Histone acetyltransferases ( HATs ) are enzymes that acetylate conserved lysine amino acids on histone proteins by transferring an acetyl group from acetyl-CoA to form ε- N -acetyllysine. DNA is wrapped around histones, and, by transferring an acetyl group to the histones, genes can be turned on and off. In general, histone acetylation increases gene expression.
146-723: In general, histone acetylation is linked to transcriptional activation and associated with euchromatin . Euchromatin, which is less densely compact, allows transcription factors to bind more easily to regulatory sites on DNA, causing transcriptional activation. When it was first discovered, it was thought that acetylation of lysine neutralizes the positive charge normally present, thus reducing affinity between histone and (negatively charged) DNA, which renders DNA more accessible to transcription factors . Research has emerged, since, to show that lysine acetylation and other posttranslational modifications of histones generate binding sites for specific protein–protein interaction domains, such as
292-427: A bromodomain , a 110-amino acid module that recognizes acetylated lysine residues and is functionally linked to the co-activators in the regulation of transcription. The ability of histone acetyltransferases to manipulate chromatin structure and lay an epigenetic framework makes them essential in cell maintenance and survival. The process of chromatin remodeling involves several enzymes, including HATs, that assist in
438-548: A vise , with the central core of the protein at the base and the N- and C-terminal segments on the sides. The p300/CBP HATs have larger HAT domains (about 500 residues) than those present in the GNAT and MYST families. They also contain a bromodomain as well as three cysteine/histidine-rich domains that are thought to mediate interactions with other proteins. The structure of p300/CBP is characterized by an elongated globular domain, which contains
584-505: A 3-basepair DNA sequence to generate 3-finger, 4-, 5-, or 6-finger arrays that recognize target sites ranging from 9 basepairs to 18 basepairs in length. Another method uses 2-finger modules to generate zinc finger arrays with up to six individual zinc fingers. The Barbas Laboratory of The Scripps Research Institute used phage display to develop and characterize zinc finger domains that recognize most DNA triplet sequences while another group isolated and characterized individual fingers from
730-488: A C-terminal activation domain that is functional in the absence of the HAT domain. In addition to those that are members of the GNAT and MYST families, there are several other proteins found typically in higher eukaryotes that exhibit HAT activity. These include p300/CBP, nuclear receptor coactivators (e.g., ACTR/SRC-1), TAF II 250, TFIIIC, Rtt109, and CLOCK . p300/CBP are metazoan -specific and contain several zinc finger regions,
876-477: A CpG island while only about 6% of enhancer sequences have a CpG island. CpG islands constitute regulatory sequences, since if CpG islands are methylated in the promoter of a gene this can reduce or silence gene transcription. DNA methylation regulates gene transcription through interaction with methyl binding domain (MBD) proteins, such as MeCP2, MBD1 and MBD2. These MBD proteins bind most strongly to highly methylated CpG islands . These MBD proteins have both
1022-505: A bacterial two-hybrid system and has been dubbed "OPEN" by its creators. This system combines pre-selected pools of individual zinc fingers that were each selected to bind a given triplet and then utilizes a second round of selection to obtain 3-finger arrays capable of binding a desired 9-bp sequence. This system was developed by the Zinc Finger Consortium as an alternative to commercial sources of engineered zinc finger arrays. It
1168-457: A bromodomain, a catalytic (HAT) domain, and regions that interact with other transcription factors. Importantly, the HAT domain shows no sequence homology to other known HATs, and it is required for p300/CBP to function in transcriptional activation. In addition, these proteins contain several HAT domain motifs (A, B, and D) that are similar to those of the GNATs. They also possess a novel motif E that
1314-721: A bromodomain, as their targets are unacetylated. The acetyl groups added by type B HATs to the histones are removed by HDACs once they enter the nucleus and are incorporated into chromatin . Hat1 is one of the few known examples of a type B HAT. Despite this historical classification of HATs, some HAT proteins function in multiple complexes or locations and would thus not easily fit into a particular class. HATs can be grouped into several different families based on sequence homology as well as shared structural features and functional roles. The Gcn5-related N -acetyltransferase (GNAT) family includes Gcn5, PCAF , Hat1, Elp3 , Hpa2, Hpa3, ATF-2 , and Nut1. These HATs are generally characterized by
1460-416: A complementary, antiparallel RNA strand called a primary transcript . In virology , the term transcription is used when referring to mRNA synthesis from a viral RNA molecule. The genome of many RNA viruses is composed of negative-sense RNA which acts as a template for positive sense viral messenger RNA - a necessary step in the synthesis of viral proteins needed for viral replication . This process
1606-436: A conserved active site lysine residue, and this modification is required for their function in vivo . Human p300 contains a highly basic loop embedded in the middle of its HAT domain that is hyperacetylated in the active form of the enzyme. It has been proposed that, upon autoacetylation, this loop is released from the electronegative substrate binding site where it sits in the inactive HAT. Acetylation of yeast Rtt109 at Lys290
SECTION 10
#17328834881161752-634: A context-dependent manner. HATs act as transcriptional co-activators or gene silencers and are most often found in large complexes made up of 10 to 20 subunits, some of which shared among different HAT complexes. These complexes include SAGA (Spt/Ada/Gcn5L acetyltransferase), PCAF, ADA (transcriptional adaptor), TFIID (transcription factor II D), TFTC (TBP-free TAF-containing complex), and NuA3/NuA4 (nucleosomal acetyltransferases of H3 and H4). These complexes modulate HAT specificity by bringing HATs to their target genes where they can then acetylate nucleosomal histones. Some HAT transcriptional co-activators contain
1898-435: A double-strand break to a desired genomic locus can be used to introduce frame-shift mutations into the coding sequence of a gene due to the error-prone nature of the non-homologous DNA repair pathway. If a homologous DNA "donor sequence" is also used then the genomic locus can be converted to a defined sequence via the homology directed repair pathway. An ongoing clinical trial is evaluating Zinc finger nucleases that disrupt
2044-505: A few. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. Initially, the term zinc finger was used solely to describe DNA-binding motif found in Xenopus laevis ; however, it is now used to refer to any number of structures related by their coordination of a zinc ion. In general, zinc fingers coordinate zinc ions with a combination of cysteine and histidine residues. Originally,
2190-513: A given DNA target from a large pool of partially randomized zinc finger arrays. This technique is difficult to use on more than a single zinc finger at a time, so a multi-step process that generated a completely optimized 3-finger array by adding and optimizing a single zinc finger at a time was developed. More recent efforts have utilized yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. A promising new method to select novel 3-finger zinc finger arrays utilizes
2336-667: A given gene can be used to alter the transcription of that gene. Fusions between engineered zinc finger arrays and protein domains that cleave or otherwise modify DNA can also be used to target those activities to desired genomic loci. The most common applications for engineered zinc finger arrays include zinc finger transcription factors and zinc finger nucleases , but other applications have also been described. Typical engineered zinc finger arrays have between 3 and 6 individual zinc finger motifs and bind target sites ranging from 9 basepairs to 18 basepairs in length. Arrays with 6 zinc finger motifs are particularly attractive because they bind
2482-438: A high degree of homology throughout their sequences. These proteins have a 400-residue N-terminal region that is absent in yeast Gcn5, but their HAT functions are evolutionarily conserved with respect to the latter. Hat1 was the first HAT protein to be identified. It is responsible for most of the cytoplasmic HAT activity in yeast, and it binds strongly to histone H4 by virtue of its association with an additional subunit, Hat2. Elp3
2628-459: A high level of specificity can be achieved in triggering specific responses. An example of this specificity is when histone H4 is acetylated at lysines 5 and 12. This acetylation pattern has been seen during histone synthesis. Another example is acetylation of H4K16, which has been associated with dosage compensation of the male X chromosome in Drosophila melanogaster . Histone modifications modulate
2774-414: A human cell ) generally bind to specific motifs on an enhancer and a small combination of these enhancer-bound transcription factors, when brought close to a promoter by a DNA loop, govern level of transcription of the target gene. Mediator (a complex usually consisting of about 26 proteins in an interacting structure) communicates regulatory signals from enhancer DNA-bound transcription factors directly to
2920-478: A hypothesized structure from the African clawed frog ( Xenopus laevis ) transcription factor IIIA . However, it has been found to encompass a wide variety of differing protein structures in eukaryotic cells. Xenopus laevis TFIIIA was originally demonstrated to contain zinc and require the metal for function in 1983, the first such reported zinc requirement for a gene regulatory protein followed soon thereafter by
3066-513: A loop and a second β-hairpin of varying length and conformation can be present between the N-terminal β-hairpin and the C-terminal α-helix. These fingers are present in a diverse group of proteins that frequently do not share sequence or functional similarity with each other. The best-characterized proteins containing treble-clef zinc fingers are the nuclear hormone receptors . The zinc ribbon fold
SECTION 20
#17328834881163212-473: A methyl-CpG-binding domain as well as a transcription repression domain. They bind to methylated DNA and guide or direct protein complexes with chromatin remodeling and/or histone modifying activity to methylated CpG islands. MBD proteins generally repress local chromatin such as by catalyzing the introduction of repressive histone marks, or creating an overall repressive chromatin environment through nucleosome remodeling and chromatin reorganization. As noted in
3358-478: A multisubunit complex in which the other subunits are necessary for them to modify histone residues around the binding site. These enzymes can also modify non-histone proteins. Histone acetyltransferases serve many biological roles inside the cell. Chromatin is a combination of proteins and DNA found in the nucleus , and it undergoes many structural changes as different cellular events such as DNA replication , DNA repair , and transcription occur. Chromatin in
3504-542: A promoter. (RNA polymerase is called a holoenzyme when sigma subunit is attached to the core enzyme which is consist of 2 α subunits, 1 β subunit, 1 β' subunit only). Unlike eukaryotes, the initiating nucleotide of nascent bacterial mRNA is not capped with a modified guanine nucleotide. The initiating nucleotide of bacterial transcripts bears a 5′ triphosphate (5′-PPP), which can be used for genome-wide mapping of transcription initiation sites. In archaea and eukaryotes , RNA polymerase contains subunits homologous to each of
3650-611: A quaternary complex. This octameric complex, in association with the 147 base pairs of DNA coiled around it, forms the nucleosome . Histone H1 locks the nucleosome complex together, and it is the last protein to bind in the complex. Histones tend to be positively charged proteins with N-terminal tails that stem from the core. The phosphodiester backbone of DNA is negative, which allows for strong ionic interactions between histone proteins and DNA. Histone acetyltransferases transfer an acetyl group to specific lysine residues on histones, which neutralizes their positive charge and thus reduces
3796-428: A segment of DNA into RNA. Some segments of DNA are transcribed into RNA molecules that can encode proteins , called messenger RNA (mRNA). Other segments of DNA are transcribed into RNA molecules called non-coding RNAs (ncRNAs). Both DNA and RNA are nucleic acids , which use base pairs of nucleotides as a complementary language. During transcription, a DNA sequence is read by an RNA polymerase , which produces
3942-487: A seven-stranded β-sheet in the center that is surrounded by nine α-helices and several loops. The structure of the central core region associated with acetyl-CoA binding is conserved with respect to GNAT and MYST HATs, but there are many structural differences in the regions flanking this central core. Overall, the structural data is consistent with the fact that p300/CBP HATs are more promiscuous than GNAT and MYST HATs with respect to substrate binding. The structure of Rtt109
4088-625: A single copy of a gene. The characteristic elongation rates in prokaryotes and eukaryotes are about 10–100 nts/sec. In eukaryotes, however, nucleosomes act as major barriers to transcribing polymerases during transcription elongation. In these organisms, the pausing induced by nucleosomes can be regulated by transcription elongation factors such as TFIIS. Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases. In eukaryotes, this may correspond with short pauses during transcription that allow appropriate RNA editing factors to bind. These pauses may be intrinsic to
4234-412: A specific sequence is an area of active research, and zinc finger nucleases and zinc finger transcription factors are two of the most important applications of this to be realized to date. Zinc fingers were first identified in a study of transcription in the African clawed frog , Xenopus laevis in the laboratory of Aaron Klug . A study of the transcription of a particular RNA sequence revealed that
4380-464: A study of brain cortical neurons, 24,937 loops were found, bringing enhancers to their target promoters. Multiple enhancers, each often at tens or hundred of thousands of nucleotides distant from their target genes, loop to their target gene promoters and can coordinate with each other to control transcription of their common target gene. The schematic illustration in this section shows an enhancer looping around to come into close physical proximity with
4526-513: A target site that is long enough to have a good chance of being unique in a mammalian genome. Engineered zinc finger arrays are often fused to a DNA cleavage domain (usually the cleavage domain of FokI ) to generate zinc finger nucleases . Such zinc finger-FokI fusions have become useful reagents for manipulating genomes of many higher organisms including Drosophila melanogaster , Caenorhabditis elegans , tobacco , corn , zebrafish , various types of mammalian cells, and rats . Targeting
Histone acetyltransferase - Misplaced Pages Continue
4672-446: A three-stranded β-sheet followed by a long α-helix parallel to and spanning one side of it. The core region, which corresponds to motifs A, B, and D of the GNAT proteins, is flanked on opposite sides by N- and C-terminal α/β segments that are structurally unique for a given HAT family. The central core and the flanking segments together form a cleft over the former, which is where histone substrates can bind prior to catalysis. While
4818-548: A variety of functions such as binding RNA and mediating protein-protein interactions, but is best known for its role in sequence-specific DNA-binding proteins such as Zif268 (Egr1). In such proteins, individual zinc finger domains typically occur as tandem repeats with two, three, or more fingers comprising the DNA-binding domain of the protein. These tandem arrays can bind in the major groove of DNA and are typically spaced at 3-bp intervals. The α-helix of each domain (often called
4964-447: A variety of ways: Some viruses (such as HIV , the cause of AIDS ), have the ability to transcribe RNA into DNA. HIV has an RNA genome that is reverse transcribed into DNA. The resulting DNA can be merged with the DNA genome of the host cell. The main enzyme responsible for synthesis of DNA from an RNA template is called reverse transcriptase . In the case of HIV, reverse transcriptase
5110-460: A water molecule for removal of a proton from the amine group on lysine, which activates it for direct nucleophilic attack on the carbonyl carbon of enzyme-bound acetyl-CoA. After the reaction, the acetylated histone is released first followed by CoA. Studies of yeast Esa1 from the MYST family of HATs have revealed a ping-pong mechanism involving conserved glutamate and cysteine residues. The first part of
5256-415: Is rifampicin , which inhibits bacterial transcription of DNA into mRNA by inhibiting DNA-dependent RNA polymerase by binding its beta-subunit, while 8-hydroxyquinoline is an antifungal transcription inhibitor. The effects of histone methylation may also work to inhibit the action of transcription. Potent, bioactive natural products like triptolide that inhibit mammalian transcription via inhibition of
5402-409: Is a particular transcription factor that is important for regulation of methylation of CpG islands. An EGR1 transcription factor binding site is frequently located in enhancer or promoter sequences. There are about 12,000 binding sites for EGR1 in the mammalian genome and about half of EGR1 binding sites are located in promoters and half in enhancers. The binding of EGR1 to its target DNA binding site
5548-494: Is also altered in response to signals. The three mammalian DNA methyltransferasess (DNMT1, DNMT3A, and DNMT3B) catalyze the addition of methyl groups to cytosines in DNA. While DNMT1 is a maintenance methyltransferase, DNMT3A and DNMT3B can carry out new methylations. There are also two splice protein isoforms produced from the DNMT3A gene: DNA methyltransferase proteins DNMT3A1 and DNMT3A2. The splice isoform DNMT3A2 behaves like
5694-404: Is also observed to acetylate H3K14 in vitro on free histones. Esa1 can also acetylate H3K14 in vitro on free histones as well as H2AK5, H4K5, H4K8, and H4K12 either in vitro or in vivo on nucleosomal histones. H2AK7 and H2BK16 are also observed to be acetylated by Esa1 in vivo . Notably, neither Sas2 nor Esa1 can acetylate nucleosomal histones in vitro as a free enzyme. This happens to be
5840-433: Is also required for it to exhibit full catalytic activity. Some HATs are also inhibited by acetylation. For example, the HAT activity of the nuclear receptor coactivator ACTR is inhibited upon acetylation by p300/CBP. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are recruited to their target promoters through physical interactions with sequence-specific transcription factors. They usually function within
5986-584: Is an oncogene found in humans. Esa1 was the first essential HAT to be found in yeast, and MOF is its homolog in fruit flies. The HAT activity of the latter is required for the twofold increased transcription of the male X chromosome ( dosage compensation ) in flies. Human HBO1 (HAT bound to ORC1) was the first HAT shown to associate with components of the origin of replication complex . MORF (MOZ-related factor) exhibits very close homology to MOZ throughout its entire length. It contains an N-terminal repression region that decreases its HAT activity in vitro as well as
Histone acetyltransferase - Misplaced Pages Continue
6132-597: Is an example of a type A HAT found in yeast. It is part of the RNA polymerase II holoenzyme and plays a role in transcriptional elongation . The MYST family of HATs is named after its four founding members MOZ , Ybf2 (Sas3), Sas2, and Tip60 . Other important members include Esa1 , MOF , MORF , and HBO1 . These HATs are typically characterized by the presence of zinc fingers and chromodomains , and they are found to acetylate lysine residues on histones H2A , H3, and H4. Several MYST family proteins contain zinc fingers as well as
6278-425: Is another nuclear receptor coactivator with HAT activity, and it also interacts with p300/CBP. A table summarizing the different families of HATs along with their associated members, parent organisms, multisubunit complexes, histone substrates, and structural features is presented below. HAT-A2 (nuclear receptor coactivators) In general, HATs are characterized by a structurally conserved core region made up of
6424-678: Is best suited for most applications. The most straightforward method to generate new zinc finger arrays is to combine smaller zinc finger "modules" of known specificity. The structure of the zinc finger protein Zif268 bound to DNA described by Pavletich and Pabo in their 1991 publication has been key to much of this work and describes the concept of obtaining fingers for each of the 64 possible base pair triplets and then mixing and matching these fingers to design proteins with any desired sequence specificity. The most common modular assembly process involves combining separate zinc fingers that can each recognize
6570-404: Is called garcinol. This compound is found within the rinds of the garcinia indica fruit, otherwise known as mangosteen . To explore the effects of garcinol on histone acetyltransferases, researchers used HeLa cells. The cells underwent irradiation, creating double-strand breaks within the DNA, and garcinol was introduced into the cells to see if it influenced the DNA damage response. If garcinol
6716-437: Is catalyzed by a viral RNA dependent RNA polymerase . A DNA transcription unit encoding for a protein may contain both a coding sequence , which will be translated into the protein, and regulatory sequences , which direct and regulate the synthesis of that protein. The regulatory sequence before ( upstream from) the coding sequence is called the five prime untranslated regions (5'UTR); the sequence after ( downstream from)
6862-447: Is characterised by two beta-hairpins forming two structurally similar zinc-binding sub-sites. The canonical members of this class contain a binuclear zinc cluster in which two zinc ions are bound by six cysteine residues. These zinc fingers can be found in several transcription factors including the yeast Gal4 protein. The zinc finger antiviral protein ( ZAP ) binds to the CpG site. It
7008-424: Is determined by its three-dimensional structure, but it can also be recognized based on the primary structure of the protein or the identity of the ligands coordinating the zinc ion. In spite of the large variety of these proteins, however, the vast majority typically function as interaction modules that bind DNA , RNA , proteins, or other small, useful molecules, and variations in structure serve primarily to alter
7154-602: Is followed by 3' guanine or CpG sites ). 5-methylcytosine (5-mC) is a methylated form of the DNA base cytosine (see Figure). 5-mC is an epigenetic marker found predominantly within CpG sites. About 28 million CpG dinucleotides occur in the human genome. In most tissues of mammals, on average, 70% to 80% of CpG cytosines are methylated (forming 5-methylCpG or 5-mCpG). However, unmethylated cytosines within 5'cytosine-guanine 3' sequences often occur in groups, called CpG islands , at active promoters. About 60% of promoter sequences have
7300-435: Is found in most GNATs, but it is not present in the majority of other known HATs. The yeast Gcn5 (general control nonderepressible-5) HAT is one of the best-characterized members of this family. It has four functional domains, including an N-terminal domain, a highly conserved catalytic (HAT) domain, an Ada2 interaction domain, and a C-terminal bromodomain. PCAF (p300/CBP-associated factor) and GCN5 are mammalian GNATs that share
7446-430: Is homologous to sequences in the HAT domains of GNATs. TFIIIC is one of the general transcription factors involved in RNA polymerase III -mediated transcription. Three components in the human protein have been shown to possess independent HAT activity ( hTFIIIC220 , hTFIIIC110 , and hTFIIIC90 ). Rtt109 is a fungal -specific HAT that requires association with histone chaperone proteins for activity. The HAT activities of
SECTION 50
#17328834881167592-735: Is hypoacetylated at a lysine 16 residue (H4K16) and this defect is due to reduced association of histone acetyltransferase, Mof, to the nuclear matrix Spinocerebellar ataxia type 1 is a neurodegenerative disease that arises as a result of a defective mutant Ataxin-1 protein. Mutant Ataxin-1 reduces histone acetylation resulting in repressed histone acetyltransferase-mediated transcription . HATs have also been associated with control of learning and memory functions. Studies have shown that mice without PCAF or CBP display evidence of neurodegeneration . Mice with PCAF deletion are incompetent with respect to learning, and those with CBP deletion seem to suffer from long-term memory loss. The misregulation of
7738-475: Is insensitive to cytosine methylation in the DNA. While only small amounts of EGR1 transcription factor protein are detectable in cells that are un-stimulated, translation of the EGR1 gene into protein at one hour after stimulation is drastically elevated. Production of EGR1 transcription factor proteins, in various types of cells, can be stimulated by growth factors, neurotransmitters, hormones, stress and injury. In
7884-566: Is known to interact with p300/CBP and PCAF, and its HAT domain is located in its C-terminal region. ACTR (also known as RAC3, AIB1, and TRAM-1 in humans) shares significant sequence homology with SRC-1, in particular in the N-terminal and C-terminal (HAT) regions as well as in the receptor and coactivator interaction domains. ACTR also interacts with p300/CBP and PCAF. The former can prevent ACTR from binding to and activating its receptor by acetylating it in its receptor interaction domain. TIF-2 (transcriptional intermediary factor 2; also known as GRIP1)
8030-403: Is not the only regulatory post-translational modification to histones that dictates chromatin structure; methylation, phosphorylation, ADP-ribosylation, and ubiquitination have also been reported. These combinations of different covalent modifications on the N-terminal tails of histones have been referred to as the histone code , and it is thought that this code may be heritable and preserved in
8176-599: Is not yet known. 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
8322-544: Is rather promiscuous with regard to substrate binding. Whereas it appears that only three to five residues on either side of the lysine to be acetylated are necessary for effective substrate binding and catalysis by members of the GNAT and p300/CBP families, more distal regions of the substrate may be important for efficient acetylation by MYST family HATs. Different HATs, usually in the context of multisubunit complexes, have been shown to acetylate specific lysine residues in histones. Gcn5 cannot acetylate nucleosomal histones in
8468-429: Is regulated by two types of mechanisms: (1) interaction with regulatory protein subunits and (2) autoacetylation. A given HAT may be regulated in multiple ways, and the same effector may actually lead to different outcomes under different conditions. Although it is clear that the association of HATs with multiprotein complexes provides a mechanism for the regulation of both HAT activity and substrate specificity in vivo ,
8614-510: Is responsible for synthesizing a complementary DNA strand (cDNA) to the viral RNA genome. The enzyme ribonuclease H then digests the RNA strand, and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure (cDNA). The cDNA is integrated into the host cell's genome by the enzyme integrase , which causes the host cell to generate viral proteins that reassemble into new viral particles. In HIV, subsequent to this,
8760-413: Is successful at inhibiting the process of non-homologous end joining , a DNA repair mechanism that shows preference in fixing double-strand breaks, then it may serve as a radiosensitizer , a molecule that increases the sensitivity of cells to radiation damage. Increases in radiosensitivity may increase the effectiveness of radiotherapy. DNA transcription Transcription is the process of copying
8906-428: Is synthesized, at which point promoter escape occurs and a transcription elongation complex is formed. Mechanistically, promoter escape occurs through DNA scrunching , providing the energy needed to break interactions between RNA polymerase holoenzyme and the promoter. In bacteria, it was historically thought that the sigma factor is definitely released after promoter clearance occurs. This theory had been known as
SECTION 60
#17328834881169052-408: Is used in mammals for antiviral defense. Various protein engineering techniques can be used to alter the DNA-binding specificity of zinc fingers and tandem repeats of such engineered zinc fingers can be used to target desired genomic DNA sequences. Fusing a second protein domain such as a transcriptional activator or repressor to an array of engineered zinc fingers that bind near the promoter of
9198-435: Is very similar to that of p300, despite there only being 7% sequence identity between the two proteins. There is a seven-stranded β-sheet that is surrounded by α-helices as well as a loop that is involved in acetyl-CoA substrate binding. Despite the conserved structure, Rtt109 and p300/CBP are functionally unique. For instance, the substrate binding site of the former is more similar to that of the GNAT and MYST HATs. In addition,
9344-570: The Krüppel factor in Drosophila . It often appears as a metal-binding domain in multi-domain proteins. Proteins that contain zinc fingers ( zinc finger proteins ) are classified into several different structural families. Unlike many other clearly defined supersecondary structures such as Greek keys or β hairpins , there are a number of types of zinc fingers, each with a unique three-dimensional architecture. A particular zinc finger protein's class
9490-458: The Mfd ATPase can remove a RNA polymerase stalled at a lesion by prying open its clamp. It also recruits nucleotide excision repair machinery to repair the lesion. Mfd is proposed to also resolve conflicts between DNA replication and transcription. In eukayrotes, ATPase TTF2 helps to suppress the action of RNAP I and II during mitosis , preventing errors in chromosomal segregation. In archaea,
9636-498: The obligate release model. However, later data showed that upon and following promoter clearance, the sigma factor is released according to a stochastic model known as the stochastic release model . In eukaryotes, at an RNA polymerase II-dependent promoter, upon promoter clearance, TFIIH phosphorylates serine 5 on the carboxy terminal domain of RNA polymerase II, leading to the recruitment of capping enzyme (CE). The exact mechanism of how CE induces promoter clearance in eukaryotes
9782-414: The "recognition helix") can make sequence-specific contacts to DNA bases; residues from a single recognition helix can contact four or more bases to yield an overlapping pattern of contacts with adjacent zinc fingers. This fold group is defined by two short β-strands connected by a turn (zinc knuckle) followed by a short helix or loop and resembles the classical Cys 2 His 2 motif with a large portion of
9928-523: The 3' end to the 5' end during transcription (3' → 5'). The complementary RNA is created in the opposite direction, in the 5' → 3' direction, matching the sequence of the sense strand except switching uracil for thymine. This directionality is because RNA polymerase can only add nucleotides to the 3' end of the growing mRNA chain. This use of only the 3' → 5' DNA strand eliminates the need for the Okazaki fragments that are seen in DNA replication. This also removes
10074-601: The BRCA1 promoter (see Low expression of BRCA1 in breast and ovarian cancers ). Active transcription units are clustered in the nucleus, in discrete sites called transcription factories or euchromatin . Such sites can be visualized by allowing engaged polymerases to extend their transcripts in tagged precursors (Br-UTP or Br-U) and immuno-labeling the tagged nascent RNA. Transcription factories can also be localized using fluorescence in situ hybridization or marked by antibodies directed against polymerases. There are ~10,000 factories in
10220-545: The CCR5 gene in CD4 human T-cells as a potential treatment for HIV/AIDS . The majority of engineered zinc finger arrays are based on the zinc finger domain of the murine transcription factor Zif268, although some groups have used zinc finger arrays based on the human transcription factor SP1. Zif268 has three individual zinc finger motifs that collectively bind a 9 bp sequence with high affinity. The structure of this protein bound to DNA
10366-479: The Cys 2 His 2 -like (the "classic zinc finger"), treble clef, and zinc ribbon. The following table shows the different structures and their key features: The Cys 2 His 2 -like fold group (C2H2) is by far the best-characterized class of zinc fingers, and is common in mammalian transcription factors. Such domains adopt a simple ββα fold and have the amino acid sequence motif : This class of zinc fingers can have
10512-524: The Eta ATPase is proposed to play a similar role. Genome damage occurs with a high frequency, estimated to range between tens and hundreds of thousands of DNA damages arising in each cell every day. The process of transcription is a major source of DNA damage, due to the formation of single-strand DNA intermediates that are vulnerable to damage. The regulation of transcription by processes using base excision repair and/or topoisomerases to cut and remodel
10658-478: The GNAT family have a conserved glutamate residue that acts as a general base for catalyzing the nucleophilic attack of the lysine amine on the acetyl-CoA thioester bond. These HATs use an ordered sequential bi-bi mechanism wherein both substrates (acetyl-CoA and histone) must bind to form a ternary complex with the enzyme before catalysis can occur. Acetyl-CoA binds first, followed by the histone substrate. A conserved glutamate residue (Glu173 in yeast Gcn5) activates
10804-458: The MYST family have HAT domains that are about 250 residues in length. Many MYST proteins also contain a cysteine-rich, zinc-binding domain within the HAT region in addition to an N-terminal chromodomain, which binds to methylated lysine residues . On a broader scale, the structures of the catalytic domains of GNAT proteins (Gcn5, PCAF) exhibit a mixed α/β globular fold with a total of five α-helices and six β-strands. The overall topology resembles
10950-502: The RNA polymerase II (pol II) enzyme bound to the promoter. Enhancers, when active, are generally transcribed from both strands of DNA with RNA polymerases acting in two different directions, producing two enhancer RNAs (eRNAs) as illustrated in the Figure. An inactive enhancer may be bound by an inactive transcription factor. Phosphorylation of the transcription factor may activate it and that activated transcription factor may then activate
11096-400: The RNA polymerase and one or more general transcription factors binding to a DNA promoter sequence to form an RNA polymerase-promoter closed complex. In the closed complex, the promoter DNA is still fully double-stranded. RNA polymerase, assisted by one or more general transcription factors, then unwinds approximately 14 base pairs of DNA to form an RNA polymerase-promoter open complex. In
11242-654: The RNA polymerase or due to chromatin structure. Double-strand breaks in actively transcribed regions of DNA are repaired by homologous recombination during the S and G2 phases of the cell cycle . Since transcription enhances the accessibility of DNA to exogenous chemicals and internal metabolites that can cause recombinogenic lesions, homologous recombination of a particular DNA sequence may be strongly stimulated by transcription. Bacteria use two different strategies for transcription termination – Rho-independent termination and Rho-dependent termination. In Rho-independent transcription termination , RNA transcription stops when
11388-1102: The XPB subunit of the general transcription factor TFIIH has been recently reported as a glucose conjugate for targeting hypoxic cancer cells with increased glucose transporter production. In vertebrates, the majority of gene promoters contain a CpG island with numerous CpG sites . When many of a gene's promoter CpG sites are methylated the gene becomes inhibited (silenced). Colorectal cancers typically have 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations. However, transcriptional inhibition (silencing) may be of more importance than mutation in causing progression to cancer. For example, in colorectal cancers about 600 to 800 genes are transcriptionally inhibited by CpG island methylation (see regulation of transcription in cancer ). Transcriptional repression in cancer can also occur by other epigenetic mechanisms, such as altered production of microRNAs . In breast cancer, transcriptional repression of BRCA1 may occur more frequently by over-produced microRNA-182 than by hypermethylation of
11534-466: The ability to acetylate multiple sites in both histones H2B and H3 when it joins other subunits to form the SAGA and ADA complexes. Moreover, the acetylation site specificity of Rtt109 is dictated by its association with either Vps75 or Asf1. When in complex with the former, Rtt109 acetylates H3K9 and H3K27, but, when in complex with the latter, it preferentially acetylates H3K56. The catalytic activity of HATs
11680-432: The absence of other protein factors. In the context of complexes like SAGA and ADA, however, Gcn5 is able to acetylate H3K14 among other sites within histones H2B, H3, and H4 (e.g., H3K9, H3K36, H4K8, H4K16). Both Gcn5 and PCAF have the strongest site preference for H3K14, either as a free histone or within a nucleosome. Hat1 acetylates H4K5 and H4K12, and Hpa2 acetylates H3K14 in vitro . In flies, acetylation of H4K16 on
11826-441: The acetyllysine-binding bromodomain . Histone acetyltransferases can also acetylate non-histone proteins, such as nuclear receptors and other transcription factors to facilitate gene expression. HATs are traditionally divided into two different classes based on their subcellular localization. Type A HATs are located in the nucleus and are involved in the regulation of gene expression through acetylation of nucleosomal histones in
11972-663: The androgen and estrogen (α) receptors, GATA-2, GATA-3 , MyoD, E2F(1-3), p73 α, retinoblastoma (Rb), NF-κB (p50, p65), Smad7 , importin-α , Ku70, YAP1 , E1A adenovirus protein, and S-HDAg ( hepatitis delta virus small delta antigen). p300/CBP have also been observed to acetylate β-catenin , RIP140 , PCNA , the DNA metabolic enzymes flap endonuclease-1 , thymine DNA glycosylase , and Werner syndrome DNA helicase , STAT6 , Runx1 (AML1) , UBF, Beta2/NeuroD, CREB , c-Jun , C/EBPβ, NF-E2 , SREBP , IRF2, Sp3 , YY1, KLF13, EVI1, BCL6 , HNF-4 , ER81 and FOXO4 (AFX) . The formation of multisubunit complexes has been observed to modulate
12118-429: The base and is flanked on opposite sides by the variable N- and C-terminal segments that mediate the majority of the interactions with the substrate peptide. It is likely that these variable regions are at least in part responsible for the observed specificity of different HATs for various histone substrates. Members of the GNAT and MYST families as well as Rtt109 exhibit greater substrate selectivity than p300/CBP, which
12264-405: The binding specificity of a particular protein. Since their original discovery and the elucidation of their structure, these interaction modules have proven ubiquitous in the biological world and may be found in 3% of the genes of the human genome. In addition, zinc fingers have become extremely useful in various therapeutic and research capacities. Engineering zinc fingers to have an affinity for
12410-461: The binding strength of a small transcription factor (transcription factor IIIA; TFIIIA) was due to the presence of zinc-coordinating finger-like structures. Amino acid sequencing of TFIIIA revealed nine tandem sequences of 30 amino acids, including two invariant pairs of cysteine and histidine residues. Extended x-ray absorption fine structure confirmed the identity of the zinc ligands: two cysteines and two histidines. The DNA-binding loop formed by
12556-743: The brain, when neurons are activated, EGR1 proteins are up-regulated and they bind to (recruit) the pre-existing TET1 enzymes that are produced in high amounts in neurons. TET enzymes can catalyse demethylation of 5-methylcytosine. When EGR1 transcription factors bring TET1 enzymes to EGR1 binding sites in promoters, the TET enzymes can demethylate the methylated CpG islands at those promoters. Upon demethylation, these promoters can then initiate transcription of their target genes. Hundreds of genes in neurons are differentially expressed after neuron activation through EGR1 recruitment of TET1 to methylated regulatory sequences in their promoters. The methylation of promoters
12702-455: The canonical pattern of interactions of zinc fingers with DNA. The binding of zinc finger is found to be distinct from many other DNA-binding proteins that bind DNA through the 2-fold symmetry of the double helix, instead zinc fingers are linked linearly in tandem to bind nucleic acid sequences of varying lengths. Zinc fingers often bind to a sequence of DNA known as the GC box . The modular nature of
12848-484: The case as well for Sas3, which is observed to acetylate H3K9 and H3K14 in vivo as well as lysine residues on H2A and H4. MOZ can also acetylate H3K14. p300/CBP acetylate all four nucleosomal core histones equally well. In vitro , they have been observed to acetylate H2AK5, H2BK12, H2BK15, H3K14, H3K18, H4K5, and H4K8. SRC-1 acetylates H3K9 and H3K14, TAF II 230 (Drosophila homolog of human TAF II 250) acetylates H3K14, and Rtt109 acetylates H3K9, H3K23, and H3K56 in
12994-518: The catalytic HAT subunit can carry out histone acetylation more effectively. In addition, the formation of multisubunit HAT complexes influences the lysine specificity of HATs. The specific lysine residues that a given HAT acetylates may become either broader or more restricted in scope upon association with its respective complex. For example, the lysine specificity of MYST family HATs toward their histone substrates becomes more restricted when they associate with their complexes. In contrast, Gcn5 acquires
13140-454: The catalytic activity of p300/CBP and PCAF in vitro . The human premature aging syndrome Hutchinson Gilford progeria is caused by a mutational defect in the processing of lamin A , a nuclear matrix protein. In a mouse model of this condition, recruitment of repair proteins to sites of DNA damage is delayed. The molecular mechanism underlying this delayed repair response involves a histone acetylation defect. Specifically, histone H4
13286-409: The cell can be found in two states: condensed and uncondensed. The latter, known as euchromatin , is transcriptionally active, whereas the former, known as heterochromatin , is transcriptionally inactive. Histones comprise the protein portion of chromatin. There are five different histone proteins: H1, H2A, H2B, H3, and H4. A core histone is formed when two of each histone subtype, excluding H1, form
13432-456: The central core domain (motif A in GNATs) is involved in acetyl-CoA binding and catalysis, the N- and C-terminal segments assist in binding histone substrates. Unique features related to the sequence and/or structure of the N- and C-terminal regions for different HAT families may help to explain some observed differences among HATs in histone substrate specificity. CoA binding has been observed to widen
13578-412: The coding sequence is called the three prime untranslated regions (3'UTR). As opposed to DNA replication , transcription results in an RNA complement that includes the nucleotide uracil (U) in all instances where thymine (T) would have occurred in a DNA complement. Only one of the two DNA strands serves as a template for transcription. The antisense strand of DNA is read by RNA polymerase from
13724-427: The coding strand (except that thymines are replaced with uracils , and the nucleotides are composed of a ribose (5-carbon) sugar whereas DNA has deoxyribose (one fewer oxygen atom) in its sugar-phosphate backbone). mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be rapidly produced from
13870-438: The context of chromatin. They contain a bromodomain , which helps them recognize and bind to acetylated lysine residues on histone substrates. Gcn5, p300/CBP , and TAF II 250 are some examples of type A HATs that cooperate with activators to enhance transcription. Type B HATs are located in the cytoplasm and are responsible for acetylating newly synthesized histones prior to their assembly into nucleosomes . These HATs lack
14016-663: The controls for copying DNA. As a result, transcription has a lower copying fidelity than DNA replication. Transcription is divided into initiation , promoter escape , elongation, and termination . Setting up for transcription in mammals is regulated by many cis-regulatory elements , including core promoter and promoter-proximal elements that are located near the transcription start sites of genes. Core promoters combined with general transcription factors are sufficient to direct transcription initiation, but generally have low basal activity. Other important cis-regulatory modules are localized in DNA regions that are distant from
14162-495: The coordination of these ligands by zinc were thought to resemble fingers, hence the name. This was followed soon thereafter by the discovery of the Krüppel factor in Drosophila by the Schuh team in 1986. More recent work in the characterization of proteins in various organisms has revealed the importance of zinc ions in polypeptide stabilization. The crystal structures of zinc finger-DNA complexes solved in 1991 and 1993 revealed
14308-417: The enhancer to which it is bound (see small red star representing phosphorylation of transcription factor bound to enhancer in the illustration). An activated enhancer begins transcription of its RNA before activating transcription of messenger RNA from its target gene. Transcription regulation at about 60% of promoters is also controlled by methylation of cytosines within CpG dinucleotides (where 5' cytosine
14454-528: The equilibrium between acetylation and deacetylation has also been associated with the manifestation of certain cancers. If histone acetyltransferases are inhibited, then damaged DNA may not be repaired, eventually leading to cell death. Controlling the chromatin remodeling process within cancer cells may provide a novel drug target for cancer research. Attacking these enzymes within cancer cells could lead to increased apoptosis due to high accumulation of DNA damage. One such inhibitor of histone acetyltransferases
14600-451: The factor. A molecule that allows the genetic material to be realized as a protein was first hypothesized by François Jacob and Jacques Monod . Severo Ochoa won a Nobel Prize in Physiology or Medicine in 1959 for developing a process for synthesizing RNA in vitro with polynucleotide phosphorylase , which was useful for cracking the genetic code . RNA synthesis by RNA polymerase
14746-413: The finger-like folds . They were first identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African clawed frog), however they are now recognised to bind DNA, RNA, protein, and/or lipid substrates . Their binding properties depend on the amino acid sequence of the finger domains and on the linker between fingers, as well as on the higher-order structures and
14892-535: The five RNA polymerase subunits in bacteria and also contains additional subunits. In archaea and eukaryotes, the functions of the bacterial general transcription factor sigma are performed by multiple general transcription factors that work together. In archaea, there are three general transcription factors: TBP , TFB , and TFE . In eukaryotes, in RNA polymerase II -dependent transcription, there are six general transcription factors: TFIIA , TFIIB (an ortholog of archaeal TFB), TFIID (a multisubunit factor in which
15038-495: The general transcription factors TFIIE and TFIIF . Other proteins include CIITA , Brm (chromatin remodeler), NF-κB (p65), TAL1/SCL , Beta2/NeuroD , C/EBPβ , IRF2 , IRF7 , YY1 , KLF13 , EVI1 , AME, ER81 , and the androgen receptor (AR) . PCAF has also been observed to acetylate c-MYC , GATA-2 , retinoblastoma (Rb) , Ku70 , and E1A adenovirus protein. It can also autoacetylate, which facilitates intramolecular interactions with its bromodomain that may be involved in
15184-629: The genome also increases the vulnerability of DNA to damage. RNA polymerase plays a very crucial role in all steps including post-transcriptional changes in RNA. As shown in the image in the right it is evident that the CTD (C Terminal Domain) is a tail that changes its shape; this tail will be used as a carrier of splicing, capping and polyadenylation , as shown in the image on the left. Transcription inhibitors can be used as antibiotics against, for example, pathogenic bacteria ( antibacterials ) and fungi ( antifungals ). An example of such an antibacterial
15330-424: The genome that are major gene-regulatory elements. Enhancers control cell-type-specific gene transcription programs, most often by looping through long distances to come in physical proximity with the promoters of their target genes. While there are hundreds of thousands of enhancer DNA regions, for a particular type of tissue only specific enhancers are brought into proximity with the promoters that they regulate. In
15476-600: The helix and β-hairpin truncated. The retroviral nucleocapsid (NC) protein from HIV and other related retroviruses are examples of proteins possessing these motifs. The gag-knuckle zinc finger in the HIV NC protein is the target of a class of drugs known as zinc finger inhibitors . The treble-clef motif consists of a β-hairpin at the N-terminus and an α-helix at the C-terminus that each contribute two ligands for zinc binding, although
15622-427: The highly conserved motif A found among GNATs that facilitates acetyl-CoA binding. A cysteine-rich region located in the N terminus of the HAT domain of MYST proteins is involved in zinc binding, which is essential for HAT activity. Tip60 (Tat-interactive protein, 60 kDa) was the first human MYST family member to exhibit HAT activity. Sas3 found in yeast is a homolog of MOZ (monocytic leukemia zinc finger protein), which
15768-461: The histone binding groove in the central core by moving the C-terminal segment of Gcn5 outward. In addition, since contacts between CoA and protein facilitate the formation of favorable histone-protein contacts, it is likely that CoA binding precedes histone binding in vivo . HATs in the GNAT family are most notably characterized by an approximately 160-residue HAT domain and a C-terminal bromodomain, which binds to acetylated lysine residues. Those in
15914-406: The histone chaperone proteins Asf1 and Vps75, which may be involved in delivering histone substrates to the enzyme for acetylation. Moreover, a general acid or base have not yet been identified for this HAT. The structures of several HAT domains bound to acetyl-CoA and histone substrate peptides reveal that the latter bind across a groove on the protein that is formed by the central core region at
16060-426: The host cell undergoes programmed cell death, or apoptosis , of T cells . However, in other retroviruses, the host cell remains intact as the virus buds out of the cell. Zinc finger A zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn ) which stabilizes the fold. It was originally coined to describe the finger-like appearance of
16206-548: The human TAF II 250 and CLOCK coactivators have not been studied as extensively. TAF II 250 is one of the TBP-associated factor subunits of TFIID , and it shares a Gly-X-Gly pattern with Gcn5 that is important for HAT activity. CLOCK is a circadian rhythm master regulator that functions with BMAL1 to carry out its HAT activity. Three important nuclear receptor coactivators that display HAT activity are SRC-1 , ACTR , and TIF-2 . Human SRC-1 (steroid receptor coactivator-1)
16352-460: The human genome. A potential drawback with modular assembly in general is that specificities of individual zinc finger can overlap and can depend on the context of the surrounding zinc fingers and DNA. A recent study demonstrated that a high proportion of 3-finger zinc finger arrays generated by modular assembly fail to bind their intended target with sufficient affinity in a bacterial two-hybrid assay and fail to function as zinc finger nucleases , but
16498-581: The key subunit, TBP , is an ortholog of archaeal TBP), TFIIE (an ortholog of archaeal TFE), TFIIF , and TFIIH . The TFIID is the first component to bind to DNA due to binding of TBP, while TFIIH is the last component to be recruited. In archaea and eukaryotes, the RNA polymerase-promoter closed complex is usually referred to as the " preinitiation complex ". Transcription initiation is regulated by additional proteins, known as activators and repressors , and, in some cases, associated coactivators or corepressors , which modulate formation and function of
16644-540: The mRNA, thus releasing the newly synthesized mRNA from the elongation complex. Transcription termination in eukaryotes is less well understood than in bacteria, but involves cleavage of the new transcript followed by template-independent addition of adenines at its new 3' end, in a process called polyadenylation . Beyond termination by a terminator sequences (which is a part of a gene ), transcription may also need to be terminated when it encounters conditions such as DNA damage or an active replication fork . In bacteria,
16790-470: The male X chromosome by MOF in the context of the MSL complex is correlated with transcriptional upregulation as a mechanism for dosage compensation in these organisms. In humans, the MSL complex carries out the majority of genome-wide H4K16 acetylation. In the context of their cognate complexes, Sas2 (SAS) and Esa1 (NuA4) also carry out acetylation of H4K16, in particular in the telomere regions of chromosomes. Sas2
16936-424: The molecular basis for how this actually occurs is still largely unknown. However, data suggests that associated subunits may contribute to catalysis at least in part by facilitating productive binding of the HAT complex to its native histone substrates. The MYST family of HATs, p300/CBP, and Rtt109 have all been shown to be regulated by autoacetylation. Human MOF as well as yeast Esa1 and Sas2 are autoacetylated at
17082-458: The need for an RNA primer to initiate RNA synthesis, as is the case in DNA replication. The non -template (sense) strand of DNA is called the coding strand , because its sequence is the same as the newly created RNA transcript (except for the substitution of uracil for thymine). This is the strand that is used by convention when presenting a DNA sequence. Transcription has some proofreading mechanisms, but they are fewer and less effective than
17228-463: The newly synthesized RNA molecule forms a G-C-rich hairpin loop followed by a run of Us. When the hairpin forms, the mechanical stress breaks the weak rU-dA bonds, now filling the DNA–RNA hybrid. This pulls the poly-U transcript out of the active site of the RNA polymerase, terminating transcription. In Rho-dependent termination, Rho , a protein factor, destabilizes the interaction between the template and
17374-419: The next cell generation. H3 and H4 histone proteins are the primary targets of HATs, but H2A and H2B are also acetylated in vivo . Lysines 9, 14, 18, and 23 of H3 and lysines 5, 8, 12, and 16 of H4 are all targeted for acetylation. Lysines 5, 12, 15, and 20 are acetylated on histone H2B, while only lysines 5 and 9 have been observed to be acetylated on histone H2A. With so many different sites for acetylation,
17520-413: The nucleoplasm of a HeLa cell , among which are ~8,000 polymerase II factories and ~2,000 polymerase III factories. Each polymerase II factory contains ~8 polymerases. As most active transcription units are associated with only one polymerase, each factory usually contains ~8 different transcription units. These units might be associated through promoters and/or enhancers, with loops forming a "cloud" around
17666-425: The number and order of these residues was used to classify different types of zinc fingers ( e.g., Cys 2 His 2 , Cys 4 , and Cys 6 ). More recently, a more systematic method has been used to classify zinc finger proteins instead. This method classifies zinc finger proteins into "fold groups" based on the overall shape of the protein backbone in the folded domain. The most common "fold groups" of zinc fingers are
17812-694: The number of fingers. Znf domains are often found in clusters, where fingers can have different binding specificities. Znf motifs occur in several unrelated protein superfamilies , varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g., some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organization, epithelial development, cell adhesion , protein folding, chromatin remodeling, and zinc sensing, to name but
17958-413: The open complex, the promoter DNA is partly unwound and single-stranded. The exposed, single-stranded DNA is referred to as the "transcription bubble". RNA polymerase, assisted by one or more general transcription factors, then selects a transcription start site in the transcription bubble, binds to an initiating NTP and an extending NTP (or a short RNA primer and an extending NTP) complementary to
18104-409: The p300/CBP HAT family and, unlike enzymes in the GNAT and MYST families, p300 does not employ a general base for catalysis. Rather, it is likely that members of the p300/CBP family use a Theorell-Chance (i.e., “hit-and-run”) acetyl transfer mechanism. Rtt109 is likely to employ a mechanism that is different from that of the other HATs. The yeast enzyme has very low catalytic activity in the absence of
18250-654: The packing of chromatin. The level of packing of the DNA is important for gene transcription, since the transcriptional machinery must have access to the promoter in order for transcription to occur. Neutralization of charged lysine residues by HATs allows for the chromatin to decondense so that this machinery has access to the gene to be transcribed. However, acetylation is not always associated with enhanced transcriptional activity. For instance, acetylation of H4K12 has been associated with condensed and transcriptionally inactive chromatin. In addition, some histone modifications are associated with both enhanced and repressed activity, in
18396-435: The piccolo NuA4 complex, it loses its dependence on the cysteine residue for catalysis, which suggests that the reaction may proceed via a ternary bi-bi mechanism when the enzyme is part of a physiologically relevant multiprotein complex. In human p300, Tyr1467 acts as a general acid and Trp1436 helps orient the target lysine residue of the histone substrate into the active site. These two residues are highly conserved within
18542-414: The presence of a bromodomain, and they are found to acetylate lysine residues on histones H2B , H3 , and H4 . All members of the GNAT family are characterized by up to four conserved motifs (A-D) found within the catalytic HAT domain. This includes the most highly conserved motif A, which contains an Arg/Gln-X-X-Gly-X-Gly/Ala sequence that is important for acetyl-CoA recognition and binding. The C motif
18688-541: The presence of either Asf1 or Vps75. In addition to the core histones, certain HATs acetylate a number of other cellular proteins including transcriptional activators , basal transcription factors , structural proteins, polyamines , and proteins involved in nuclear import. Acetylation of these proteins can alter their ability to interact with their cognate DNA and/or protein substrates. The idea that acetylation can affect protein function in this manner has led to inquiry regarding
18834-649: The previous section, transcription factors are proteins that bind to specific DNA sequences in order to regulate the expression of a gene. The binding sequence for a transcription factor in DNA is usually about 10 or 11 nucleotides long. As summarized in 2009, Vaquerizas et al. indicated there are approximately 1,400 different transcription factors encoded in the human genome by genes that constitute about 6% of all human protein encoding genes. About 94% of transcription factor binding sites (TFBSs) that are associated with signal-responsive genes occur in enhancers while only about 6% of such TFBSs occur in promoters. EGR1 protein
18980-475: The product of a classical immediate-early gene and, for instance, it is robustly and transiently produced after neuronal activation. Where the DNA methyltransferase isoform DNMT3A2 binds and adds methyl groups to cytosines appears to be determined by histone post translational modifications. On the other hand, neural activation causes degradation of DNMT3A1 accompanied by reduced methylation of at least one evaluated targeted promoter. Transcription begins with
19126-418: The promoter of a target gene. The loop is stabilized by a dimer of a connector protein (e.g. dimer of CTCF or YY1 ), with one member of the dimer anchored to its binding motif on the enhancer and the other member anchored to its binding motif on the promoter (represented by the red zigzags in the illustration). Several cell function specific transcription factors (there are about 1,600 transcription factors in
19272-408: The reaction involves the formation of a covalent intermediate in which a cysteine residue becomes acetylated following nucleophilic attack of this residue on the carbonyl carbon of acetyl-CoA. Then, a glutamate residue acts as a general base to facilitate transfer of the acetyl group from the cysteine to the histone substrate in a manner analogous to the mechanism used by GNATs. When Esa1 is assembled in
19418-500: The reformation of nucleosomes and are required for DNA damage repair systems to function. HATs have been implicated as accessories to disease progression, specifically in neurodegenerative disorders. For instance, Huntington's disease is a disease that affects motor skills and mental abilities. The only known mutation that has been implicated in the disease is in the N-terminal region of the protein huntingtin (htt) . It has been reported that htt directly interacts with HATs and represses
19564-492: The regulation of its HAT activity. p300/CBP have many non-histone substrates, including the non-histone chromatin proteins HMG1 , HMG-N1/HMG14 , and HMG-I(Y), the transcriptional activators p53, c-Myb , GATA-1 , EKLF , TCF , and HIV Tat, the nuclear receptor coactivators ACTR, SRC-1, and TIF-2, and the general transcription factors TFIIE and TFIIF. Other substrates include the transcription factors Sp1, KLF5 , FOXO1 , MEF2C , SRY , GATA-4 , and HNF-6 , HMG-B2 , STAT3 ,
19710-424: The residues in the active site of each enzyme are distinct, which suggests that they employ different catalytic mechanisms for acetyl group transfer. The basic mechanism catalyzed by HATs involves the transfer of an acetyl group from acetyl-CoA to the ε-amino group of a target lysine side-chain within a histone. Different families of HATs employ unique strategies in order to effect such a transformation. Members of
19856-472: The role of acetyltransferases in signal transduction pathways and whether an appropriate analogy to kinases and phosphorylation events can be made in this respect. PCAF and p300/CBP are the main HATs that have been observed to acetylate a number of non-histone proteins. For PCAF, these include the non-histone chromatin ( high-mobility group (HMG) ) proteins HMG-N2/HMG17 and HMG-I(Y) , the transcriptional activators p53 , MyoD , E2F(1-3) , and HIV Tat , and
20002-474: The strong interactions between the histone and DNA. Acetylation is also thought to perturb interactions between individual nucleosomes and act as interaction sites for other DNA-associated proteins. There can be different levels of histone acetylation as well as other types of modifications, allowing the cell to have control over the level of chromatin packing during different cellular events such as replication, transcription, recombination, and repair. Acetylation
20148-497: The substrate specificity of HATs. In general, while recombinant HATs are able to acetylate free histones, HATs can acetylate nucleosomal histones only when they are in their respective in vivo HAT complexes. Some of the proteins that associate with HATs in these complexes function by targeting the HAT complex to nucleosomes at specific regions in the genome . For instance, it has been observed that HAT complexes (e.g. SAGA, NuA3) often use methylated histones as docking sites so that
20294-689: The success rate was somewhat higher when sites of the form GNNGNNGNN were targeted. A subsequent study used modular assembly to generate zinc finger nucleases with both 3-finger arrays and 4-finger arrays and observed a much higher success rate with 4-finger arrays. A variant of modular assembly that takes the context of neighboring fingers into account has also been reported and this method tends to yield proteins with improved performance relative to standard modular assembly. Numerous selection methods have been used to generate zinc finger arrays capable of targeting desired sequences. Initial selection efforts utilized phage display to select proteins that bound
20440-431: The transcription initiation complex. After the first bond is synthesized, the RNA polymerase must escape the promoter. During this time there is a tendency to release the RNA transcript and produce truncated transcripts. This is called abortive initiation , and is common for both eukaryotes and prokaryotes. Abortive initiation continues to occur until an RNA product of a threshold length of approximately 10 nucleotides
20586-456: The transcription start site sequence, and catalyzes bond formation to yield an initial RNA product. In bacteria , RNA polymerase holoenzyme consists of five subunits: 2 α subunits, 1 β subunit, 1 β' subunit, and 1 ω subunit. In bacteria, there is one general RNA transcription factor known as a sigma factor . RNA polymerase core enzyme binds to the bacterial general transcription (sigma) factor to form RNA polymerase holoenzyme and then binds to
20732-513: The transcription start sites. These include enhancers , silencers , insulators and tethering elements. Among this constellation of elements, enhancers and their associated transcription factors have a leading role in the initiation of gene transcription. An enhancer localized in a DNA region distant from the promoter of a gene can have a very large effect on gene transcription, with some genes undergoing up to 100-fold increased transcription due to an activated enhancer. Enhancers are regions of
20878-524: The zinc finger motif allows for a large number of combinations of DNA and RNA sequences to be bound with high degree of affinity and specificity, and is therefore ideally suited for engineering protein that can be targeted to and bind specific DNA sequences. In 1994, it was shown that an artificially-constructed three-finger protein can block the expression of an oncogene in a mouse cell line. Zinc fingers fused to various other effector domains, some with therapeutic significance, have since been constructed. Such
21024-454: Was established in vitro by several laboratories by 1965; however, the RNA synthesized by these enzymes had properties that suggested the existence of an additional factor needed to terminate transcription correctly. Roger D. Kornberg won the 2006 Nobel Prize in Chemistry "for his studies of the molecular basis of eukaryotic transcription ". Transcription can be measured and detected in
21170-674: Was its importance that "the zinc-finger motif" was cited in the Scientific Background to the 2024 Nobel Prize in Chemistry (awarded to David Baker , Demis Hassabis , and John M. Jumper for computational protein design and protein structure prediction). Zinc finger (Znf) domains are relatively small protein motifs that contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not, instead binding other metals such as iron, or no metal at all. For example, some family members form salt bridges to stabilise
21316-399: Was solved in 1991 and stimulated a great deal of research into engineered zinc finger arrays. In 1994 and 1995, a number of groups used phage display to alter the specificity of a single zinc finger of Zif268. There are two main methods currently used to generate engineered zinc finger arrays, modular assembly, and a bacterial selection system, and there is some debate about which method
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