Genetics Chapter 15
Transcriptional activation with nucleosomes
-1. Binding of the activator -Activator protein binds to the enhancer sequence. The enhancer may be close to the transcriptional start site or far away, 2. Chromatin remodeling and histone modification -an activator protein recruites a remodeling complex and a histone modifying enzyme. The various methods of histone modification will be completed here as necessary. 3. Formation of preinitiation complex -General transcription factors and RNA polymerase II are able to bind to the core promoter and form a pre-initiation complex 4. Elongation -During elongation, histones ahead of the RNA polymerase are modified or evicted as needed until the gene is done being transcribed.
Replacement with histone variants
-A few of these genes have accumulated mutations that alters the amino acid sequence -These are termed histone variants -Some histone variants are incorporated into a subset of nucleosomes to create specialized chromatin
Histone eviction
-A histone octamer is completely removed from the DNA which allows for open to DNA to transcribe
-Chromatin Immunoprecipitation Sequencing has revealed a common pattern of nucleosome organization
-A nucleosome free region (NFR) is found at the beginning and the end of many genes. Nucleosomes tend to be precisely positioned near the beginning and end of a gene, but are less regularly distributed elsewhere.
Helix-loop-helix motif
-A short alpha helix is connected to a longer alpha helix by a loop.
Histone remodeling and histones
-ATP-dependent chromatin remodeling refers to dynamic changes in chromatin structure These changes range from a few nucleosomes to large scale changes Carried out by diverse multiprotein machines that reposition and restructure nucleosomes
Zinc-finger motif
-Each zinc finger is composed of one alpha helix and two antiparallel beta sheets. A zinc ion, shown in red, holds the zinc finger together.
Benefits of gene regulation
-For example to respond to changes in environment nutrient availability environmental stresses In plants and animals, multicellularity and a more complex cell structure also demand a much greater level of gene regulation
Heterodimers
-Heterodimers are formed by two different transcription factors
Histone proteins
-Histone proteins are basic -They contain many positively-charged amino acids Lysine and arginine -These bind to the phosphates along the DNA backbone -Histone proteins have a globular domain and a flexible, charged amino terminus or 'tail' -There are five types of histones H2A, H2B, H3 and H4 are the core histones Two of each make up the octamer H1 is called the linker histone Binds to DNA in the linker region Less tightly bound to DNA than core histones May help compact adjacent nucleosomes
Homoedimers
-Homodimers are formed by two identical transcription factors;
Enhancers and Silencers
-Many response elements are orientation independent or bidirectional -They can function in the forward or reverse orientation -Most response elements are located within a few hundred nucleotides upstream of the promoter However, some are found at various other sites Several thousand (even 100,000) nucleotides away Downstream from the promoter Even within introns!
Combinatorial Control
-Most Eukaryotic genes are regulated by many factors -One or more activator proteins may stimulate transcription -One or more repressor proteins may inhibit transcription -Activators and repressors may be modulated by: binding of small effector molecules protein-protein interactions covalent modifications Section 15.2 -Regulatory proteins may alter nucleosomes near the promoter Section 15.3 -DNA methylation may inhibit transcription prevent binding of an activator protein recruiting proteins that compact the chromatin -Various combinations of these factors can contribute to the regulation of a single gene
Histone Code
-Over 50 enzymes have been identified in mammals that selectively modify the amino terminal tails of histones -acetylation, methylation and phosphorylation are common (see Figure 15.10) -These modifications affect the level of transcription May influence interactions between nucleosomes -Occur in patterns that are recognized by proteins Called the histone code -The pattern of modifications provide binding sites for proteins that specify alterations to be made to chromatin structure -These proteins bind based on the code and affect transcription
Methylation affects transcription in two ways
-Prevent or enhance binding of regulatory transcription factors to the promoter. -Special proteins bind to methylated DNA. Methyl-CpG-binding proteins -These proteins recruit other proteins, such as histone deacetylases What is the effect of deacetylation? -will give the positive charge back to the nucleosome which will tighten the DNA around the histone which will decrease expression.
Regulatory Transcription Factors
-Regulatory transcription factors recognize cis regulatory elements located near the core promoter These sequences are known as control elements, regulatory elements or regulatory sequences
Steroid Hormones and Regulatory Transcription Factors
-Regulatory transcription factors that respond to steroid hormones are termed steroid receptors The hormone actually binds to the transcription factor -The ultimate action of a steroid hormone is to affect gene transcription -Steroid hormones are produced by endocrine glands -Secreted into the bloodstream Then taken up by cells that respond to the hormone
CREB Protein
-The CREB protein is another regulatory transcriptional factor CREB is an acronym for cAMP response element-binding CREB protein becomes activated in response to cell-signaling molecules that cause an increase in the cytoplasmic concentration of cAMP Cyclic adenosine monophosphate The CREB protein recognizes a response element with the consensus sequence 5'-TGACGTCA-3' This has been termed a cAMP response element (CRE)
Transcriptional activation via TFIID
-The activator/coactivator complex recruites TFIID to the promoter and activates its function. Transcription will be enhancedl.
tissue-specific genes
-The expression of these genes may be silenced by the methylation of CpG islands -Methylation may influence the binding of transcription factors -Methyl-CpG-binding proteins may recruit factors that lead to compaction of the chromatin
Transcriptional repression via TFIID
-The repressor protein inhibits the binding of TFIID to the core promoter or inhibits its function. Transcription is repressed.
gene regulation
-The term gene regulation means that the level of gene expression can vary under different conditions -The benefit of regulating genes is that encoded proteins will be produced only when required Gene
Glucocorticoid Response Elements
-These function as enhancers GREs are located near dozens of different genes, so the hormone can activate many genes
Constitutive
-They have essentially constant levels of expression Frequently, constitutive genes encode proteins that are continuously necessary for the survival of the organism
Structural Features of Regulatory Transcription Factors
-Transcription factor proteins contain regions, called domains, that have specific functions -One domain could be for DNA-binding Another could provide a binding site for effector molecules -A motif is a domain, or a portion of a domain, that has a very similar structure in many different proteins
Transcription Factors
-Transcription factors are proteins that influence the ability of RNA polymerase to transcribe a given gene
Transcriptional activation via mediator
-Transcriptional activator stimulates the function of mediator -This enables RNA pol to form a preinitiation complex -It then proceeds to the elongation phase of transcription the interaction between the activator protein and the mediator results in the phosphorylation of the carboxyl-terminus domain of RNA polymerase. Some general transcription factors are then released and thus complex proceeds to the elongation phase of transcription.
Effect of acetyation
-acetylation removes the positive from the histone proteins which makes the negatively charged DNA less attracted to it which loosens the DNA around the histone -histone acetylotransferases looses the DNA around the histone -Histone deacetylases tighten the DNA around the histone
Leucine zipper motif
-the leucine zipper promotes teh dimerization of two transcription factor proteins. Two alpha helices are intertwined via the leucines.
The binding of regulatory transcription factors to control elements affects the transcription of an associated gene
1. A regulatory protein that increases the rate of transcription is termed an activator The sequence it binds is called an enhancer 2. A regulatory protein that decreases the rate of transcription is termed a repressor The sequence it binds is called a silencer
Two different confirmations of chromatin
1. Closed conformation Chromatin is very tightly packed Transcription may be difficult or impossible 2. Open conformation Chromatin is accessible to transcription factors Transcription can take place
Gene regulation is necessary to insure
1. Expression of genes in an accurate pattern during the various developmental stages of the life cycle Some genes are only expressed during embryonic stages, whereas others are only expressed in the adult 2. Differences among distinct cell types Nerve and muscle cells look so different because of gene regulation rather than differences in DNA content
2 main types of transcription factors
1. General transcription factors Required for the binding of the RNA pol to the core promoter and its progression to the elongation stage Are necessary for basal transcription 2. Regulatory transcription factors Serve to regulate the rate of transcription of target genes They influence the ability of RNA pol to begin transcription of a particular gene 2-3% of human genes encode transcription factors
Cells respond to steroid hormones in different ways
1. Glucocorticoids These influence nutrient metabolism in most cells They promote glucose utilization, fat mobilization and protein breakdown 2. Gonadocorticoids These include estrogen and testosterone They influence the growth and function of the gonads
Four different important motives
1. Helix-turn-helix motif 2. Helix-loop-helix motif 3. Zinc Finger motif 4. Leucine zipper motif
Three common interactions that communicate the effects of regulatory transcription factors are (GMRP)
1. TFIID-direct or through coactivators 2. Mediator 3. recruiting proteins that affect nucleosome composition
Process of CREB protein activation within the cell
1. The extracellular signaling molecule binds to a plasma membrane receptor 2. which activates a G protein which activates adenlyl cyclase which catalyzes the synthesis of cAMP (which acts as a second messenger and activates protein kinase A). 3. protein kinase A travels into the nucleus and phosphorylates several different cellular proteins, including CREB protein. 4. Phosphorylated CREB binds to DNA and stimulates transcription 5. Unphosphorylated CREB can bind to DNA, but cannot activate RNA pol
Stepwise action of glucocorticoid hormones
1. The hormone enters the cytosol of a cell by diffusing through the plasma membrane. 2. Once inside, the hormone specifically binds to glucocorticoid receptors. Prior to hormone binding, the glucocorticoid receptor is complexed with proteins known as heat shock proteins (HSP) 3. After the hormone binds to the glucocorticoid, HSP90 is released. 4. This exposed a nuclear localization signal (NLS) a sequence of amino acids within the protein that directs the protein into the nucleus. 5. Two glucorcorticoid receptors form a homodimer and then travel through a nuclear pore into the nucleus 6. Binds to a glucocorticoid response element (GRE) which functions as an enhancer and are next to various genes 7.The binding of the glucocorticoid recepter (homodimer) to the GRE stimulates the transcription and translation of the nearby gene into a functional product.
Different types of methylation in DNA strands
1. unmethylated 2. hemimethylated 3. double methylated
Transcriptional repression via mediator
1.Transcriptional repressor inhibits the function of mediator 2. Transcription is repressed Explained: The repressor protein interacts with the mediator in a way that does not let it phosphorylate RNA polymerase which represses it from going into elongation phase.
DNA methylation is heritable
=Methylated DNA sequences are inherited during cell division May explain genomic imprinting (Chapter 5) Specific genes are methylated in gametes from mother or father Pattern of one copy of the gene being methylated and the other not is maintained in the resulting offspring
ATP-dependent Chromatin remodeling
ATP-dependent chromatin remodeling. -Energy of ATP hydrolysis is used to drive change in location and/or composition of nucleosomes -Makes the DNA more or less amenable to transcription -like change in the relative positions of a few nucleosomes or.... -change in the spacing of nucleosomes over a long distance.
DNA methylation
DNA methylation is a change in chromatin structure that silences gene expression Carried out by the enzyme DNA methyltransferase It is common in some eukaryotic species, but not all
DNA methyltransferase
DNA methyltransferase converts hemi-methylated to fully- methylated DNA
DNA methylation usually inhibits the transcription of eukaryotic genes
Especially when it occurs in the vicinity of the promoter In vertebrates and plants, many genes contain CpG islands near their promoters These CpG islands are 1,000 to 2,000 nucleotides long Contain high number of CpG sites
INSULATORS
Since eukaryotic gene regulation can occur over long distances, it is important to limit regulation to one particular gene, but not to neighboring genes Insulators are segments of DNA that insulates a gene from the regulatory effects of other genes Some act as barriers to chromatin remodeling Others block the effects of enhancers May do this by chromosome looping
housekeeping genes
The CpG islands are unmethylated Genes tend to be expressed in most cell types
Modulation of Regulatory Transcription Factor Functions BEFppCM
There are three common ways that the function of regulatory transcription factors can be modulated 1. Binding of a small effector molecule 2. Protein-protein interactions 3. Covalent modification
Constitutive Genes
tRNA, rRNA, ribosomal proteins, RNA polymerase, housekeeping genes (enzymes catalyzing metabolic processes) Always expressed in most cells
Helix-turn-helix motif
the protein secondary structure known as an alpha helix is frwquenctly found in transcription factors. The alpha helix is so common because the alpha helix is the proper width to bind into the major groove of the DNA double helix. -Helix-turn-helix motif: Two alpha helices are connected by a turn. The alpha helices bind to the DNA within the major groove.