Misplaced Pages

#689310

49-458: The TaqMan probe principle relies on the 5´–3´ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore -based detection. As in other quantitative PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR; however,

98-475: A K-homology (KH) domain . In eukaryotes, three different "S1" proteins are bound to the ring, whereas in archaea either one or two different "S1" proteins can be part of the exosome (although there are always three S1 subunits attached to the complex). This ring structure is very similar to that of the proteins RNase PH and PNPase . In bacteria, the protein RNase PH, which is involved in tRNA processing, forms

147-612: A 3’ to 5’ exonuclease subunit, one of the three separately encoded core proteins of the DNA polymerase III holoenzyme. In contrast to E. coli and S. typhimurium , where the polymerase and editing functions are encoded by separate genes, in the bacterial species Buchnera aphidicola the DNA polymerase encoded by the DNA III (polC) gene contains both DNA polymerase and 3’ to 5’ exonuclease domains. An evolutionary divergence (about 0.25 to 1.2 billion years ago), appears to have been associated with

196-477: A 5' exonuclease (human gene Xrn2) to degrade the newly formed transcript downstream, leaving the polyadenylation site and simultaneously shooting the polymerase. This process involves the exonuclease's catching up to the pol II and terminating the transcription. Pol I then synthesizes DNA nucleotides in place of the RNA primer it had just removed. DNA polymerase I also has 3' to 5' and 5' to 3' exonuclease activity, which

245-494: A crucial role in the activity of the yeast exosome complex. While a human homologue of the protein exists, no evidence was found for a long time that its human homologue was associated with the human exosome complex. In 2010, however, it was discovered that humans have three Rrp44 homologues and two of these can be associated with the exosome complex. These two proteins most likely degrade different RNA substrates due to their different cellular localization, with one being localized in

294-709: A distinctive set of proteins is precipitated. Already years before the exosome complex was identified, this pattern was termed the PM/Scl complex . Immunofluorescence using sera from these patients usually shows a typical staining of the nucleolus of cells, which sparked the suggestion that the antigen recognized by autoantibodies might be important in ribosome synthesis . More recently, recombinant exosome proteins have become available and these have been used to develop line immunoassays (LIAs) and enzyme linked immunosorbent assays (ELISAs) for detecting these antibodies. In these diseases, antibodies are mainly directed against two of

343-404: A hexameric ring consisting of six identical RNase PH proteins. In the case of PNPase, which is a phosphorolytic RNA-degrading protein found in bacteria and the chloroplasts and mitochondria of some eukaryotic organisms, two RNase PH domains, and both an S1 and KH RNA binding domain are part of a single protein, which forms a trimeric complex that adopts a structure almost identical to that of

392-473: A minor groove binder (MGB) moiety, dihydrocyclopyrroloindole tripeptide (DPI 3 ), in order to increase its binding affinity to the target sequence; MGB-conjugated probes have a higher melting temperature (T m ) due to increased stabilization of van der Waals forces. As the Taq polymerase extends the primer and synthesizes the nascent strand (from the single-stranded template), the 5' to 3' exonuclease activity of

441-468: A proof reading exonuclease, nsp14-ExoN, that is part of the viral genome is responsible for recombination that is implicated in novel strain emergence. Exosome complex The exosome complex (or PM/Scl complex , often just called the exosome ) is a multi- protein intracellular complex capable of degrading various types of RNA (ribonucleic acid) molecules. Exosome complexes are found in both eukaryotic cells and archaea , while in bacteria

490-418: A simpler complex called the degradosome carries out similar functions. The core of the exosome contains a six-membered ring structure to which other proteins are attached. In eukaryotic cells, the exosome complex is present in the cytoplasm , nucleus , and especially the nucleolus , although different proteins interact with the exosome complex in these compartments regulating the RNA degradation activity of

539-611: A simpler complex that includes the protein PNPase , called the degradosome . The exosome is a key complex in cellular RNA quality control. Unlike prokaryotes, eukaryotes possess highly active RNA surveillance systems that recognise unprocessed and mis-processed RNA-protein complexes (such as ribosomes ) prior to their exit from the nucleus. It is presumed that this system prevents aberrant complexes from interfering with important cellular processes such as protein synthesis . In addition to RNA processing, turnover and surveillance activities,

SECTION 10

#1733085396690

588-410: A specific type of function or requirement. Exonuclease I breaks apart single-stranded DNA in a 3' → 5' direction, releasing deoxyribonucleoside 5'-monophosphates one after another. It does not cleave DNA strands without terminal 3'-OH groups because they are blocked by phosphoryl or acetyl groups. Exonuclease II is associated with DNA polymerase I, which contains a 5' exonuclease that clips off

637-424: A time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the endonuclease , which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA : 5′ to 3′ exonuclease (Xrn1) , which

686-401: Is a 3' to 5' hydrolyzing enzyme that catalyzes linear double-stranded DNA and single-stranded DNA, which requires Ca2+ . This enzyme is extremely important in the process of homologous recombination . Exonuclease VIII is 5' to 3' dimeric protein that does not require ATP or any gaps or nicks in the strand, but requires a free 5' OH group to carry out its function . In Escherichia coli

735-411: Is a dependent decapping protein ; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease. In both archaea and eukaryotes , one of the main routes of RNA degradation is performed by the multi-protein exosome complex , which consists largely of 3′ to 5′ exoribonucleases . RNA polymerase II is known to be in effect during transcriptional termination; it works with

784-502: Is a general transcription regulatory complex in budding yeast that is found to be associated with mRNA metabolism, transcription initiation, and mRNA degradation. CCR4 has been found to contain RNA and single-stranded DNA 3' to 5' exonuclease activities. Another component associated with the CCR4-Not is CAF1 protein, which has been found to contain 3' to 5' or 5' to 3' exonuclease domains in

833-621: Is primarily a 3'-5' exoribonuclease , meaning that it degrades RNA molecules from their 3' end . Exoribonucleases contained in exosome complexes are either phosphorolytic (the RNase PH-like proteins) or, in eukaryotes, hydrolytic (the RNase R and RNase D domain proteins). The phosphorolytic enzymes use inorganic phosphate to cleave the phosphodiester bonds – releasing nucleotide diphosphates . The hydrolytic enzymes use water to hydrolyse these bonds – releasing nucleotide monophosphates . In archaea,

882-408: Is used in editing and proofreading DNA for errors. The 3' to 5' can only remove one mononucleotide at a time, and the 5' to 3' activity can remove mononucleotides or up to 10 nucleotides at a time. In 1971, Lehman IR discovered exonuclease I in E. coli . Since that time, there have been numerous discoveries including: exonuclease, II, III , IV, V , VI, VII , and VIII. Each type of exonuclease has

931-453: The 3' or from the 5' end of the RNA, the exosome complex is essential for cell survival. When the expression of exosome proteins is artificially reduced or stopped, for example by RNA interference , growth stops and the cells eventually die. Both the core proteins of the exosome complex, as well as the two main associated proteins, are essential proteins. Bacteria do not have an exosome complex; however, similar functions are performed by

980-518: The 3' untranslated region of mRNAs interact with the exosome complex. In the nucleus , the exosome is required for the correct processing of several small nuclear RNA molecules. Finally, the nucleolus is the compartment where the majority of the exosome complexes are found. There it plays a role in the processing of the 5.8S ribosomal RNA (the first identified function of the exosome) and of several small nucleolar RNAs . Although most cells have other enzymes that can degrade RNA, either from

1029-503: The PM/Scl overlap syndrome , an autoimmune disease in which patients have symptoms from both scleroderma and either polymyositis or dermatomyositis . Autoantibodies can be detected in the serum of patients by a variety of assays. In the past, the most commonly used methods were double immunodiffusion using calf thymus extracts, immunofluorescence on HEp-2 cells or immunoprecipitation from human cell extracts. In immunoprecipitation assays with sera from anti-exosome positive sera,

SECTION 20

#1733085396690

1078-468: The Taq polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in

1127-477: The dnaQ gene encodes the ε subunit of DNA polymerase III . The ε subunit is one of three core proteins of the DNA polymerase complex. It acts as a 3’→5’ DNA directed proofreading exonuclease that removes incorrectly incorporated bases during replication. Similarly, in Salmonella typhimurium bacteria, the 3’ to 5’ editing function employed during DNA replication is also encoded by a gene, dnaQ , which specifies

1176-450: The fluorescence emitted by the fluorophore when excited by the cycler’s light source via Förster resonance energy transfer (FRET). As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals. TaqMan probes are designed such that they anneal within a DNA region amplified by a specific set of primers. (Unlike the diagram, the probe binds to single stranded DNA.) TaqMan probes can be conjugated to

1225-467: The mouse and Caenorhabditis elegans . This protein has not been found in yeast, which suggests that it is likely to have an abnormal exonuclease domain like the one seen in a metazoan. Yeast contains Rat1 and Xrn1 exonuclease. The Rat1 works just like the human type (Xrn2) and Xrn1 function in the cytoplasm is in the 5' to 3' direction to degrade RNAs (pre-5.8s and 25s rRNAs) in the absence of Rat1. In beta Coronaviruses , including SARS-CoV-2 ,

1274-525: The PCR. TaqMan probe-based assays are widely used in quantitative PCR in research and medical laboratories : 1. TaqMan RT-PCR resources - primer databases, software, protocols Archived 2010-11-25 at the Wayback Machine 2. Beacon Designer - Software to design real time PCR primers and probes including SYBR Green primers, TaqMan Probes, Molecular Beacons. Exonuclease Exonucleases are enzymes that work by cleaving nucleotides one at

1323-398: The RNA primer contained immediately upstream from the site of DNA synthesis in a 5' → 3' manner. Exonuclease III has four catalytic activities: Exonuclease IV adds a water molecule, so it can break the bond of an oligonucleotide to nucleoside 5' monophosphate. This exonuclease requires Mg 2+ in order to function and works at higher temperatures than exonuclease I. Exonuclease V

1372-457: The Rrp41 subunit of the complex is a phosphorolytic exoribonuclease. Three copies of this protein are present in the ring and are responsible for the activity of the complex. In eukaryotes, none of the RNase PH subunits have retained this catalytic activity, meaning the core ring structure of the human exosome has no enzymatically active protein. Despite this loss of catalytic activity, the structure of

1421-683: The TaqMan probe significantly increases the specificity of the detection. TaqMan probes were named after the videogame Pac-Man ( Taq Polymerase + PacMan = TaqMan) as its mechanism is based on the Pac-Man principle. TaqMan probes consist of a fluorophore covalently attached to the 5’-end of the oligonucleotide probe and a quencher at the 3’-end. Several different fluorophores (e.g. 6-carboxyfluorescein , acronym: FAM , or tetrachlorofluorescein, acronym: TET) and quenchers (e.g. tetramethyl rhodamine , acronym: TAMRA) are available. The quencher molecule quenches

1470-426: The autobodies are found mainly in patients that have characteristics of several different autoimmune diseases, the clinical symptoms of these patients can vary widely. The symptoms that are seen most often are the typical symptoms of the individual autoimmune diseases and include Raynaud's phenomenon , arthritis , myositis and scleroderma . Treatment of these patients is symptomatic and is similar to treatment for

1519-414: The budding yeast Saccharomyces cerevisiae , an often-used model organism . Not long after, in 1999, it was realized that the exosome was in fact the yeast equivalent of an already described complex in human cells called the PM/Scl complex , which had been identified as an autoantigen in patients with certain autoimmune diseases years earlier (see below ). Purification of this "PM/Scl complex" allowed

TaqMan - Misplaced Pages Continue

1568-475: The complex in eukaryotic organisms. One of these is Rrp44 , a hydrolytic RNase, which belongs to the RNase R family of hydrolytic exoribonucleases (nucleases that use water to cleave the nucleotide bonds). In addition to being an exoribonucleolytic enzyme, Rrp44 also has endoribonucleolytic activity, which resides in a separate domain of the protein. In yeast, Rrp44 is associated with all exosome complexes and has

1617-418: The complex to substrates specific to these cell compartments. Substrates of the exosome include messenger RNA , ribosomal RNA , and many species of small RNAs . The exosome has an exoribonucleolytic function, meaning it degrades RNA starting at one end (the 3′ end in this case), and in eukaryotes also an endoribonucleolytic function, meaning it cleaves RNA at sites within the molecule. Several proteins in

1666-593: The complex. The exosome is involved in the degradation and processing of a wide variety of RNA species. In the cytoplasm of cells, it is involved in the turn-over of messenger RNA (mRNA) molecules. The complex can degrade mRNA molecules that have been tagged for degradation because they contain errors, through interactions with proteins from the nonsense mediated decay or non-stop decay pathways. In alternative fashion, mRNAs are degraded as part of their normal turnover . Several proteins that stabilize or destabilize mRNA molecules through binding to AU-rich elements in

1715-407: The core exosome is highly conserved from archaea to humans, suggesting that the complex performs a vital cellular function. In eukaryotes, the absence of the phosphorolytic activity is compensated by the presence of the hydrolytic enzymes, which are responsible for the ribonuclease activity of the exosome in such organisms. As stated above, the hydrolytic proteins Rrp6 and Rrp44 are associated with

1764-504: The current time, the most sensitive way to detect these antibodies is by using a peptide , derived from the PM/Scl-100 protein, as the antigen in an ELISA , instead of complete proteins. By this method, autoantibodies are found in up to 55% of patients with the PM/Scl overlap syndrome, but they can also be detected in patients with either scleroderma, polymyositis, or dermatomyositis alone. As

1813-507: The cytoplasm ( DIS3L1 ) and the other in the nucleus ( DIS3 ). The second common associated protein is called Rrp6 (in yeast) or PM/Scl-100 (in human). Like Rrp44, this protein is a hydrolytic exoribonuclease, but in this case of the RNase D protein family. The protein PM/Scl-100 is most commonly part of exosome complexes in the nucleus of cells, but can form part of the cytoplasmic exosome complex as well. Apart from these two tightly bound protein subunits, many proteins interact with

1862-420: The exosome are the target of autoantibodies in patients with specific autoimmune diseases (especially the PM/Scl overlap syndrome ) and some antimetabolic chemotherapies for cancer function by blocking the activity of the exosome. In addition, mutations in exosome component 3 cause pontocerebellar hypoplasia and spinal motor neuron disease . The exosome was first discovered as an RNase in 1997 in

1911-551: The exosome complex in both the cytoplasm and nucleus of cells. These loosely associated proteins may regulate the activity and specificity of the exosome complex. In the cytoplasm, the exosome interacts with AU-rich element (ARE) binding proteins (e.g. KRSP and TTP), which can promote or prevent degradation of mRNAs. The nuclear exosome associates with RNA binding proteins (e.g. MPP6/Mpp6 and C1D/Rrp47 in humans/yeast) that are required for processing certain substrates. In addition to single proteins, other protein complexes interact with

1960-467: The exosome in yeast and in humans, besides Rrp6, two different proteins, Dis3 and Dis3L1 can be associated at the position of the yeast Rrp44 protein. Although originally the S1 domain proteins were thought to have 3'-5' hydrolytic exoribonuclease activity as well, the existence of this activity has recently been questioned and these proteins might have just a role in binding substrates prior to their degradation by

2009-464: The exosome is important for the degradation of so-called cryptic unstable transcripts (CUTs) that are produced from thousands of loci within the yeast genome. The importance of these unstable RNAs and their degradation are still unclear, but similar RNA species have also been detected in human cells. The exosome complex is the target of autoantibodies in patients with various autoimmune diseases . These autoantibodies are mainly found in people with

TaqMan - Misplaced Pages Continue

2058-506: The exosome. Because of this high similarity in both protein domains and structure, these complexes are thought to be evolutionarily related and have a common ancestor . The RNase PH-like exosome proteins, PNPase and RNase PH all belong to the RNase PH family of RNases and are phosphorolytic exoribonucleases , meaning that they use inorganic phosphate to remove nucleotides from the 3' end of RNA molecules. Besides these nine core exosome proteins, two other proteins often associate with

2107-642: The exosome. One of those is the cytoplasmic Ski complex , which includes an RNA helicase (Ski2) and is involved in mRNA degradation. In the nucleus, the processing of rRNA and snoRNA by the exosome is mediated by the TRAMP complex , which contains both RNA helicase (Mtr4) and polyadenylation (Trf4) activity. As stated above, the exosome complex contains many proteins with ribonuclease domains. The exact nature of these ribonuclease domains has changed across evolution from bacterial to archaeal to eukaryotic complexes as various activities have been gained and lost. The exosome

2156-457: The identification of more human exosome proteins and eventually the characterization of all components in the complex. In 2001, the increasing amount of genome data that had become available allowed the prediction of exosome proteins in archaea, although it would take another 2 years before the first exosome complex from an archaeal organism was purified. The core of the complex has a ring structure consisting of six proteins that all belong to

2205-430: The individual autoimmune disease, often involving either immunosuppressive or immunomodulating drugs. The exosome has been shown to be inhibited by the antimetabolite fluorouracil , a drug used in the chemotherapy of cancer . It is one of the most successful drugs for treating solid tumors . In yeast cells treated with fluorouracil, defects were found in the processing of ribosomal RNA identical to those seen when

2254-429: The product until it is completely degraded. This allows the nucleotides to be recycled. Xrn1 is linked to a co-transcriptional cleavage (CoTC) activity that acts as a precursor to develop a free 5' unprotected end, so the exonuclease can remove and degrade the downstream cleavage product (DCP). This initiates transcriptional termination because one does not want DNA or RNA strands building up in their bodies. CCR4-Not

2303-430: The proteins of the complex, called PM/Scl-100 (the RNase D like protein) and PM/Scl-75 (one of the RNase PH like proteins from the ring) and antibodies recognizing these proteins are found in approximately 30% of patients with the PM/Scl overlap syndrome. Although these two proteins are the main target of the autoantibodies, other exosome subunits and associated proteins (like C1D) can be targeted in these patients. At

2352-600: The same class of RNases, the RNase PH -like proteins. In archaea there are two different PH-like proteins (called Rrp41 and Rrp42), each present three times in an alternating order. Eukaryotic exosome complexes have six different proteins that form the ring structure. Of these six eukaryotic proteins, three resemble the archaeal Rrp41 protein and the other three proteins are more similar to the archaeal Rrp42 protein. Located on top of this ring are three proteins that have an S1 RNA binding domain (RBD). Two proteins in addition have

2401-429: The separation of the DNA polymerase gene function from the 3’ to 5’ exonuclease editing gene function in the lineage that led to E. coli and S. typhimurium . The 3' to 5' human type endonuclease is known to be essential for the proper processing of histone pre-mRNA, in which U7 snRNP directs the single cleavage process. Following the removal of the downstream cleavage product (DCP) Xrn1 continues to further breakdown

#689310