Eukaryotic Transcription is a complex process that eukaryotic cells use to copy the genetic information stored in DNA into RNA replica units. Gene transcription occurs in both eukaryotic and prokaryotic cells. Unlike prokaryotic RNA polymerases that initiate transcription of all different types of RNA, RNA polymerase in eukaryotes (including humans) comes in three variations, each encoding a different type of gene. The eukaryotic cells have a nucleus that separates the transcription and translation processes. Eukaryotic transcription occurs within the nucleus where DNA is packed into the nucleosome and higher chromatin structure of the skeleton. The complexity of the eukaryotic genome requires the variation and complexity of large gene expression controls.
Video Eukaryotic transcription
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Transcription is the process of copying the genetic information stored in the DNA strand into a removable complementary RNA strand. Eukaryotic transcription takes place in the cell nucleus and takes place in three successive stages: initiation, elongation, and termination. The transcriptional engine that catalyzes this complex reaction has a nucleus of three multi-subunit RNA polymerases. RNA polymerase I is responsible for copying RNAs that encode genes into structural components of the ribosome.
Protein coding genes are transcribed to messenger RNA (mRNAs) that carry information from DNA to protein synthesis sites. Although mRNAs have great diversity, mRNAs are not the most abundant RNA species made in cells. Called RNA is non-coding account for most of cell transcription output. This non-coding RNA performs important cellular functions.
Maps Eukaryotic transcription
RNA polymerase
Eukaryotes have three nuclear RNA polymerases, each with different roles and traits
RNA polymerase I (Pol I) catalyzes the transcription of all rRNA genes except 5S. These rRNA genes are arranged into one transcriptional unit and transcribed into continuous transcripts. These precursors are then processed into three rRNAs: 18S, 5.8S, and 28S. Transcription of the rRNA gene occurs in a special structure of a nucleus called nucleolus, in which transcribed rRNAs are combined with proteins to form ribosomes.
RNA polymerase II (Pol II) is responsible for the transcription of all mRNAs, multiple snRNAs, siRNAs, and all miRNAs. Many transcripts of Pol II exist temporarily as single strand precursor RNAs (pre-RNA) are further processed to produce mature RNA. For example, precursor mRNA (pre-mRNA) is extensively processed prior to exit to the cytoplasm through nuclear pores for protein translation.
RNA polymerase III (Pol III) transcribes a small, non-coding RNA, including tRNA, 5S rRNA, U6 snRNA, SRP RNA, and other stable stable RNAs such as ribonuclease P RNA.
RNA Polymerase I, II, and III each contain 14, 12, and 17 subunits. The three eukaryotic polymerases have five core subunits showing homology with ,,?,? I ,? II , and? subunit of E. coli RNA polymerase. Similar subunits (RBP6) are used by all three eukaryotic polymers, while similar subunits are used by Poles I and III. The three eukaryotic polymerases share four common subunits among themselves. The remaining subunits are unique for each RNA polymerase. The additional subunits found in Pol I and Pol III relative to Pol II, are homologous with the Pol II transcription factor.
The crystalline structure of RNA polymerase I and II provides an opportunity to understand the interactions between subunits and molecular mechanisms of eukaryotic transcription in atomic detail.
The carboxyl terminal (CTD) domain of RPB1, the largest subunit of RNA polymerase II, plays an important role in uniting the machinery necessary for the synthesis and processing of Pol II transcripts. Long and structurally structured, CTD contains several repetitions of YSPTSPS heptapeptide sequences that are subject to phosphorylation and other posttranslational modifications during the transcription cycle. These modifications and arrangements are operational codes for CTD to control the initiation of transcription, elongation and termination and for some RNA transcription and processing.
Initiation
The initiation of gene transcription in eukaryotes occurs at certain steps. First, the RNA polymerase along with the common transcription factor binds to the promoter region of the gene to form a closed complex called a complex preinitiation. The subsequent transition from the complex from closed to open state results in the fusion or separation of two DNA strands and the placement of the template strand to the active site of the RNA polymerase. Without the need for primers, RNA polymerase can initiate the synthesis of new RNA chains using DNA template strands to guide the selection of ribonucleotides and polymerization chemistry. However, many of the initiated syntheses are canceled before the transcript reaches a significant length (~ 10 nucleotides). During this abortive cycle, polymerases continue to make and release short transcripts capable of producing transcripts exceeding ten nucleotides in length. Once this threshold is reached, RNA polymerase passes the promoter and transcription to the elongation phase.
eukaryotic promoters and common transcription factors
The transcribed genes of Pol-II contain an area around the transcriptional binding site (TSS) and place the pre-initiation complex. This area is referred to as the core promoter because of its important role in transcription initiation. Different classes of sequence elements are found in the promoter. For example, the TATA box is a highly sustainable sequence of DNA recognition for the TATA box binding protein, TBP, which binds the initiation of complex transcriptional assembly in many genes.
The eukaryotic gene also contains regulatory sequences outside the core promoter. This cis-acting control element binds a transcriptional or repressor activator to increase or decrease the transcription of the core promoter. Well-characterized regulatory elements include enhancers, silencers, and insulators. This arrangement sequence can be spread over large genome distances, sometimes located hundreds of kilobases from the core promoter.
A common transcription factor is a group of proteins involved in transcriptional and regulatory initiation. These factors usually have DNA binding domains that bind certain sequence elements of the core promoter and help recruit RNA polymerase to the transcriptional initial site. Common transcription factors for RNA polymerase II include TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH.
Complex assembly preinitiation
To prepare for transcription, a complete set of common transcription factors and RNA polymerase need to be assembled on the core promoter to form a ~ 2 million dalton preinitiation complex. For example, for a promoter that contains a TATA box near TSS, the TATA box recognition by the TBD subunit of TFIID starts the assembly of the transcription complex. Subsequent proteins included are TFIIA and TFIIB, which stabilize the DNA-TFIID complex and recruit Pol II in association with TFIIF and additional transcription factors. TFIIF serves as a bridge between TATA-bound TBP and polymerase. One of the last transcription factors to be recruited into the complex preinitiation is TFIIH, which plays an important role in melting and escaping promoters.
Promoter melts and opens complex formations
For pol II-transcribed genes, and unlike bacterial RNA polymerases, melting promoters require ATP hydrolysis and are mediated by TFIIH. TFIIH is a protein of ten subunits, including ATPase activity and protein kinase. While the upstream DNA promoter is held in a fixed position by TFIID, TFIIH pulls down the double-stranded DNA into the polymerase gap, encouraging the separation of DNA strands and the transition of the preinitiation complex from closed to open state. TFIIB helps in the formation of an open complex by binding to the melting DNA and stabilizing the transcription bubbles.
Initiation that failed
After the initiation complex opens, the first ribonucleotide is brought to the active site to initiate the polymerization reaction in the absence of a primer. This produces a newborn RNA chain that forms hetero-duplex with DNA template strands. However, before entering the elongation phase, the polymerase can stop prematurely and release short cut transcripts. This process is called a failed initiation. Many failed initiation cycles can occur before the transcript grows long enough to promote the escape polymerase of the promoter. During the failed initiation cycle, the RNA polymerase remains attached to the promoter and pulls the downstream DNA into its catalytic gap in a motion-like motion.
Runaway promoter
When the transcript reaches the length of the threshold of ten nucleotides, it enters the RNA outlet. The polymerase breaks up its interaction with the promoter element and any regulatory proteins associated with the initiation complex that are no longer needed. Runaway promoters in eukaryotes require ATP hydrolysis and, in the case of Pol II-phosphorylation of CTD. Meanwhile, the transcription bubble collapses to 12-14 nucleotides, providing the kinetic energy necessary to escape.
Elongation
After escaping the promoter and releasing most of the transcription factors for initiation, the polymerase acquires new factors for the next phase of transcription: elongation. Transcription of extension is process process. Double-stranded DNA entering from the front of the enzyme will be opened to exploit the mold strand for RNA synthesis. For each base pair of DNA separated by an advanced polymerase, a hybrid RNA: DNA base pairs are immediately formed. DNA strands and newborn RNA chains come out of separate channels; two strands of DNA reunite at the end of the transcription bubble while a single stranded RNA appears alone.
Extension factor
Among the proteins that are recruited to the polymerase are elongation factors, so called because they stimulate the elongation of transcription. There are various classes of extension factors. Several factors can improve overall transcription rates, some may help polymerase through temporary pause sites, and some may help the polymerase to transcribe via chromatin. One of the extension factors, P-TEFb, is very important. P-TEFb phosphorylates the second residue (Ser-2) from the repeated CTD (YSPTSPS) of the bound Pol II. P-TEFb also phosphorylates and activates SPT5 and TAT-SF1. SPT5 is a universal transcription factor that helps to recruit the 5'-capping enzyme to Pol II with a phosphorylated CTD in Ser-5. TAF-SF1 recruits components from RNA splicing machines to CT-phosphorylated Serials. P-TEFb also helps suppress temporary polymerase delays when meeting a particular sequence immediately after initiation.
Transcription fidelity
Transcriptional allegiance is achieved through various mechanisms. RNA polymerase select the correct nucleoside triphosphate (NTP) substrate to prevent transcriptional errors. Only NTPs are actually paired with coding bases in DNA that are inserted into the active center. RNA polymerase performs two known evidence-reading functions to detect and remove incorrectly operating nucleotides: pyrophosphorylytic editing and hydrolytic editing. The first eliminates the wrong ribonucleotides incorporated by a simple reversal of the polymerization reaction, while the latter involves backtracking of the polymerase and splitting the segment of the RNA product containing the error. The TFIIS elongation factor stimulates the ribonuclease activity inherent in the polymerase, which allows the erroneous incorrect base generation to be integrated by limited local RNA degradation. Note that all reactions (synthesis of phosphodiester bonds, pyrophosphorolysis, hydrolysis of phosphodiester bonds) are carried out by RNA polymerase using a single active center.
Pause, limp, and retreat
Transcription extension is not a smooth journey along the DNA rails. For proofreading, polymerase is made to back-up, deleting some of the RNAs it has created and transcribing again. In general, RNA polymerase does not transcribe through genes at a constant velocity. Instead it stops periodically on a certain order, sometimes for long periods of time before continuing transcription. In extreme cases, for example, when a polymerase meets a defective nucleotide, it stops completely. More often, the elongated polymerase stops near the promoter. Proximal-stop promoters during early elongation are commonly used mechanisms for managing genes that are readily disclosed rapidly or in a coordinated manner. Pausing is mediated by a complex called NELF (negative elongation factor) in association with DSIF (DRB-induced induction factor containing SPT4/SPT5). The blockage is released after the polymerase receives an activation signal, such as Ser-2 phosphorylation of the CTD tail by P-TEFb. Other elongation factors such as ELL and TFIIS stimulate the rate of elongation by limiting the length of time the polymerase stops.
RNA processing
Polymerase elongation is associated with a set of protein factors required for different types of RNA processing. The mRNA closes immediately after it emerges from the RNA-channel out of the polymerase. After restriction, Ser-5 defos- phorylation in CTD repetition may be responsible for the separation of the capping machine. Furthermore Ser-2 phosphorylation causes recruitment of RNA splicing machines that catalyze non-coding intron removal to produce mature mRNA. Alternative grafting extends the complementary protein in eukaryotes. As with 5'-capping and splicing, CTD is involved in recruiting enzymes responsible for 3'-polyadenylation, a final RNA processing process coupled with transcription termination.
Termination
The last stage of transcription is termination, leading to complete transcript dissociation and release of RNA polymerase from template DNA. The process is different for each of the three RNA polymerases. The disconnection mechanism is the least understood of the three stages of transcription.
Factor-dependent
Termination of pre-rRNA gene transcription by polymerase Pol I is performed by systems requiring certain transcription termination factors. The mechanism used has some resemblance to the rho-dependent termination in prokaryotes. The eukaryotic cells contain hundreds of ribosomal DNA replications, sometimes distributed over several chromosomes. Termination of transcription occurs in the area of ​​the ribosomal intergenic spacer containing some transcription termination sites upstream from the Polus I stop site. Through unknown mechanism, the 3'-end transcript is split, producing a large primary rRNA molecule that is further processed into rRNA 18S, 5.8 S and 28S are mature.
When Pol II reaches the end of the gene, two protein complexes are carried by CTD, CPSF (cleavage specificity factor and polyadenylation) and CSTF (split stimulation factor), recognizing the poly-A signal in transcribed RNA. Poly-A-bound CPSF and CSTF recruit other proteins to perform RNA division and then polyadenylation. Polymerase Poly-A adds about 200 adenine to the 3 'end of RNA that is cleaved without a template. Poly-A long tail is unique to transcripts made by Pol II
In the process of terminating transcription by Pol I and Pol II, the elongation complex does not dissolve immediately after RNA is cleaved. The polymerase continues to move along the template, producing the second RNA molecule associated with the elongation complex. Two models have been proposed to explain how termination is achieved eventually. The allosteric model states that when transcription takes place through a sequence of terminations, it causes the dismantling of the extension factor and/or the assembly of the termination factor that causes the elongation complex conformation change. The torpedo model shows that the 5 'to 3' exonuclease lowers the second RNA when it emerges from the elongation complex. Polymerase is released as a very exonuclease procession following it. It is proposed that the emerging view will express the merging of these two models.
Factor-independent
RNA polymerase III can stop transcription efficiently without involving additional factors. The Pol III termination signal consists of strain thymines (on the nontemplate strand) located within 40bp downstream of the 3 'end of the mature RNA. The poly-T termination signal stops Pol III and causes it to retreat to the nearest RNA hairpin to become a "dead end" complex. Consistent with the allosteric cessation mechanism, RNA hairpin allosterically opens Pol III and causes the elongation complex to collapse. The broad structure is embedded in III Pol III transcripts so it is responsible for a factor-independent release of Pol III at the end of the gene. RNA-duplex hanging is an ancient mechanism that begins with the last universal ancestor.
Eukaryotic transcription control
The regulation of gene expression in eukaryotes is achieved through the interaction of several levels of control that act locally to enable or deactivate individual genes in response to cell-specific needs and globally to maintain the expression pattern of chromatin-wide genes that make up cell identity. Since the eukaryotic genome is wrapped around histones to form nucleosomes and high-level chromatin structures, the substrate for the transcriptional engine is generally partially concealed. Without regulatory proteins, many genes are expressed at low levels or not expressed at all. Transcription requires displacement of the positioned nucleosome to allow the transcriptional machine to gain access to DNA.
All steps in transcription are subject to some level of regulation. Intrate transcription is particularly the main level at which gene expression is regulated. Targeting the first step of rate limitation is the most efficient in terms of energy costs for the cell. The initiation of transcription is governed by cis-acting elements (enhancers, silencers, insulators) within the DNA regulation region, and specific sequence trans-acting factors acting as activators or repressors. Gene transcription can also be set up post-initiation by targeting the elongated polymerase movement.
Global control and epigenetic regulation
The eukaryotic genome is structured in a compact chromatin structure that allows only access that is set to DNA. The chromatin structure can be globally "open" and more transcription possible, or globally "condensed" and inactive transcription. The first (euchromatin) is packed lightly and rich in genes under active transcription. The latter (heterochromatin) belongs to the gene-poor regions such as telomeres and centromere but also areas with normal gene density but are transcriptionally silenced. Transcription can be silenced by histone modification (deaceltylation and methylation), RNA interference, and/or DNA methylation.
The pattern of gene expression that defines the identity of cells is inherited through cell division. This process is called epigenetic regulation. DNA methylation can be inherited through methylation maintenance actions that alter the strands of newborn DNA generated by replication. In mammalian cells, DNA methylation is the principal marker of areas that are transcriptionally silenced. Specific proteins can recognize markers and recruit histone deacetylases and methylases to rebuild silencing. The modification of the histone nucleosome can also be inherited during cell division, however, it is unclear whether it can work independently without direction by DNA methylation.
Gene-specific activation
The two main tasks of transcription initiation are to provide RNA polymerase with access to the promoter and to assemble a common transcription factor with polymerase into a transcription initiation complex. Various mechanisms of initiating transcription by overriding the inhibitory signals on the gene promoter have been identified. Eukaryotic genes have acquired a wide range of regulatory regimes that cover a large number of regulatory sites and spread overall kilobase (sometimes hundreds of kilobase) from promoters - both upstream and downstream. Regular binding sites are often grouped together into units called enhancers. Enhancers can facilitate highly cooperative actions of some transcription factors (which are enhososomes). Remote enhancers allow transcription regulation at some distance. Insulators located between enhancers and promoters help determine which genes can or can not be affected by enhancers.
Eukaryotic transcription activators have a DNA binding function and a separate activation. After binding the cis element, an activator may recruit polymerase directly or recruit other factors required by the transcription machine. Activators can also recruit a nucleosome converter that converts chromatin around the promoter and thus aids initiation. Some activators can work together, either by recruiting one or two interdependent components from a transcriptional machine, or by helping each other bind to their DNA site. This interaction can synergize multiple input signals and generate complex transcription responses to meet mobile needs.
Gene-specific repression
Eukaryotic transcriptional repressors share some of the mechanisms used by their prokaryotic counterparts. For example, by binding sites on DNA that overlap with activator binding sites, repressors may inhibit activator binding. But more often, eukaryotic oppressors inhibit the function of activators by covering the activation domain, preventing nuclear localization, promoting degradation, or disabling it through chemical modification. The repressor can directly inhibit the initiation of transcription by binding to the upstream site of the promoter and interacting with the transcription machine. Repressors can indirectly suppress transcription by recruiting histone modifiers (deacetylases and methylases) or nucleosome remodeling enzymes that affect the accessibility of DNA. Emphasizing histone and DNA modification is also the basis of transcriptional silencing that can spread along chromatin and kill some genes.
Extension and termination control
The elongation phase begins after the assembly of the elongation complex has been completed, and continues until the cessation sequence is found. The post-initiation movement of RNA polymerase is another class target of important regulatory mechanisms. For example, trans Tat activators affect elongation rather than initiation during HIV transcription settings. In fact, many eukaryotic genes are regulated by releasing blocks to the elongation of transcription called proximal-promoter pause. Pausing may affect the chromatin structure of the promoter to facilitate gene activity and lead to rapid or synchronous transcriptional response when cells are exposed to the activation signal. Pausing is associated with the binding of two negative renewal factors, DSIF (SPT4/SPT5) and NELF, to the elongation complex. Other factors may also affect the stability and duration of the stopping polymerase. The release pause was triggered by P-TEFb kinase recruitment.
Termination of transcription also emerges as an important field of transcriptional regulation. Termination is paired with efficient polymerase recycling. Factors associated with transcription termination can also mediate the repetition of genes and thus determine the efficiency of re-initiation.
Improved DNA coupled with transcription
When transcription is captured by a lesion on a transcribed gene strand, DNA repair proteins are recruited to suspended RNA polymerase to initiate a process called coupled-transcription repair. The center of this process is a common transcription factor TFIIH that has ATPase activity. TFIIH causes conformational changes in the polymerase, to expose the transcription bubbles trapped inside, in order for the DNA repair enzyme to gain access to the lesion. Thus, RNA polymerase acts as a cellular sensing protein in order to target enzyme repair in genes that are actively transcribed.
Eukaryotic transcription is more complex than prokaryotic transcription. For example, in the eukaryotes of genetic material (DNA), and therefore transcription, mainly localized to the nucleus, where it is separated from the cytoplasm (where translation occurs) by nuclear membranes. This allows for the temporal regulation of gene expression through RNA sequestration in the nucleus, and allows for selective transport of mature RNA into the cytoplasm. Bacteria do not have a distinct nucleus that separates DNA from ribosomes and mRNAs that translate into proteins as soon as they are transcribed. Merging between the two processes provides an important mechanism for prokaryotic gene regulation.
At the initiation level, RNA polymerase in prokaryotes (especially bacteria) binds strongly to the promoter region and initiates a high basal transcription rate. No ATP hydrolysis is required for close-to-open transitions, the dissolution of the promoter is driven by a binding reaction that supports the melting conformation. Chromatin greatly inhibits transcription in eukaryotes. The installation of a large multi-protein preinitiation complex is required for the promoter's specific initiation. Promoter melt in eukaryotes requires hydrolysis of ATP. As a result, eukaryotic RNA polymerase exhibits low levels of low transcriptional initiation.
src: oregonstate.edu
Transcriptional rules on cancer
In vertebrates, the majority of gene promoters contain CpG islands with many CpG sites. When many CpG promoter gen sites gene dimethylated into silence. Colorectal cancer usually has 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations. However, transcription silencing may be more important than a mutation that causes progression to cancer. For example, in colorectal cancers of 600 to 800 genes transcriptionally silenced by CpG island methylation (see transcriptional regulation on cancer). Transcriptional suppression of cancer may also occur with other epigenetic mechanisms, such as changes in the expression of microRNA. In breast cancer, BRCA1 transcriptional suppression may occur more frequently with microRNA-182 overrespression compared with BRCA1 promoter hypermethylation (see BRCA1 Expression low on breast and ovarian cancers).
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