Unit 3. The Nucleus PDF

Unit 3. The nucleus. SECTION II: CELL STRUCTURE AND FUNCTION. INDEX 3.1. The cell nucleus and the DNA 3.2. Nuclear envelope 3.3. DNA replication 3.4. DNA transcription 3.5. Traffic between the nucleus and the cytoplasm 3.6. Nuclear bodies 3.1. The cell nucleus and the DNA Funct...

Unit 3. The nucleus. SECTION II: CELL STRUCTURE AND FUNCTION. INDEX 3.1. The cell nucleus and the DNA 3.2. Nuclear envelope 3.3. DNA replication 3.4. DNA transcription 3.5. Traffic between the nucleus and the cytoplasm 3.6. Nuclear bodies 3.1. The cell nucleus and the DNA Functions: Serves as a storehouse for genetic information At the genomic level: ✓ DNA replication takes place ✓ RNA transcription and processing Regulates gene expression, by regulating the transport of transcription factors from the cytoplasm to the nucleus. Video “DNA structure” https://www.youtube.com/watch?v=yjEOcOEOLq0&list=PLBd5pZfVmZ5aFrKMcJ 53vOfOocA2Ay8hA&index=90 Chromosomes and chromatin Eukaryotic genomes are more complex than prokaryotic. Furthermore, DNA is organized not on single chromosomes but on multiple chromosomes, each of which contains a linear DNA molecule. DNA binds to small proteins (histones) that package it in an orderly fashion in the cell nucleus. Primary characteristic (Ex: the length of human DNA is 2 meters, but it must fit in a core of 5-10 micrometers). Heterochromatin and euchromatin are two major categories of chromatin. Heterochromatin has condensed chromatin structure and is inactive for transcription Euchromatin has loose chromatin structure and active for transcription. Chromatin at the interphase. Electron micrograph of nucleus in interphase. Euchromatin is distributed throughout the nucleus. Heterochromatin is indicated by the triangles and the nucleolus by an arrow. Chromatin is a complex structure of DNA and proteins and its degree of condensation will depend on the activity of each chromosomal region and the moment of the cell cycle. There are different levels of DNA packaging.  Level 1: the DNA double helix (about 147 bp) coils around a core or octamer of histones (two molecules of each of the histones H2A, H2B, H3 and H4) in a structure known as a nucleosome. The binding of the H1 binding histone to the core nucleosome forms a chromatin subunit called the chromatosome (166 bp, two turns of the superhelix). H1 is located where the DNA enters and leaves the nucleosome. Chromatosome There is a distance of about 200 bp between two consecutive nucleosomes, which makes this structure look like necklace beads under an electron microscope: “beads on a string” structure. The structure of nucleosomes linked together gives rise to an 11nm chromatin fiber.  Level 2: The 11 nm fiber is rewound to form a 30 nm chromatin fiber, in a structure containing about 6 nucleosomes per turn. Most of the euchromatin at the interphase is in the form of 30 nm fibers or somewhat more condensed (60-130 nm). Level 3: The 30 nm chromatin fiber undergoes different degrees of packaging. First, it forms loops linked at its base to a protein scaffold structure. Chromatin loops are attached to this scaffold by sequences rich in A and T called SAR (scaffold- attachement regions) or MAR (matrix- attachment regions). This results in a 300 nm fiber. During mitosis, the 300 nm fiber is recondensed to form a 600-700 nm fiber (or chromatid). Chromosomes: The degree of packaging of DNA reaches 10,000 times Degree of DNA packaging: Level 1: 2nm fiber (dsDNA) → 11nm (necklace beads) 6 x Level 2: 11 nm fiber (necklace beads) → 30 nm (solenoid structure) 40 x Level 3: 30 nm (solenoid structure) → 300 nm (hairpins) 2,000 x 300 nm (hairpins) → 600-700 nm (chromatids) 10,000 x The interaction between histones and DNA must be dynamic The histones that make up the nucleosome have an amino- terminal tail that can undergo modifications: the Lys can be acetylated or methylated; and Ser's can be phosphorylated. These modifications constitute a histone code that can be 'read' by proteins that participate in the replication or expression of genetic material. Histone acetylation is associated with transcriptional activation. Consequently, acetylated nucleosomes associate with more transcriptionally active chromosomal regions. Acetylation of histones relaxes chromatin and facilitates transcription, while deacetylation Methylation can be associated with both active and promotes more compact chromatin. repressed chromatin. Chromosomes are the consequence of nuclear DNA packaging together with various proteins in their maximum degree of condensation Each eukaryotic species has a characteristic number of chromosomes (number n). In most cells there are two sets of chromosomes (diploid or 2n cells), one from the father and one from the mother. Gametes have n chromosomes and are haploid cells. Humans have 46 chromosomes (2n) and each of the chromosome sets is made up of 23 chromosomes (n). Of these, 22 are made up of strictly homologous chromosomes, in terms of size, shape and genetic information (autosomes, which are numbered 1-22). The X and Y sex chromosomes, although they behave as homologues, do not have the same information, shape, or size. Females are homogametic (two X chromosomes) and males are heterogametic (XY chromosomes).  In the nucleus in metaphase, both copies of the replicated DNA are held together by the centromere or primitive constriction. Each of these copies is a chromatid. The two chromatids that make up a metaphase chromosome (sister chromatids) will be delivered to daughter cells at the end of cell division. The centromere divides the chromatids into two arms, which can be the same or different in length. The ends of the chromatids are called telomeres. The centromere is a specialized region of the chromosome whose role is to ensure the correct distribution of duplicated chromosomes to daughter cells during mitosis. - sister chromatid association sites. - binding site for the mitotic spindle. In yeast the sequences of the centromeres are short, about 125 bp. In mammals they are made up of hundreds of Kb of repetitive DNA, which is sometimes chromosome specific.  Telomeres are made up of specialized structures of DNA and proteins. Functions: - maintain the structural integrity of chromosomes (if these are lost, the chromosomes can fuse with each other or degrade); - position each chromosome in the nucleus; - ensure complete DNA replication. The sequences that make up telomeres are similar, presenting repeats of a single DNA sequence containing groups of G residues on one strand. In humans: TTAGGG, it is repeated in tandem up to a variable length of between 3-20Kb.  Structure of a telomere: Telomeric DNA forms a loop on itself to form a circular structure with a protein complex (shelterin) that protects the ends of chromosomes against degradation. DNA polymerase cannot replicate these chromosomal endings. A special enzyme, telomerase, replicates telomeric DNA. Genes and genomes Genome: set of genetic material of an organism. Gene: DNA (structural and regulatory) required to encode a gene product (mature RNA or protein) Extragenic DNA: does not encode any protein. Recent research suggests that they are not devoid of functionality, such as regulation of gene expression. All cells have the same genome (except gametes), but the expression is differential in each cell type. Haploid nuclear human genome = 3200 Mb  DNA quantity paradox Amount of DNA = Complexity Amount of DNA = number of genes Complexity = number of genes  Gene structure: Introns and exons exon intron Fragment of a messenger RNA that Non-coding sequence of a gene, which survives the assembly process although it is initially transcribed into (splicing) to become part of the messenger RNA, is lost during the mature messenger RNA. The exons assembly process, and therefore is not make up both the coding region present in mature messenger RNA. and the untranslated transcribed regions that flank the coding region.  It gives the genome enormous potential and flexibility, since the same gene will be able to synthesize different and related products. alternative splicing Generation of different mature RNA transcripts from the same gene, obtained by deleting one or more exons in the splicing processes, which take place in RNA maturation. Alternative splicing occurs in RNA, which codes for many proteins, so that by alternating the exons used, a set of related proteins can be generated that are often differentially expressed throughout development or in different tissues. Alternative splicing allows 21,000 human protein coding genes to specify nearly 85,000 different proteins.  Complexity in Human DNA: Types of Sequences in our genome 21000 Genes (exons, introns, regulatory sequences). ◦ Regulatory sequences: promoters, silencers, enhancers Extragenic DNA. ◦ DNA which is transcribed to noncoding RNA ◦ Repetitive DNA sequences: ◦ Tandem repeats: satellite DNA ◦ Sparse repeats: LINES, SINES, LTR, transposons ◦ Gene duplication and pseudogenes Noncoding RNA. - tRNA and rRNA, known for a long time, they play fundamental roles in protein synthesis.. - micro RNA (miRNA): double-stranded RNA with approx. 22 nt. One of the strands associates with the RNA-induced silencing complex (RISC) and the miRNA directs the RISC to target (complementary) mRNAs, where they inhibit translation or stimulate mRNA degradation. It is estimated that each miRNA can target up to 100 different mRNAs. They act in various developmental processes, regulate cell proliferation and survival, and their abnormal expression is associated with heart disease and cancer, although their biological functions are not fully understood. - Long non-coding RNAs (long ncRNAs, lncRNA): non-coding RNA of more than 200 nt, important regulators of gene expression in eukaryotes. More than 50,000 in humans. Example: Xist RNA, 17Kb that blocks the transcription of one of the two X chromosomes in females. - snRNA. - snoRNA. Repetitive DNA sequences. - Single sequence repeats: Tandem sets made up of thousands of copies of short sequences (1-500 nt). They are not transcribed or contain functional genetic information, but they are important for the structure of the chromosome. They represent about 40% of the total genomic DNA in Drosophila and approximately 10% in humans. This is satellite DNA, so called because when separating genomic DNA by density gradient centrifugation, it appears as "satellite" bands of the main band. AT rich sequences are less dense. GC-rich sequences are denser. 3.2. Nuclear envelope Structure of the nuclear envelope Nuclear pore complex NUCLEAR ENVELOPE It acts as a selective barrier that prevents the free traffic of molecules between the nucleus and cytoplasm. Nuclear pore complexes constitute the only communication channels between the nucleus and the cytoplasm, allowing the controlled exchange of molecules between both compartments. It maintains both compartments metabolically independent. Maintains the internal composition of the nucleus. Key role in the regulation of gene expression in eukaryotes Controlled transcription by regulating the transport of transcription factors to the nucleus. Post-transcriptional mechanisms (e.g. alternative splicing) Nuclear envelope structure: 2 nuclear membranes: o Outer membrane o Inner membrane The nuclear pore complex Nuclear lamina (underlies the INM)) Electron micrographs of the nucleus. The inner and outer nuclear membranes join in the nuclear pore complexes (arrows). Continuity of the outer nuclear membrane with the endoplasmic reticulum.  Outer nuclear membrane ✓ Continuation with the membrane of the endoplasmic reticulum ✓ There is communication between the nuclear intermembrane space and the lumen of the endoplasmic reticulum. ✓ Functionally it is similar to that of the endoplasmic reticulum. ✓ It has ribosomes attached to its surface and membrane proteins that bind to the cytoskeleton. Inner nuclear membrane ✓ It has integral membrane proteins specific to the nucleus (some bind the nuclear lamina).  Nuclear pore complex ✓ Inner and outer membranes join together in nuclear pore complexes ✓ They are the only channels that allow small polar molecules and macromolecules to pass through the nuclear envelope. ✓ It is a complex structure responsible for the selective trafficking of proteins and RNA between the nucleus and the Electron micrograph showing nuclear pores. cytoplasm. Many nuclear pores (arrows) are seen in this preparation made by nuclear envelope freeze- fracture.  Nuclear lamina ✓ Fiber network that provides structural support to the nucleus ✓ Made up of lamin A, B and C proteins. ✓ All lamins are fibrous proteins between 60-80 kDa. ✓ Lamins are joined together forming filaments. Electron micrograph of the nuclear lamina. The lamina is a network of filaments below the inner nuclear membrane.  The first level of association is the interaction between two lamins to form a dimer in which the -helix areas of two polypeptides wrap around each other forming a structure called a coiled coil. The dimers associate with each other (head to tail) to form filaments that in turn interact with each other to form the nuclear lamina.  The interaction of the lamina with the inner nuclear membrane is facilitated by the post- translational addition of lipids and the interaction with integral proteins of the inner nuclear membrane, such as emerin, the lamin B receptor (LBR) or the SUN proteins. The SUN proteins bind to the KASH proteins of the outer nuclear envelope, creating the LINC complex that connects the nuclear lamina with the cytoskeleton. Lamina and inner nuclear membrane proteins also interact, via emerin and LBR, with chromatin- associated proteins. NUCLEAR PORE COMPLEX The nuclear pore complex has octamer symmetry organized around a large central channel. The central channel of the pore is where small polar molecules, ions, proteins and RNAs are transported. Approximate diameter of 120 nm with a molecular weight of 125 million Da (30 times that of a ribosome). In vertebrates the complex is made up of Electron micrograph of nuclear pore complexes multiple copies of about 30 different proteins (nucleoporins).  8 protein spokes are assembled around the central channel. These spokes are attached to a cytoplasmic ring and another nuclear ring. This spoke-ring structure is anchored to the nuclear envelope at the fusion sites between the inner and outer nuclear membranes. Cytoplasmic filaments extend from the cytoplasmic ring. Filaments that extend from the nuclear ring form the nuclear basket. During the passage of macromolecules, the opening of the pore channel can be modified from 9 nm to 40 nm. 3.3. DNA replication Mechanism that allows DNA to duplicate (that is, to synthesize an identical copy). This duplication of genetic material occurs according to a semi-conservative mechanism, which indicates that the two complementary polymers of the original DNA, when separated, each serve as a template for the synthesis of a new complementary strand of the template strand, so that each new double helix contains one of the original DNA strands and a new one. Thanks to the complementation between the bases that make up the sequence of each of the chains, DNA has the important property of reproducing identically, which allows genetic information to be transmitted from a mother cell to daughter cells and is the basis of the inheritance of genetic material. DNA polymerase Both eukaryotes and prokaryotes contain different DNA polymerases with different roles in DNA replication and repair. - In bacteria, DNA polymerase III is the main polymerase responsible for replication. - In eukaryotes, DNA polymerases ,  and  function in the replication of nuclear DNA, and DNA polymerase  is responsible for the replication of mitochondrial DNA. All DNA polymerases share two fundamental properties: - They synthesize DNA only in the 5 'to 3' direction, adding a dNTP to the 3'OH group of a growing chain. - They can add a new deoxyribonucleotide only to an existing strand (primer) that is hydrogen bonded to the template strand; they are not capable of directing de novo synthesis by catalyzing the polymerization of free dNTPs. Video “DNA POLYMERASE ” https://www.youtube.com/watch?v=6hcK-- 4S68U&list=PLBd5pZfVmZ5aFrKMcJ53vOfOocA2Ay8hA&index=94 Origin of replication The DNA molecule opens like a zipper, by breaking the hydrogen bonds between complementary bases at certain points: the origins of replication, where replication begins. The initiator proteins recognize specific nucleotide sequences at these points and facilitate the attachment of other proteins that will allow the separation of the two DNA strands, forming two replication forks. A large number of enzymes and proteins are involved in the molecular mechanism of replication, forming the so-called replication complex or replisome. These proteins and enzymes are homologous in eukaryotes and archaea, but differ in bacteria. ORIGIN OF REPLICATION IN PROKARYOTES. 1 single origin. - Binding of a initiator protein to specific sequences within the origin. It begins to unwind the DNA and recruits the other proteins involved. - The helicase along with the single-stranded DNA- binding proteins continue to unwind and present the DNA. 2 replication forks are formed that move in opposite directions along the circular chromosome. ORIGIN OF REPLICATION IN EUKARYOTES In eukaryotes, multiple origins are needed to replicate the long chromosomes in a reasonable time. The E. coli genome (4 x 106 bp) replicates from a single origin of replication in 30 minutes. If mammalian genomes (3 x 109 bp) were to replicate from a single origin, they would require around 3 weeks (30,000 minutes). The problem is exacerbated by the fact that the rate of DNA replication in mammalian cells is 10 times slower than in E. coli, probably due to DNA packaging in chromatin, However, the genome of mammalian cells replicates in a few hours, thanks to the presence of multiple origins of replication. Replication fork Place of the DNA molecule in replication where the parental DNA strands are separated and two new daughter strands are synthesized. Problem: Since the two complementary strands of the DNA double helix are arranged in opposite directions (antiparallel), the continuous synthesis of two new strands in the replication fork would require that one of the strands be synthesized in a 5 'to 3' direction and the other in 3 'to 5'. But DNA polymerase only polymerizes in the 5 'to 3' direction (moving along the parent strand in 3 'to 5’ direction). Solution: Okazaki fragments (1-3 kb). DNA ligase joins the fragments of the lagging strand https://www.youtube.com/watch?v=IjVLhoyfGAM  DNA polymerase cannot initiate de novo synthesis, it needs a primer that provides a 3'OH group to which to add the next nucleotide. RNA can be synthesized de novo. The enzyme primase synthesizes short RNA fragments (3-10 nt) complementary to the template lagged strand in the replication fork. Okazaki fragments are synthesized by extension of these by DNA polymerase. RNA primers must be removed and replaced by DNA. In prokaryotes, this is done by DNA polymerase I, and in eukaryotes RNAse H degrades the RNA strand and DNA polymerase  fills in the gaps. The resulting DNA fragments are joined by DNA ligase. Sliding-clamps (PCNA, proliferative cell nuclear antigen, in eukaryotes) associate with prokaryotic polymerase III or eukaryotic  polymerase to position them in the primer and maintain their association with the stable template as replication progresses. Clamp-loaders (RFC, replication factor C, in eukaryotes) bind sliding-clamp proteins to DNA at the primer and template binding site. They are then released and DNA polymerase binds to the sliding-clamp protein. Helicases: They catalyze the unwinding of parental DNA, associated with the hydrolysis of ATP, at the head of the replication fork. Single-stranded DNA-binding proteins stabilize the uncoiled DNA template strand, keeping it in an extended single-stranded state for it to be copied by the polymerase. Topoisomerases reduce the molecular stress caused by the supercoiling of DNA. Action of Topoisomerases during DNA replication (A) Once the two strands of DNA are unwound, the DNA at the head of the replication fork is forced to rotate in the opposite direction so that the circular molecules are wound on themselves. (B) This problem is solved by Topoisomerases, which catalyze the reversible breaking and joining of DNA strands. The transient breaks introduced by these enzymes act as twirling links that allow the two strands of DNA to rotate freely on one another. Model of the replication fork of E. coli. A helicase, a primase, and two DNA polymerase III molecules carry out the coordinated synthesis of the leading and lagging strands of DNA. Both molecules of the DNA polymerase form a complex with the clamp-loading protein, which is bound to the helicase at the replication fork. Topoisomerase acts as a swivel at the head of the hairpin, and polymerase I together with a ligase removes the RNA primers and binds the Okazaki fragments behind the hairpin. VIDEO “REPLICATION” https://www.youtube.com/watch?v=PbCPGjs4X- 4&list=PLBd5pZfVmZ5aFrKMcJ53vOfOocA2Ay8hA&index=92 DNA maintenance FIDELITY OF THE REPLICATION The accuracy of DNA replication is critical for cell reproduction. Error frequency: less than one incorrect base for every 109 incorporated nucleotides. Mechanisms by which DNA polymerase achieves it: - It helps to select the correct base, although how it does this has not been figured out. - Double reading activity. Exonuclease activity of polymerase III and polymerase δ and ε allows to hydrolyze DNA in the 3'-5 'direction, contrary to DNA synthesis, which allows an incorrect base to be cleaved at the end of the growing DNA chain. Telomerase DNA polymerase involved in telomere formation, with the rare peculiarity that it is only capable of synthesizing oligonucleotides with the telomeric sequence. The enzyme contains a 159-nucleotide RNA oligonucleotide, which is essential for its activity and which provides the template for replication of the telomere sequence (so it is actually a type of reverse transcriptase). VIDEO “ TELOMERE REPLICATION” https://www.youtube.com/watch?v=2NS0jBPurWQ  3.4. DNA transcription DNA strands have different functions in transcription. The synthesis of the RNA molecule is carried out using the antisense strand as a template, from which its complementary sequence is synthesized. Thus, the base sequence of the RNA transcript is identical to the sequence of the sense strand, with the difference that the RNA contains uracils instead of thymine and hydroxyl groups at the 2 'carbon of pentoses.  RNA polymerase is the main enzyme involved in transcription RNA polymerase is the enzyme responsible for RNA synthesis. Catalyzed reaction: (NMP) n: RNA strand with "n" nucleotides monophosphate (NMP) NTP: nucleotide triphosphate PPi: pyrophosphate differences and similarities between replication & transcription  INITIATION Transcription begins with the binding of RNA polymerase to the promoter. The promoter region extends from tens or hundreds of bases before the transcription initiation site to a few bases beyond the initiation point. Although promoters are highly varied, there are consensus sequences that are frequently repeated in different genes. The DNA unwinds and the polymerase undergoes conformational and chemical changes (phosphorylation) that induce the initiation of transcription.  ELONGATION Most of the transcription factors are released at the beginning of the elongation. RNA polymerase advances synthesizing in the 5 'to 3' direction. The 3'OH group of the forming RNA reacts with a phosphate of the incoming ribonucleoside triphosphate, forming a new phosphodiester bond.  TERMINATION RNA synthesis ends when RNA polymerase recognizes certain DNA sequences that lie at the end of genes. The termination mechanism has been extensively studied in prokaryotes, where sequences and factors that participate in the arrest of elongation and in the release of the transcription machinery are known. However, in eukaryotes the details of the process are so far little known. Video “Transcription” https://www.youtube.com/watch?v=SMtWvDbfHLo Transcription in eukaryotes Eukaryotic cells have three RNA polymerases (I, II, III) that transcribe different classes of genes. RNA polymerases must interact with additional proteins to initiate and regulate transcription. Transcription takes place on chromatin; regulation of chromatin structure is important in regulating gene expression. Video “RNA pol II” https://www.youtube.com/watch?v=GdKfadJGId4  RNA polymerase II synthesizes mRNA and has been the focus of most transcription studies. It requires initiation factors, called Transcription factors. General transcription factors are proteins involved in transcription of polymerase II promoters. Other transcription factors join DNA sequences that control the expression of individual genes. About 10% of the genes in the human genome encode transcription factors, emphasizing the importance of these proteins. Promoters contain several important sequence elements: The TATA box. Resembles the –10 sequence of bacterial promoters. Consensus sequence: TATAA. Located 25 to 30 nucleotides upstream of the transcription start site Initiator element (Inr). It encompasses the transcription start site. TFIIB recognition elements (BRE). About 35 nucleotides upstream of the transcription start site. Downstream elements DCE, MTE, and DPE.  Five general transcription factors are required for initiation of transcription in vitro: TFIID is composed of multiple subunits, including TATA-binding protein (TBP) and other subunits (TAFs) that bind to the Inr, DCE, MTE, and DPE sequences. Several other transcription factors (TFIIB, TFIIF, TFIIE, and TFIIH) bind in association with the RNA polymerase II to form the transcription preinitiation complex. STEPS: 1. The formation of the transcription complex begins by the binding of the transcription factor TFIID. A subunit of this factor, the TATA-binding protein or TBP, binds to the TATA box; other subunits (TBP or TAF associated factors) bind to the Inr element and downstream promoter elements. 2. TFIIB then binds TBP and BRE sequences 3. The binding of the TFIIF-associated polymerase is followed. 4. Finally, the TFIIE and TFIIH proteins associate with this complex. Two subunits of TFIIH are helicases, which unwind DNA around the initiation site. Another subunit is a protein kinase that phosphorylates Serine residues in the C-terminal domain of the main subunit of polymerase II, which induces the release of the polymerase and initiation of transcription. Formation of a polymerase II preinitiation complex in vitro CTD is the C-terminal domain of the largest subunit of RNA polymerase. RNA polymerase II/Mediator complexes and transcription initiation Within a cell, additional factors are required to initiate transcription. These include Mediator, a protein complex of 20+ subunits; it interacts with both general transcription factors and polymerase. The Mediator complex: stimulates basal transcription it is key in the association of general transcription factors with the specific transcription factors of certain genes that regulate gene expression. Mediator proteins are released from the polymerase upon assembly of the preinitiation complex and phosphorylation of the C-terminal domain. The phosphorylated CTD then binds to other proteins that facilitate transcription elongation and are involved in mRNA processing. Transcription of the ribosomal RNA gene RNA polymerase I transcribes rRNA genes, which are present in tandem repeats. Transcription yields a large 45S pre- rRNA, which is processed to yield the 28S, 18S, and 5.8S rRNAs. Promoters of rRNA genes are recognized by two transcription factors that recruit RNA polymerase I to form an initiation complex: UBF (upstream binding factor) and SL1 (selectivity factor 1) Transcription of RNA polymerase III genes Genes for tRNAs, 5S rRNA, and some of the snRNAs are transcribed by polymerase III. They are expressed from three types of promoters, to which different transcription factors are bounded: 5S rRNA gene promoter: TFIIIA initiates the assembly of the transcription complex by binding to specific sequences in the promoter of the 5S rRNA gene. Then TFIIIC, TFIIIB and the polymerase bind the promoter. tRNA gene promoter: TFIIIC binds and attracts TFIIIB and polymerase. Promoters of DNA encoding snRNA: SNAP and TFIIIB bind cooperatively and the polymerase is recruited. Regulation of transcription in eukaryotes Mechanisms: - The binding of proteins to specific regulatory sequences - Modifications of chromatin structure The transcription of a gene is subject to a strict control, carried out through regulatory sequences, such as promoters, which are located at the beginning of the gene. Housekeeping genes: genes in the genome that are expressed in all cells of the body (gene A). Inducible genes: only expressed in certain cell types and in a variable way (genes B, C, D). In some cases the expression of a gene is specific to a single cell type (gene C). A. PROMOTERS AND ENHANCERS Genes transcribed by RNA polymerase II have two main promoter elements, which serve as specific binding sites for general transcription factors: - TATA sequence (joined by TBP from TFIID) - Inr (joined by TAF from TFIID) Other sequences serve as binding sites for a wide variety of regulatory factors that control the expression of individual genes. E. g.: Promoter sequences CCAAT and GGGCGG [GC box] are present in many eukaryotic genes. Eukaryotic promoter Many genes in eukaryotic cells are also controlled by sequences located long distances (sometimes hundreds of kb) from the transcription start site: enhancers. Enhancers: Regulatory sequences located farther from the start site to which transcription factors bind. The ability of enhancers to act even at a distance from the transcription initiation sites is due to the formation of loops in DNA. Example: in addition to a TATA sequence and a set of 6 GC sequences, 2 repeats of 72 bp located further upstream are required to carry out efficient transcription.  The activity of enhancers does not depend on their distance or orientation with respect to the transcription start site. Action of enhancers Enhancers, like promoters, bind transcription factors that then regulate RNA polymerase. DNA looping allows a transcription factor bound to a distant enhancer to interact with proteins associated with the RNA polymerase/Mediator complex at the promoter. Enhancers are responsible for the control of gene expression during development and differentiation, as well as in the cellular response to hormones and growth factors. Enhancers represent 10% or more of human genomic DNA. There are many more enhancers than genes, which emphasizes the importance of these elements. Many human disease-related mutations affect enhancers rather than protein coding sequences. Enhancers usually have multiple sequence elements that bind different regulatory proteins that work together to regulate gene expression. E. g.: The immunoglobulin heavy-chain enhancer has at least nine sequence elements that are protein-binding sites. The immunoglobulin enhancer ❖ TRANSCRIPTION FACTORS Proteins that bind to specific DNA sequences, thus controlling the transcription of genetic information. A defining characteristic of transcription factors is that they contain one or more DNA- binding domains (DBDs), which bind to specific DNA sequences. Types: Basal (general) transcription factors: they are part of the preinitiation complex that interacts directly with RNA polymerase in the promoter. Other transcription factors: differentially regulate the expression of various genes by joining the enhancers. These transcription factors are crucial to ensure that genes are expressed in the correct cell at the right time and in the amount needed, depending on the body's requirements. There are activators and repressors of transcription. Additional proteins such as chromatin remodelers, histone acetyltransferases, deacetylases, kinases and methyltransferases, also play crucial roles in gene regulation, but they lack DBDs and therefore are not classified as transcription factors.  Transcriptional activators bind to regulatory Gene expression is also regulated by repressors, DNA sequences and stimulate transcription. which inhibit transcription. B. CHROMATIN AND EPIGENETICS Because eukaryotic DNA is packaged in chromatin, chromatin structure is a critical aspect of gene expression. Chromatin limits availability of DNA for transcription, affecting both transcription factor binding and action of RNA polymerase. Actively transcribed genes are in relatively decondensed chromatin. Chromatin can be altered by histone modifications and nucleosome rearrangements. Many histone modifications are stably inherited when cells divide, providing a mechanism for the transmission of gene expression patterns to daughter cells. LOCATION OF CHROMATIN AND TRANSCRIPTIONAL ACTIVITY Euchromatin: most of the chromatin of interphase cells. Decondensed and transcriptionally active. Preferably located inside the nucleus. Heterochromatin: Highly condensed chromatin that is not transcribed. Frequently associated with the nuclear envelope or the periphery of the nucleolus. Constitutive heterochromatin: DNA segments that are always and in all cell types in condensed form. Eg: Highly repetitive sequences in centromeres and telomeres. Facultative heterochromatin: Genes that are not transcribed in that cell type or at that time in the cell cycle. Chromosomes rich in genes are located in the center of the nucleus, while those with few genes are located in the periphery. Histone modifications: These modifications occur at specific amino acid residues in the histone tails. Histone acetylation: The amino-terminal end of histones extends outside the nucleosome. It is Amino-terminal rich in lysine and can be modified by end of a histone acetylation. Actetyl groups are added by histone acetyltransferase (HAT) and removed by histone deacetylase (HDAC). Acetylation neutralizes the positive charge of lysine, relaxing chromatin structure and increasing availability of the DNA template for transcription. Transcriptional activators and repressors are associated with HAT and HDAC respectively. Chromatin remodeling factors They are protein complexes that alter contacts between DNA and histones. They can reposition nucleosomes, change conformation of nucleosomes, or eject nucleosomes from the DNA. Like histone modifying enzymes, remodeling factors can be incorporated into DNA in association with transcriptional activators or repressors. DNA methylation Another mechanism for epigenetic control of transcription: transcriptional repression Addition of methyl groups at C5 of cytosine residues preceding guanines (CG dinucleotide) Methylation patterns are maintained after DNA replication DNA methylation plays a role in genomic imprinting: expression of some genes depends on whether they come from the mother or the father. Example: Gene H19 is transcribed only from the maternal copy. It is methylated during development of male, but not female, germ cells. Noncoding RNA Transcription can also be regulated by noncoding RNA molecules: miRNAs (20–30 nucleotides) act by the RNA interference pathway to inhibit translation or induce degradation of homologous mRNAs. Long noncoding RNAs (lncRNAs) (>200 nucleotides): Form complexes with proteins that modify chromatin and recruit these complexes to their sites of transcription, thereby regulating expression of neighboring genes. RNA processing Bacterial mRNAs are used immediately for protein synthesis while still being transcribed. But they are an exception; most RNAs must be processed in various ways. - Ribosomal RNA - Transfer RNA - Eukaryotic messenger RNA  Ribosomal RNAs of both prokaryotes and eukaryotes are derived from a single long pre- rRNA molecule. In prokaryotes, this is cleaved to form three rRNAs (16S, 23S, and 5S). Eukaryotes have four rRNAs; 5S rRNA is transcribed from a separate gene.  tRNAs also start as long precursors (pre-tRNAs) in prokaryotes and eukaryotes. Cleavage of the 5′ end of pre- tRNAs by the enzyme RNase P. RNase P is a ribozyme—an enzyme in which RNA rather than protein catalyzes the reaction. Cleavage of the 3′ end by a conventional RNAase protein. Addition of a 3’ CCA terminus, the site of amino acid attachment. Bases are also modified at specific positions. About 10% of the bases are modified.  In eukaryotes, pre-mRNAs are extensively modified before export from the nucleus. Throughout processing, transport, translation, and degradation, mRNA molecules are associated with proteins to form messenger ribonucleoprotein particles (mRNPs). Transcription and processing are coupled. The C-terminal domain (CTD) of RNA polymerase II plays a key role by serving as a binding site for the enzymes involved in mRNA processing. 1. The 5′ end of the transcript is modified by addition of a 7-methylguanosine cap. The 5′ cap stabilizes the RNA, and aligns it on the ribosome during translation. 2. At the 3′ end, a poly-A tail is added by polyadenylation. Poly-A tails regulate the translation and stability of mRNA. 3. Introns (noncoding sequences) are removed from pre-mRNA by splicing. Splicing proceeds in two steps: 1. Cleavage at the 5′ splice site (SS) and joining of the 5′ end of the intron to an adenine within the intron (branch point). The intron forms a loop. 2. Cleavage at the 3′ SS and simultaneous ligation of the exons excises the intron loop. Alternative splicing: Most pre-mRNAs have multiple introns, thus different mRNAs can be produced from the same gene. This is one way of controlling gene expression, and increases the diversity of proteins that can be encoded. Splicing takes place in large complexes, called spliceosomes, which have five types of small nuclear RNAs (snRNAs)—U1, U2, U4, U5, and U6. The snRNAs are complexed with proteis to form small nuclear ribonucleoprotein particles (snRNPs). Video “splicing” https://www.youtube.com/watch?v=CdwLKwseP9Q Clinical importance of the correct recognition of splicing sequences It is estimated that 15% of genetic diseases are due to mutations that affect splicing sites. Example: β-thalassemia A single nucleotide mutation in the first intron of the hemoglobin β chain gene generates a defective protein, causing anemia (poor oxygen transport). 3.5. Traffic between the nucleus and the cytoplasm Selective transport of proteins to and from the nucleus Regulation of protein transport to the nucleus RNA transport  Depending on the size of the molecules, they can pass through the nuclear pore complex by one of two different mechanisms: ✓ Passive diffusion: small molecules and proteins with molecular weight less than 40 kDa passively diffuse through the pore in both directions. ✓ Selective transport. Most RNAs and proteins cross the nuclear pore complex through a selective transport process, in which these molecules are recognized and selectively transported in the specific direction. SELECTIVE TRANSPORTATION OF PROTEINS FROM AND TO THE NUCLEUS Proteins necessary for nuclear functions must enter the nucleus from synthesis sites in the cytoplasm. Histones In addition, many proteins undergo a continuous DNA polymerases transfer between the nucleus and the cytoplasm. RNA polymerases The proteins responsible for the structure and function of the genome are 'tagged' to be Transcription factors destined for the nucleus with specific aa … sequences, called nuclear localization signals (NLS), which are recognized by nuclear transport receptors. IMPORT TO THE NUCLEUS. Experiment. https://www.youtube.com/watch?v=kxMwkGwp6ZY&list=PL Bd5pZfVmZ5aFrKMcJ53vOfOocA2Ay8hA&index=28  Alan Smith et al. (1984) characterized in detail the first nuclear localization signal by studying the T antigen of the simian virus SV40. T antigen is a virus-encoded protein that initiates viral DNA replication in infected cells. Given its function, this protein is located in the nucleus. The first experiments showed that the Lys-128 mutation prevented the accumulation of T antigen in the nucleus, accumulating instead in the cytoplasm. This suggested that Lys-128 was part of a nuclear localization signal (NLS). Subsequently, it was shown that a 7 aa sequence, from residue 126 to 132, was responsible for the nuclear localization of the T antigen.  Through targeted deletion experiments, to eliminate aa. of the protein, they found that deletions comprising residues between 1-125 or between 133 and the carboxyl terminal end of the T antigen, normally accumulated in the nucleus. In contrast, the deletions that affected from aa. 126 to 132 caused the retention of the T antigen in Intact nuclear localization signal the cytoplasm of cells. In addition, they found that the sequence of residues 126-132 added, through the creation of chimeras, to cytoplasmic proteins (beta- galactosidase and pyruvate kinase), caused their accumulation in the nucleus (right images). Inactivated nuclear localization signal  NLS have now been identified in many other proteins. The NLS of the SV40 T antigen has been found to be a prototype for similar sequences of other nuclear proteins. Most of these sequences, like the T antigen, are short, rich in Lys and Arg and aa. responsible for nuclear signaling are contiguous with each other. In other cases, the aa. they are together, but not necessarily contiguous. As is the case with nucleoplasmin (a protein that participates in the assembly of chromatin) in which the NLS is a bipartite sequence, formed by a Lys-Arg sequence separated by 10 aa. of another sequence Lys-Lys-Lys-Lys. Two proteins play a fundamental role in the transport of proteins to the nucleus: Importins: recognize the NLS of the protein-cargo and transport it from the cytoplasm to the nucleus. Ran proteins: Guanosine di/triphosphate (GDP/GTP) binding protein. Its conformation and activity is regulated by the fact of being bound to GTP or GDP. High concentration of Ran / GTP in the nucleus, which determines the directionality of nuclear transport. The enzymes that stimulate the exchange of GDP for GTP on Ran are located on the nuclear side of the nuclear envelope; while those that stimulate GTP hydrolysis are found on the cytoplasmic side. 1. Importin recognizes the NLS of the cargo protein. 2. The cargo-importin complex binds to the proteins of the cytoplasmic filaments of the nuclear pore complex and is transported through the pore. 3. In the nucleus, Ran / GTP binds importin, disrupting the cargo-importin complex and releasing the cargo protein. 4. The importin-Ran / GTP complex is re-exported through the nuclear pore. 5. The GTPase activating protein (Ran-GAP) associated with cytoplasmic filaments hydrolyzes GTP in Ran to GDP, releasing importin. 6. Ran / GDP is transported back to the nucleus associated with its own import receptor: NTF2. 7. In the nucleus, Ran-GEF (chromatin-bound) causes Ran- bound GDP to be exchanged for GTP, thus regenerating Ran / GTP. https://www.youtube.com/watch?v=ZGPpKk-6-K0 To be exported to the cytoplasm, proteins are tagged with a specific aa sequence called the nuclear export signal (NES). They are often sequences rich in leucine. These signals are recognized by receptors within the nucleus (exportins) that direct the transport of proteins to the cytoplasm through the nuclear pore complex. The cargo-exportin complex binds to Ran / GTP, which directs the movement of proteins with NES from the nucleus to the cytoplasm. Once transport to the cytosolic side has occurred, the hydrolysis of GTP causes dissociation of the target protein, which is released into the cytosol. Exportins as well as Ran / GDP are recycled through the nuclear pore complex. REGULATION OF TRANSPORTATION OF PROTEINS TO THE NUCLEUS Mechanisms of regulation of transport to the nucleus: A. Some cytoplasmic proteins mask the NLS, causing the proteins to remain in the cytoplasm. Ex. The transcription factor NF-kB in unstimulated cells is bound to an inhibitory protein (IkB) that masks the NLS. In stimulated cells, IkB is phosphorylated and degraded, allowing the transport of NF-kB to the nucleus. B. Other proteins are restricted from entering the nucleus by phosphorylation. Ex. The yeast transcription factor Pho4 is phosphorylated on a serine residue adjacent to the NLS, preventing it from binding to its importin. Regulated dephosphorylation exposes NLS and allows Pho4 to be transported to the nucleus at the appropriate stage of the cell cycle. RNA TRANSPORT RNAs involved in protein synthesis are exported from the nucleus. RNAs are transported through the nuclear envelope as ribonucleoprotein complexes (RNPs). Export of tRNA, miRNA and rRNA: mediated by specific exportins and Ran/GTP. tRNA and miRNA precursors: exportin-t and exportin-5 respectively, which bind directly to RNAs. rRNAs first associate with ribosomal proteins in the nucleolus, and nascent 40S and 60S ribosomal subunits are transported separately to the cytoplasm by exportin Crm1. mRNA export. Exportins and Ran are not involved. After mRNA processing, an exporter complex made up of various proteins transports the mRNA. A helicase associated with the cytoplasmic face of CNP releases the mRNA into the cytoplasm. Other small noncoding RNAs act in the nucleus. The snRNAs involved in the splicing of pre-mRNAs. snRNAs are initially transported to the cytoplasm by exportin Crm1, where they bind to proteins to form functional RNPsn that are carried to the nucleus by importin snuportin. The snoRNAs are involved in rRNA processing. The snoRNAs remain in the nucleus. Okamura, M.; Inose, H.; Masuda, S. RNA Export through the NPC in Eukaryotes. Genes 2015, 6, 124-149. 3.6. Nuclear bodies Differentiated organelles in the nucleus. They compartmentalize the nucleus and serve to concentrate RNA and proteins that function in certain processes in the nucleus. They do not have membranes, but are maintained thanks to interactions between proteins and between proteins and RNA. Therefore, they are dynamic structures capable of exchanging their content with the rest of the nucleus. Active field of research: its functions are not fully known. CAJAL BODIES Described by Ramón y Cajal in 1906. They intervene in the final stages of the maturation of the snRNP (small nuclear ribonucleoproteins, formed by snRNA associated with proteins): modification of snRNAs by ribose methylation pseudouridylation: conversion (isomerization) of uridine to a different isomeric form, pseudouridine. SPECKLES The components of the splicing system are concentrated in 20-50 discrete structures called speckles. After assembly and maturation in the Cajal bodies, the snRNP are transferred to the nuclear speckles, where splicing factors are also found. Genes that are actively transcribed are distributed throughout the nucleus, but components of the splicing machinery are concentrated in these discrete subnuclear domains. These structures are the storage site for the Speckles. The localization of these components components responsible for splicing, and from here in discrete domains has been carried out by they are recruited to the actively transcribed genes, immunofluorescence using antibodies against where the pre-mRNA processing takes place. ribonucleoproteins (RNPsn) and splicing factors. NUCLEOLUS Largest structure in the nucleus of eukaryotic cells. It is best known as the site of ribosome biogenesis. The transcription and processing of the 28S, 5.8S and 18S rRNA takes place, as well as the assembly of ribosomes. The three rRNAs are transcribed as a single unit in the nucleolus by the RNApolI, giving rise to the 45S pre-rRNA, which is processed into these 3 rRNAs. 5S rRNA is transcribed outside the nucleolus by RNApolIII.  Continuously growing mammalian cells contain between 5 and 10 million ribosomes, which must be synthesized each time the cell divides. Cells contain multiple copies of rRNA genes in order to meet the transcription demand of a large number of rRNA molecules. The human genome contains approximately 200 copies of the gene encoding the 5.8S, 18S and 28S rRNAs, and approximately 2000 copies of the gene encoding 5S rRNA. The 5.8S, 18S, and 28S rRNA genes are arranged in tandem on 5 different human chromosomes (13, 14, 15, 21, and 22). The genes for 5S rRNA are located in tandem on chromosome 1. The nucleolus is organized around regions of the chromosomes that contain the genes for the 5.8S, 18S, and 28S rRNAs, called nucleolar organization regions.  Transcription of rRNA genes. Three rRNA genes separated by spacer DNA that is not transcribed. The high density of the growing RNA chains is due to the large number of RNA polymerase molecules, present at a maximum density of approximately one polymerase per 100 bp of template DNA. The nucleolus consist of three distinct regions. These areas possibly reflect the progression of the rRNA transcription, processing, and ribosome assembly steps. the fibrillar center (FC), where the genes that encode rRNA are located, which are transcribed at the interface with the DFC. the dense fibrillar component (DFC), where the Structure of the nucleolus. Fibrillar center (FC), the dense fibrillar component (DFC) pre-rRNA is processed. and the granular component (G). the granular component (G), where the ribosomal subunits are assembled. The size of the nucleolus depends on the metabolic activity of the cell, with large nucleolus present in cells that are actively engaged in protein synthesis. This variation is mainly due to differences in the size of the granular component, which reflects the rate of ribosome assembly. rRNA transcription and processing The large 45S pre-rRNA is processed through a series of cleavages.  In addition to cleavages, pre-rRNA processing involves important nucleotide modifications: Addition of methyl groups to ribose residues. Conversion of uridine to pseudouridine. The processing of pre-rRNA requires the intervention of proteins and RNA located in the nucleolus. Nucleolous contain more than 300 proteins and approximately 200 small nulceolar RNAs (snoRNA) that are involved in the processing of pre-rRNA. The snoRNAs together with the proteins form snoRNPs that bind to the pre-rRNA to form a processing complex. The snoRNAs contain short sequences (15 nt. approx.) complementary to the rRNA and by base pairing it directs the enzymes that catalyze the modification of the rRNA (e.g. methylation) to the appropriate places. RIBOSOME ASSEMBLY The formation of ribosomes involves the assembly of pre-rRNA with ribosomal proteins and 5S rRNA. The genes encoding ribosomal proteins are transcribed outside the nucleolus by RNA polymerase II and translated in the cytoplasm. Ribosomal proteins are transported to the nucleolus where they assemble with the pre-rRNA to form preribosomal particles. The genes for 5S rRNA are transcribed by RNA polymerase III outside the nucleolus, and also assemble in the nucleolus to form the preribosomal particles. The association of ribosomal proteins with rRNA begins while synthesis of rRNA occurs, and more than half of ribosomal proteins are bound to pre-rRNA prior to processing. The remaining ribosomal proteins and the 5S RNA are incorporated into the preribosomal particles while cleavage of the rRNA takes place.