunit of the CRSP (cofactor required for SP1 activation) complex, which, along with TFIID, is required for efficient activation by SP1. This protein is also a component of other multisubunit complexes e.g. thyroid hormone receptor-(TR-) associated proteins which interact with TR and facilitate TR function on DNA templates in conjunction with initiation factors and cofactors. This protein also acts as a metastasis suppressor. Two alternatively spliced transcript variants encoding different isoforms have been described for this gene.
Upon binding DNA, the protein cyclic GMP-AMP Synthase (cGAS) triggers reaction of GTP and ATP to form cyclic GMP-AMP (cGAMP). cGAMP binds to Stimulator of Interferon Genes (STING) which triggers phosphorylation of IRF3 via TBK1. IRF3 can then go to the nucleus to trigger transcription of inflammatory genes. This pathway plays a critical role in mediating immune defense against double-stranded DNA viruses.