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Transcription of DNA and further splicing of mRNA is regulated by various transcription factors, small nuclear RNAs and so on; similarly such related mechanisms as transposition of transposons.
All such factors and mechanisms, including those linked to inter-cellular signalling mediated by peptides and various so-called epigenetic enzymatic processes, require and are dependent upon transcription itself, in the first place of DNA codons.
What mechanism then is supposed ultimately to control the expression, eventually as peptides translated from mRNA, of information encoded in DNA; if it is not peptides thus encoded? That is, since transcription depends on transcription factors which themselves require transcription requiring further transcription factors, does this mean that in the entirety of its function, DNA controls itself?
How then can DNA and its transcription be conceived as a such a self-controlling function?
Transcription of DNA is ultimately controlled by "various transcription factors, small nuclear RNAs and so on" in the cell.
The molecules which make up the "various transcription factors", etc. are already present in the cell when a new transcription begins. As you note, most all of these molecules are themselves dependent on the transcription of DNA, but also of the availability of component molecules (e.g. nucleotides, amino acids) and of processing mechanisms such as ribosomes.
So, a new instance of transcription requires a working cellular environment, and that environment was created and maintained as directed by DNA molecules at an earlier time. Usually those DNA molecules were exactly the same as the ones from which the transcription is proceeding. Occasionally, those DNA molecules would have been physically different, but adequately compatible, such as the source chromosomes which were copied during mitosis to create the current DNA molecule.
What Ultimately Controls DNA Transcription? - Biology
For a cell to function properly, necessary proteins must be synthesized at the proper time. All cells control or regulate the synthesis of proteins from information encoded in their DNA. The process of turning on a gene to produce RNA and protein is called gene expression. Whether in a simple unicellular organism or a complex multi-cellular organism, each cell controls when and how its genes are expressed. For this to occur, there must be a mechanism to control when a gene is expressed to make RNA and protein, how much of the protein is made, and when it is time to stop making that protein because it is no longer needed.
The regulation of gene expression conserves energy and space. It would require a significant amount of energy for an organism to express every gene at all times, so it is more energy efficient to turn on the genes only when they are required. In addition, only expressing a subset of genes in each cell saves space because DNA must be unwound from its tightly coiled structure to transcribe and translate the DNA. Cells would have to be enormous if every protein were expressed in every cell all the time.
The control of gene expression is extremely complex. Malfunctions in this process are detrimental to the cell and can lead to the development of many diseases, including cancer.
Learning Outcomes
- Discuss why every cell does not express all of its genes
- Compare prokaryotic and eukaryotic gene regulation
New mechanism for terminating transcription of DNA into RNA in bacteria
The protein, known as NusG, pauses the transcription machinery at specific DNA sequences to facilitate what is called "intrinsic termination" and prevent unwanted transcription that could disrupt cellular function.
A new study, led by Penn State researchers, shows that NusG and the related protein, NusA, together facilitate termination at about 88% of the intrinsic terminators in the bacteria Bacillus subtilis. Understanding this process expands our basic knowledge of this key cellular function and could eventually aid in the development of antibiotics that target and disrupt gene regulation in bacteria. A paper describing the research appears online in the journal eLife.
"For a cell to access the genetic information stored in DNA, it first must be transcribed into RNA by an enzyme called RNA polymerase," said Zachary F. Mandell, a graduate student at Penn State and first author of the paper. "This process is highly coordinated to ensure that the right genes are expressed at the right times and at appropriate levels for the cell to function properly. We are interested in understanding the mechanisms that allow the cell to stop transcription at precise locations along the genome."
Proper regulation of gene expression occurs in three basic stages. First, transcription is initiated by RNA polymerase that binds to the DNA at the beginning of the sequence that is being transcribed. NusA and NusG then bind to RNA polymerase during the elongation process to make an RNA copy of the DNA sequence. Finally, the transcription must be terminated at the appropriate spots in the genome.
"Termination of transcription is especially important in bacteria because the genes are packed tightly together along the genome, such that failure to terminate transcription at the right locations could lead to inappropriate gene expression," said Paul Babitzke, professor of biochemistry and molecular biology at Penn State and the leader of the research team.
The mechanisms for termination in bacteria traditionally have been classified as either "factor-dependent," which relies on a protein called Rho, or intrinsic termination, which was thought to be "factor-independent." Intrinsic termination relies on an RNA hairpin structure that forms in the RNA molecule being produced, which causes the RNA to be released from the transcription machinery.
NusG and NusA are both proteins classified as transcription factor that help regulate gene expression and are part of the complex of proteins that read and transcribe DNA during elongation. NusA interacts with the RNA molecule being produced and NusG can bind to the DNA to pause elongation. Based on previous research, NusA is thought to play a role in intrinsic termination by aiding in the formation of the RNA hairpin, but the role of NusG in termination had not been established.
To further explore the role of these two proteins in intrinsic termination, the research team produced strains of bacteria that lacked NusA, lacked NusG, and lacked both NusA and NusG. They then used a technique that they invented called "Term-Seq," in which they can preserve and identify the ends of all the RNA molecules produced in their bacterial strains. The RNA ends from the mutant strains could then be compared to RNA molecules from bacteria with normally functioning NusA and NusG.
"We found that some intrinsic termination sites were dependent on NusA, some on NusG, some on either NusA or NusG, and some required both," said Mandell. "We were somewhat surprised by how big of a role these two proteins play in intrinsic termination. A total of 88% of all intrinsic termination sites relied on NusA or NusG in some capacity. Intrinsic termination is clearly not completely 'factor-independent.'"
The researchers are still investigating the precise role that the Nus proteins play in transcription termination.
"We think that NusA helps directly in the formation of the hairpins required for intrinsic termination and that NusG is pausing elongation at termination sites to give the hairpin additional time to form," said Babitzke.
Histone modifications
Chromatin architecture, nucleosomal positioning, and ultimately access to DNA for gene transcription, is largely controlled by histone proteins. Each nucleosome is made of two identical subunits, each of which contains four histones: H2A, H2B, H3, and H4. Meanwhile, the H1 protein acts as the linker histone to stabilize internucleosomal DNA and does not form part of the nucleosome itself.
Histone proteins undergo post-translational modification (PTM) in different ways, which impacts their interactions with DNA. Some modifications disrupt histone-DNA interactions, causing nucleosomes to unwind. In this open chromatin conformation, called euchromatin, DNA is accessible to binding of transcriptional machinery and subsequent gene activation. In contrast, modifications that strengthen histone-DNA interactions create a tightly packed chromatin structure called heterochromatin. In this compact form, transcriptional machinery cannot access DNA, resulting in gene silencing. In this way, modification of histones by chromatin remodeling complexes changes chromatin architecture and gene activation.
Figure 1: The most common histone modifications. To find out more see our full histone modifications poster
Together, these histone modifications make up what is known as the histone code, which dictates the transcriptional state of the local genomic region. Examining histone modifications at a particular region, or across the genome, can reveal gene activation states, locations of promoters, enhancers, and other gene regulatory elements.
Histone modifications in detail
Acetylation
Acetylation is one of the most widely studied histone modifications since it was one of the first discovered to influence transcriptional regulation. Acetylation adds a negative charge to lysine residues on the N-terminal histone tails that extend out from the nucleosome. These negative charges repel negatively charged DNA, which results in a relaxed chromatin structure. The open chromatin conformation allows transcription factor binding and significantly increases gene expression (Roth et al., 2001)
Histone acetylation is involved in cell cycle regulation, cell proliferation, and apoptosis and may play a vital role in regulating many other cellular processes, including cellular differentiation, DNA replication and repair, nuclear import and neuronal repression. An imbalance in the equilibrium of histone acetylation is associated with tumorigenesis and cancer progression.
Acetyl groups are added to lysine residues of histones H3 and H4 by histone acetyltransferases (HAT) and removed by deacetylases (HDAC). Histone acetylation is largely targeted to promoter regions, known as promoter-localized acetylation. For example, acetylation of K9 and K27 on histone H3 (H3K9ac and H3K27ac) is usually associated with enhancers and promoters of active genes. Low levels of global acetylation are also found throughout transcribed genes, whose function remains unclear.
Methylation
Methylation is added to the lysine or arginine residues of histones H3 and H4, with different impacts on transcription. Arginine methylation promotes transcriptional activation (Greer et al., 2012) while lysine methylation is implicated in both transcriptional activation and repression depending on the methylation site. This flexibility may be explained by the fact that that methylation does not alter histone charge or directly impact histone-DNA interactions, unlike acetylation.
Lysines can be mono-, di-, or tri-methylated, providing further functional diversity to each site of methylation. For example, both mono- and tri-methylation on K4 of histone H3 (H3K4me1and H3K4me3) are activation markers, but with unique nuances: H3K4me1 typically marks transcriptional enhancers, while H3K4me3 marks gene promoters. Meanwhile, tri-methylation of K36 (H3K36me3) is an activation marker associated with transcribed regions in gene bodies.
In contrast, tri-methylation on K9 and K27 of histone H3 (H3K9me3 and H3K27me3) are repressive signals with unique functions: H3K27me3 is a temporary signal at promoter regions that controls development regulators in embryonic stem cells, including Hox and Sox genes. Meanwhile, H3K9me3 is a permanent signal for heterochromatin formation in gene-poor chromosomal regions with tandem repeat structures, such as satellite repeats, telomeres, and pericentromeres. It also marks retrotransposons and specific families of zinc finger genes (KRAB-ZFPs). Both marks are found on the inactive chromosome X, with H3K27me3 at intergenic and silenced coding regions and H3K9me3 predominantly in coding regions of active genes.
Histone methylation is a stable mark propagated through multiple cell divisions, and for many years was thought to be irreversible. However, it was recently discovered to be an actively regulated and reversible process.
Methylation: histone methyltransferases (HMTs)
- Lysine
- SET domain-containing (histone tails)
- Non-SET domain-containing (histone cores)
- PRMT (protein arginine methyltransferases) family
Demethylation: histone demethylases
- Lysine
- KDM1/LSD1 (lysine-specific demethylase 1)
- JmjC (Jumonji domain-containing)
- PAD4/PADI4
Phosphorylation
Histone phosphorylation is a critical intermediate step in chromosome condensation during cell division, transcriptional regulation, and DNA damage repair (Rossetto et al., 2012, Kschonsak et al., 2015). Unlike acetylation and methylation, histone phosphorylation establishes interactions between other histone modifications and serves as a platform for effector proteins, which leads to a downstream cascade of events.
Phosphorylation occurs on all core histones, with differential effects on each. Phosphorylation of histone H3 at serine 10 and 28, and histone H2A on T120, are involved in chromatin compaction and the regulation of chromatin structure and function during mitosis. These are important markers of cell cycle and cell growth that are conserved throughout eukaryotes. Phosphorylation of H2AX at S139 (resulting in γH2AX) serves as a recruiting point for DNA damage repair proteins (Lowndes et al., 2005, Pinto et al., 2010) and is one of the earliest events to occur after DNA double-strand breaks. H2B phosphorylation is not as well studies but is found to facilitate apoptosis-related chromatin condensation, DNA fragmentation, and cell death (Füllgrabe et al., 2010).
Ubiquitylation
All histone core proteins can be ubiquitylated, but H2A and H2B are most commonly and are two of the most highly ubiquitylated proteins in the nucleus (Cao et al., 2012). Histone ubiquitylation plays a central role in the DNA damage response.
Monoubiquitylation of histones H2A, H2B, and H2AX is found at sites of DNA double-strand breaks. The most common forms are monoubiquitylated H2A on K119 and H2B on K123 (yeast)/K120 (vertebrates). Monoubiquitylated H2A is also associated with gene silencing, whereas H2B is also associated with transcription activation.
Poly-ubiquitylation is less common but is also important in DNA repair-- polyubiquitylation of H2A and H2AX on K63 provides a recognition site for DNA repair proteins, like RAP80.
Enzymatic regulation
Like other histone modifications, monoubiquitylation of H2A and H2B is reversible and is tightly regulated by histone ubiquitin ligases and deubiquitylating enzymes.
Monoubiquitylation
Polyubiquitylation
Quick Reference Guide to Histone Modifications
Most common histone modifications and where to find them:
Histone modification Function Location
H3K4me1 Activation Enhancers
H3K4me3 Activation Promoters
H3K36me3 Activation Gene bodies
H3K79me2 Activation Gene bodies
H3K9Ac Activation Enhancers, promoters
H3K27Ac Activation Enhancers, promoters
H4K16Ac Activation Repetitive sequences
H3K27me3 Repression Promoters, gene-rich regions
H3K9me3 Repression Satellite repeats, telomeres, pericentromeres
Gamma H2A.X DNA damage DNA double-strand breaks
H3S10P DNA replication Mitotic chromosomes
Studying histone modifications by ChIP
ChIP uses antibodies to isolate a protein or modification of interest, along with the DNA to which it is bound (figure 5). The DNA is then sequenced and mapped to the genome to identify the protein or modification’s location and abundance.
Figure 2: Histone modification ChIP. Antibodies bind directly to modified histone tails. Immunoprecipitation and DNA purification allow for the isolation and identification of the genomic regions that the modifications occupy.
Utilizing antibodies against specific histones and histone modifications in ChIP experiments can reveal the specific locations of
- Higher order chromatin structures, eg H3K9me3 marks heterochromatin and satellite repeats
- Active or silenced genes and genetic programs, eg AH3K9ac marks gene activation
- Genetic elements like promoters and enhancers, eg H3K27me3 marks promoters in gene-rich regions, H3K4me1 marks active enhancers
If the function of a histone modification is known, ChIP can identify specific genes and regions with this histone modification signature and the corresponding function across the genome. These genes and regions can then be further examined for their role in the biological process of interest. Using ChIP against H3K4me1, for example, will reveal the locations and sequences of active enhancers throughout the genome, pointing to genes and genetic programs of interest.
Alternatively, if the function of the histone modification is not known, ChIP can identify sequences, genes, and locations with this signature, which can then be used to infer the function of the modification. This technique was pivotal in decoding much of the histone code and is still valuable in ascertaining the function of newly discovered modifications like ubiquitylation and other novel markings.
Histone modifying enzymes: writers and erasers
Histone modifications are dynamically added and removed from histone proteins by specific enzymes (table 1). The balance between these writers and erasers dictates which marks are present on histones, and at what levels, to ultimately control whether specific genetic programs and the cellular processes they orchestrate, are turned on or off.
Table 1. The major categories of histone writers and erasers.
Modification
Histone acetyltransferases (HATs)
Histone deacetylases (HDACs)
Histone methyltransferases (HMTs/KMTs) and protein arginine methyltransferases (PRMTs)
For more details on the readers, writers, and erasers of histone modifications take a look at our epigenetic modification’s poster.
Identifying modification pathways and the specific writers and erasers at play can reveal:
- Relevant cellular pathways, genetic programs and physiological effects for further investigation. For example, histone deacetylases (HDACs) activate immune developmental pathways, while histone acetyltransferases (HATs) play a crucial role in differentiation and proliferation.
- Imbalances between writers and erasers that alter genetic programming and underlie disease processes. Characterizing such imbalances, and the specific enzymes involved, can provide insights into disease pathology, from cancers to autoimmune disorders.
- New drug targets and therapeutic strategies. Once an imbalance is identified, drugs can be developed to impact the activity of these enzymes and correct the imbalance, offering new therapeutic strategies against diseases that have thus far evaded medical efforts. For example, many HDAC inhibitors are in development as novel drugs against cancers and inflammatory diseases like arthritis and type I diabetes.
For drug development efforts, compounds can easily be screened for their impact on writer and eraser activity.
Characterizing histone methylation pathways
In general, histone methyltransferase (HMT) assays are challenging to develop, and most have several drawbacks due to assay design. Typical HMT assays utilize 3 H-SAM as a methyl donor and measure S-adenosylhomocysteine (SAH) as a general by-product of the methylation reaction. However, this requires
- Handling radioactive material
- High sensitivity to overcome low kcat (turnover typically < 1 min-1) and KM values for the methyl donor, SAM
- Prior purification of enzyme/protein complexes to assess activity of specific HMTs
Abcam HMT activity assays overcome these difficulties, assessing the activity of specific HMTs with antibodies that detect the specific methylated product, providing:
- Easy colorimetric or fluorometric detection, without radioactivity
- Compatibility with nuclear extracts, or purified proteins (assay is specific for the modification of interest)
- Data in 3 hours
For more information on our histone methylation assays click here.
Characterizing demethylase activity
Histone demethylase activity assays typically measure the formation of formaldehyde, a by-product of demethylation. They are therefore susceptible to interference from detergents, thiol groups and a range of ions. Similar to methylation assays, these assays are not specific for any demethylase and can only be performed with purified protein.
Abcam’s histone demethylase assays circumvent these issues by directly measuring the formation of the demethylated product, providing:
- Increased sensitivity (20–1,000 fold) over formaldehyde-based assays
- More accurate data without interference from thiols, detergents or ions
- Compatibility with nuclear extracts or purified protein (due to the assay’s specificity for the modification of interest)
- Measures demethylase activity from a broad range of species including mammalian cells/tissues, plants, and bacteria
- Fast microplate format with simple colorimetric or fluorometric readouts
- Data in 3 hours
For more information on our histone demethylase assays click here.
Characterizing histone acetylation pathways
Abcam offers kits to analyze overall, as well as H4-specific, HAT activity. These assays measure the HAT-catalyzed transfer of acetyl groups from the Acetyl-CoA donor to histone peptides, which generates the acetylated peptide and CoA-SH. The CoA-SH byproduct is then be measured via colorimetric or fluorometric methods:
- Colorimetric assays- CoA-SH serves as an essential coenzyme for producing NADH, which reacts with soluble tetrazolium dye to generate a product that can be detected spectrophotometrically. This assay is ideal for kinetic studies, with continuous detection.
- Fluorometric assays- CoA-SH reacts with a developer and Probe to generate a product that is detected fluorometrically.
Characterizing histone deacetylase activity
HDAC proteins fall into four major groups (class I, class IIA, class IIB, class III, class IV) based on function and DNA sequence similarity. Classes I, IIA, and IIB are considered "classical" HDACs whose activities are inhibited by trichostatin A (TSA), whereas class III is a family of NAD + -dependent proteins (sirtuins (SIRTs)) not affected by TSA. Class IV is considered an atypical class on its own, based solely on DNA sequence similarity to the others.
Each of these classes are associated with different cellular programs and may be assayed individually with various fluorometric assays. For example, SIRTs are typically associated with cancers and neurological diseases. Detecting SIRT activity, or identifying drugs that impact SIRT activity, may point to novel diagnostics or therapeutic strategies for these diseases.
Fluorometric assays utilize an acetylated peptide substrate with a fluorophore and quencher at its amino and carboxyl terminals. Once the substrate is deacetylated, it can be cleaved by a peptidase, releasing the fluorophore from the quencher. The subsequent increase in fluorescence intensity of the fluorophore is directly proportional to deacetylase activity.
Inhibiting writers and erasers
I can be useful to inhibit these modifying enzymes using small molecules and then assess downstream consequences to probe the involvement and biological functions of histone modifications. Thus, inhibitors of writers and erasers are vital tools for understanding the roles of epigenetic modification pathways. They are also essential for the validation of “druggable” targets in the context of pre-clinical studies both in academic and industry contexts.
Histone modification readers/translators
Histone modifications regulate the physical properties of chromatin, and its corresponding transcriptional state, either directly (eg acetyl groups that repel negatively charged DNA to create open chromatin conformation) or via protein adaptors termed effectors. Effector proteins recognize and bind to specific epigenetic marks, and subsequently, recruit molecular machinery to alter chromatin structure. These epigenetic readers determine the functional outcome of histone modifications by translating the histone code into action.
Effector domains recognize specific histone modifications
Effector proteins recognize and bind to histone modification marks through effector domains, known as modules (Table 2).
Table 2. Recognition of histone marks by modules or histone-binding proteins.
What Ultimately Controls DNA Transcription? - Biology
Recent advances in methods to isolate TCR complexes revealed a sequential and cooperative assembly of CSB, CSA, and UVSSA onto DNA damage-stalled RNAPII.
The UV-induced ubiquitylation of a single lysine residue in the largest subunit of RNAPII (RPB1-K1268) by CRL4 E3 ligases, including CRL4 CSA , is a key regulator of TCR and, together with the ubiquitylation of UVSSA (K414), promotes the transfer of TFIIH onto RNAPII to initiate DNA repair.
The CS protein-dependent ubiquitylation of RPB1-K1268 acts as a molecular clock that promotes DNA repair at early time-points, but leads to the removal of DNA damage-stalled RNAPII when repair fails.
Rather than a DNA repair disorder, emerging evidence suggests that Cockayne syndrome is caused by a deficiency in processing RNAPII, resulting in its prolonged arrest at DNA lesions, ultimately causing neurodegeneration.
DNA lesions pose a major obstacle during gene transcription by RNA polymerase II (RNAPII) enzymes. The transcription-coupled DNA repair (TCR) pathway eliminates such DNA lesions. Inherited defects in TCR cause severe clinical syndromes, including Cockayne syndrome (CS). The molecular mechanism of TCR and the molecular origin of CS have long remained enigmatic. Here we explore new advances in our understanding of how TCR complexes assemble through cooperative interactions between repair factors stimulated by RNAPII ubiquitylation. Mounting evidence suggests that RNAPII ubiquitylation activates TCR complex assembly during repair and, in parallel, promotes processing and degradation of RNAPII to prevent prolonged stalling. The fate of stalled RNAPII is therefore emerging as a crucial link between TCR and associated human diseases.
An essay on the transcription process
With particular emphasis on the biochemistry, discuss the machinery involved in the transcriptional process.
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https://brainmass.com/biology/cell-biochem/an-essay-on-the-transcription-process-2527Solution Preview
The mechanism of transcription: regulation of the conversion of DNA into RNA
A central dogma of biology is that DNA is transcribed into single-stranded RNA, which is then translated into proteins. Translation of proteins is dependent on fine control of genes. Through the action of various factors that influence the transcription of a gene, a particular protein is ultimately translated. This fine control allows external factors such as changes in the environment or other stresses to dictate what proteins a cell produces.
The genome of every cell codes for every possible protein needed in any cell type in the entire organism but most cells need only a subset of these proteins to conduct the specific functions of an individual cell type. Hence, a cell will only translate proteins only as they are necessary and in response to different cues. There must be some sort of control that allows transcription of genes specific to an environmental stress. There must also be a means of controlling the rate of transcription of a particular gene in a timely manner.
This essay reviews the machinery and the various control mechanisms involved in transcription that are employed by the cell. The process of transcription is a complex mutlistep process that has various checkpoints of control that are examined in this essay. As well, this essay also investigates a few techniques that are used in the study of transcription and gene regulation via transcriptional control.Background
During transcription a single-stranded piece of RNA is made from a double-stranded piece of DNA. In eukaryotes a gene usually contains coding sequences or exons, interspersed with non-coding sequences or introns. After transcription, the non-coding sequences are removed to form message RNA (mRNA), which is then transferred to the cytoplasm for translation into proteins. A gene also contains specific sequences before and after the transcribed region that are involved in the regulation of transcription. Introns and exons along with these regulatory sequences make up the structure of a gene (Fig. 1). The transcription process involves numerous proteins that act in a concerted fashion to allow RNA polymerase to work (see Shilatifard, 1998). These proteins are all subject to regulation and are points of control in the transcription of a gene.
Three different RNA polymerases (pol I, II, III) synthesize different types of RNA from a DNA template. Pol I, II, and III transcribe ribosomal RNA (rRNA), message RNA (mRNA), and transfer RNA (tRNA), respectively. This review will focus mostly on Pol II and its involvement in the transcription of DNA into mRNA. The Pol II basal machinery binds to a region called the promotor, which is upstream of the DNA transcriptional start site. The promotor region is an important sequence as the strength of binding of the basal machinery to this region determines how frequently a gene will be transcribed. The frequency of gene transcription, as will be discussed further, is dependent on transcription factors and their control. The transcriptional start site is typically designated as +1 and sequences upstream of the transcriptional start site are given a negative designation. The promotor region is found upstream of the transcriptional start site and includes the TATA box, a highly conserved septamer (5'-TATAAAA-3') that is found at about position -25 (Workman and Roeder, 1987).
Transcription can be broken down into three stages: pre-initiation and initiation, followed by elongation, and then termination. These three steps are outlined below and summarized in Figure 2. Pre-initiation and initiation steps happen close together, do not have clear boundaries and will be discussed here as a single stage. Elongation is the actual synthesis of the mRNA strand by the addition of the ribonucleotides adenine, guanine, cytosine and uracil. During synthesis of RNA, instead of adding thymine, uracil residues are added instead. Termination involves the release of Pol II from the DNA template allowing it to make another RNA transcript.Pre-Initiation and Initiation
The pre-initiation and initiation steps involve protein interactions that unwind the chromatin DNA and facilitate the interaction of the exposed DNA template with RNA polymerase (Ghosh and Van Duyne, 1996). The pre-initiation step involves the formation of the basal machinery from several general transcription factors that allow the binding of Pol II and the initiation of transcription.
One of the first factors to bind does so at the TATA box. The TATA box binding protein (TBP) recognises the septamer and binds at the minor groove of the DNA double helix. TBP looks like a saddle with its inner surface binding to the DNA leaving the outer surface free to interact with other proteins. In fact, eleven other proteins interact, with TBP. TBP together with eight other TBP associated factors (TAFs) forms a large complex known as TFIID (Transcription factor D for pol II) (Ghosh and Van Duyne, 1996). TBP and TFIID are used interchangeably throughout the literature but will be referred to as TBP herein.
The second factor to bind is TFIIA (transcription Factor A for pol II), which binds directly to TBP and aids in stabilizing the interaction between TBP and the DNA (Maldonado et al., 1990). Nogales (2000) suggests from structural observations that TFIIA increases the surface area of the basal complex that interacts with the DNA. TFIIA also functions as an anti-repressor, blocking proteins that de-stabilize the TBP-DNA interaction (Coleman et al., 1999). TBP can form a dimer through its DNA binding domain and thus prevent any unwanted gene transcription. TFIIA can block the dimerization of TBP by promoting its disassociation leaving TBP in monomer form (Coleman et al., 1999). As a result, TFIIA facilitates TBP binding to the DNA promotor and transcription of the gene.
After TFIIA binding, a third factor called TFIIB binds to the growing basal complex. TFIIB binds to TBP directly and aids in stabilizing the binding to the TATA box. Both TFIIB and TFIIA .Solution Summary
This solution is an essay detailing the conversion of DNA to RNA. This solution includes all the necessary peer-reviewed citations in the text with full references. This is a 5000 word solution and includes two figures to illustrate the process.
DNA structure influences the function of transcription factors
A depiction of the double helical structure of DNA. Its four coding units (A, T, C, G) are color-coded in pink, orange, purple and yellow. Credit: NHGRISubstances known as transcription factors often determine how a cell develops as well as which proteins it produces and in what quantities. Transcription factors bind to a section of DNA and control how strongly a gene in that section is activated. Scientists had previously assumed that gene activity is controlled by the binding strength and the proximity of the binding site to the gene. Researchers at the Max Planck Institute for Molecular Genetics in Berlin have now discovered that the DNA segment to which a transcription factor binds can assume various spatial arrangements. As a result, it alters the structure of the transcription factor itself and controls its activity. Neighbouring DNA segments have a significant impact on transcription factor shape, thus modulating the activity of the gene.
For a car to move, it is not enough for a person to sit in the driver's seat: the driver has to start the engine, press on the accelerator and engage the transmission. Things work similarly in the cells of our body. Until recently, scientists had suspected that certain proteins only bind to specific sites on the DNA strand, directing the cell's fate in the process. The closer and more tightly they bind to a gene on the DNA, the more active the gene was thought to be. These proteins, known as transcription factors, control the activity of genes.
A team of scientists headed by Sebastiaan Meijsing at the Max Planck Institute for Molecular Genetics have now come to a different conclusion: The researchers discovered that transcription factors can assume various shapes depending on which DNA segment they bind to. "The shape of the bond, in turn, influences whether and how strongly a gene is activated," Meijsing explains.
Consequently, transcription factors can bind to DNA segments without affecting a nearby gene. As in our car analogy, the mere presence of a "driver" is evidently not sufficient to set the mechanism in train. Other factors must also be involved in determining how strongly a transcription factor activates a gene.
Glucocorticoid receptor is also a transcription factor
One example is glucose production in the liver. If the blood contains too little glucose, the adrenal glands release glucocorticoids, which act as chemical messengers. These hormones circulate through the body and bind to glucocorticoid receptors on liver cells. The receptors simultaneously act as transcription factors and regulate gene activity in the cells. In this way, the liver is able to produce more glucose, and the blood sugar level rises again.
"Sometimes glucocorticoid receptor binding results in strong activation of neighbouring genes, whereas at other times little if anything changes," Meijsing reports. The scientists found that the composition of DNA segments to which the receptors bind help determine how strongly a gene is activated. However, these segments are not in direct contact with the receptors acting as transcription factors they only flank the binding sites. Yet, that is evidently enough to have a significant influence on the interaction.
"The structure of the interface between the transcription factor and genome segments must therefore play a key role in determining gene activity. In addition, adjacent DNA segments influence the activity of the bound transcription factors. These mechanisms ultimately ensure that liver cells produce the right substances in the right amounts," Meijsing says.
The findings could also have medical applications. Many DNA variants associated with diseases belong to sequences that evidently control the activity of transcription factors. "Scientists had previously assumed that these segments exert an effect by inhibiting the binding of transcription factors, thus impeding the activity of neighbouring genes," Meijsing says. "Our findings have now shown that some of these segments may not influence the contact directly but nevertheless reduce the activation state of the associated transcription factor."
DNA structure influences the function of transcription factors
Substances known as transcription factors often determine how a cell develops as well as which proteins it produces and in what quantities. Transcription factors bind to a section of DNA and control how strongly a gene in that section is activated. Scientists had previously assumed that gene activity is controlled by the binding strength and the proximity of the binding site to the gene. Researchers at the Max Planck Institute for Molecular Genetics in Berlin have now discovered that the DNA segment to which a transcription factor binds can assume various spatial arrangements. As a result, it alters the structure of the transcription factor itself and controls its activity. Neighbouring DNA segments have a significant impact on transcription factor shape, thus modulating the activity of the gene.
For a car to move, it is not enough for a person to sit in the driver's seat: the driver has to start the engine, press on the accelerator and engage the transmission. Things work similarly in the cells of our body. Until recently, scientists had suspected that certain proteins only bind to specific sites on the DNA strand, directing the cell's fate in the process. The closer and more tightly they bind to a gene on the DNA, the more active the gene was thought to be. These proteins, known as transcription factors, control the activity of genes.
A team of scientists headed by Sebastiaan Meijsing at the Max Planck Institute for Molecular Genetics have now come to a different conclusion: The researchers discovered that transcription factors can assume various shapes depending on which DNA segment they bind to. "The shape of the bond, in turn, influences whether and how strongly a gene is activated," Meijsing explains.
Consequently, transcription factors can bind to DNA segments without affecting a nearby gene. As in our car analogy, the mere presence of a "driver" is evidently not sufficient to set the mechanism in train. Other factors must also be involved in determining how strongly a transcription factor activates a gene.
Glucocorticoid receptor is also a transcription factor
One example is glucose production in the liver. If the blood contains too little glucose, the adrenal glands release glucocorticoids, which act as chemical messengers. These hormones circulate through the body and bind to glucocorticoid receptors on liver cells. The receptors simultaneously act as transcription factors and regulate gene activity in the cells. In this way, the liver is able to produce more glucose, and the blood sugar level rises again.
"Sometimes glucocorticoid receptor binding results in strong activation of neighbouring genes, whereas at other times little if anything changes," Meijsing reports. The scientists found that the composition of DNA segments to which the receptors bind help determine how strongly a gene is activated. However, these segments are not in direct contact with the receptors acting as transcription factors they only flank the binding sites. Yet, that is evidently enough to have a significant influence on the interaction.
"The structure of the interface between the transcription factor and genome segments must therefore play a key role in determining gene activity. In addition, adjacent DNA segments influence the activity of the bound transcription factors. These mechanisms ultimately ensure that liver cells produce the right substances in the right amounts," Meijsing says.
The findings could also have medical applications. Many DNA variants associated with diseases belong to sequences that evidently control the activity of transcription factors. "Scientists had previously assumed that these segments exert an effect by inhibiting the binding of transcription factors, thus impeding the activity of neighbouring genes," Meijsing says. "Our findings have now shown that some of these segments may not influence the contact directly but nevertheless reduce the activation state of the associated transcription factor."
Original publication: Stefanie Schöne, Marcel Jurk, Mahdi Bagherpoor Helabad, Iris Dror, Isabelle Lebars, Bruno Kieffer, Petra Imhof, Remo Rohs, Martin Vingron, Morgane Thomas-Chollier, Sebastiaan H. Meijsing
Sequences flanking the core binding site modulate glucocorticoid receptor structure and activity
Nature Communications 1 September, 2016Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.
Gene expression is controlled by a number of features – regulation of transcription and translation:
In eukaryotes, transcription or target genes can be stimulated or inhibited when specific transcriptional factors move from the cytoplasm into the nucleus. As only target genes are transcribed, it means that specific proteins are made. Each type of body cell has different target cells so they give different characteristics i.e. a nerve cell is different to a red blood cell. Transcription factors can change the rate of transcription and the process is as follows:
- The transcription factors move in by diffusion into the nucleus from the cytoplasm.
- When in the nucleus they may bind to promoter sequence (the sequence which is the start of the target gene).
- The transcription factors either increase or decrease the rate of transcription depending if they have bound onto the promoter sequence.
Some transcription factors are called activators where they increase the rate of transcription. This is done by the transcription factors helping the RNA polymerase to bind to the promoter sequence to activate transcription. Others are called repressors where they decrease the rate of transcription. This is done by the transcription factors binding to the promoter sequence preventing RNA polymerase from binding. This stops transcription.
Oestrogen can initiate the transcription of target genes. NB: Sometimes it can cause a transcription factor to be a repressor. You don’t need to know this for the AQA exam. A transcription factor may be bound to an inhibitor stopping it from binding to the promoter sequence. Oestrogen binds to the transcription factor making an oestrogen-oestrogen receptor complex and changes the site where the inhibitor is joined on (called DNA binding site). This means that the inhibitor is detached allowing the transcription factor to attach to the promoter sequence. NB: You don’t need to know the name of the inhibitor. Also the DNA binding site on the transcription factor stays changed whilst the oestrogen has bound to it.
In eukaryotes and some prokaryotes, translation of the mRNA produced from target genes can be inhibited by RNA interference known as RNAi. Short RNA molecules such as micro RNA, known as miRNA, and small interference RNA, known as siRNA, form an RNA Induced Silencing Complex, known as RISC, with proteins. NB: The small RNA molecules known to be double stranded in the revision guides or in textbooks this is confusing so it is better to start the process as miRNA and siRNA being single stranded. RNA forms a complex with a protein which is an enzyme called RNA hydrolase. miRNA does not form a complex with RNA hydrolase but another protein. These RNA molecules can each make a RISC with more then one protein and the proteins involved do not need to be known for AQA. The complexes each attach to their target mRNA sequence and preventing translation in different ways. This is how it is done for each small RNA molecules:
- siRNA/miRNA in plants:
- The bases on the siRNA attach to the bases on the mRNA by complementary base pairing.
- RNA hydrolase hydrolyses the mRNA strand into fragments preventing translation to occur as the whole polypeptide chain will not be made
NB: It is not necessary to know that the fragments are degraded in the processing body. If you want to learn this there is no harm.
- miRNA in mammals:
- The bases on the miRNA attach to the bases on the mRNA by complementary base pairing.
- Ribosomes are prevented from attaching to the mRNA strand stopping translation from occurring.
NB: Again here, it is not necessary to know that mRNA is degraded or stored in the processing body.
Epigenetics involves heritable changes in gene function, without changes to the DNA base sequence. These changes are caused by changes in the environment (more exposure to pollution) that inhibit transcription by:
- Increased methylation of DNA:A methyl group (known as an epigenetic mark) attaches to cytosine that has to be part of the nucleotide that is attached to guanine by a phosphodiester bond. NB: You may be confused right now but look at the diagram below of one strand of DNA and notice which of the cytosine nucleotides the methyl group joins on to. Notice that the nucleotide on the far right of the strand and the third one from the left does not have a methyl group as they are not next to a nucleotide with guanine as the base. The joining of the methyl group should not be confused by joining on to cytosine which is complementary to guanine on the other strand as this is wrong. Also the methyl group – CH3 – does not change the base sequence but the structure. As the structure has changed, it has become harder for enzymes to attach to the DNA stopping the expression of a gene. If the tumour suppressor gene is not transcribed it can cause cancer.
- Decreased of associated histones: An acetyl group – COCH3 – is another epigenetic mark which attaches to histone proteins to make the chromatin (mixture of DNA wound around histone proteins) less condensed for easy genetic expression to occur. The problem originates when histone deacetylase breaks the bond between the histone protein and acetyl group. The DNA becomes highly condensed making hard for enzymes to carry out the gene expression. NB: Histone deacetylase can be abbreviated into HDAC but it is best that you stay with the full name.
Epigenetic changes to the DNA are fortunately reversible therefore they are good targets by drugs to stop the effects of epigenetic occurring. These drugs can either stop DNA methylation or can inhibit histone deacetylase allowing the acetyl groups to remain attached to the DNA.
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