Eukaryotic Gene Expression John O. Thomas

Transcription

1 Eukaryotic Gene Expression John O. Thomas I) RNA polymerases A) There are four RNA polymerases in human cells. 1) RNA polymerase I, located in the nucleolar region of the nucleus, is responsible for the synthesis of the precursors to 5.8S, 18S and 28S ribosomal RNA (rrna). 2) RNA polymerase II, located in the nucleus, is responsible for the synthesis of the precursors to messenger RNA (mrna). 3) RNA polymerase III, located in the nucleus, is responsible for the synthesis of some small RNAs such as transfer RNA (trna) and 5S ribosomal RNA. 4) Mitochondrial RNA polymerase, located in the mitochondria, is responsible for the synthesis of RNAs encoded by mitochondrial DNA (mtdna). B) Events involved in the synthesis of RNA. 1) Initiation of RNA synthesis. (a) The amount of RNA produced is controlled primarily by the rate at which RNA synthesis is initiated. (b) Initiation of RNA synthesis is highly regulated. (c) The first nucleoside is added as a triphoshate. Unless the RNA is processed, its 5' end will have a triphosphate 2) Extension of the growing RNA chain. (a) Progresses by the addition of nucleoside triphosphates (b) The reaction progresses in the 5' to 3' direction. (c) The nucleotide to be added is determined by complementarity to the template strand. (d) Pyrophosphate is produced as a reaction product. The hydrolysis of pyrophosphate to two phosphates is an important driving force of the reaction. Two high energy phosphoanhydride bonds are hydrolyzed for each lower energy phosphodiester formed. (e) Sequences toward the 3' end of the template strand are "upstream" (f) Sequences toward the 5' end of the template strand are "downstream" Start of Transcription Incoming nucleoside triphosphate Incorporated nucleotide 2pi RNA OH pppc-oh pppapcpgpu pppapcpgpupc OH 3' H 2 O 5'...CpApGpGpTpGpCpApGpTpGpCpTp......CpApGpGpTpGpCpApGpTpGpCpTp... + ppi phosphate Upstream DNA template strand (other strand not shown) Downstream pyrophosphate II) Genes that encode RNAs as final products A) Large amounts of ribosomal RNAs and transfer RNAs are required by most cells. In order to generate these large amounts, there are multiple copies of the genes encoding ribosomal RNAs and transfer RNAs. B) For genes that encode proteins, the product of the gene is amplified at two stages: transcription, where each gene gives rise to many copies of mrna, and translation, 1

2 where each mrna gives rise to many copies of a protein. For rrnas and trnas there is only one stage of amplification (transcription). This further increases the need for multiple copies of the rrna and trna genes. III) Eukaryotic Messenger RNA Structure A) A typical eukaryotic mrna encodes a single polypeptide chain. This is in contrast to bacterial mrnas, which typically encode more than one polypeptide chain. B) Eukaryotic mrnas have a "cap" at the 5' end. 1) The cap is not encoded by the gene. 2) The cap structure is added post-transcriptionally. 3) Features of the cap: (a) A G residue linked through a 5'-5' triphosphate. (b) Methylation at the 7 position of G gives the cap a positive charge. (c) Methylation of the 2 -OH of the 1st (and sometimes 2nd) mrna residues enhances the chemical stability of the cap (prevents 2' -OH catalyzed hydrolysis of the phosphodiester). 4) The cap is important for translation and for the stability of the mrna. The 7 position of G is methylated. The resulting positive charge is essential for translation of most mrnas. The first base is usually adenine. A 5'-5' triphosphate Important features of the mrna cap. Methylation of the 2' OH of the first and second nucleotides stabilizes the cap by inhibiting cleavage of the adjacent 3'-5' phosphodiester. The second base may also be methylated C) A typical mrna contains a 5 untranslated region that is important for regulation of protein synthesis (this will be discussed in the lectures on translation). D) Translation is initiated at an AUG codon that is usually within a hundred based of the cap. E) The coding sequence is an open reading frame, a series of triplet codons bounded by the initiator AUG at the 5 end and a stop codon at the 3 end. By definition there can be no stop codon in the open reading frame. A long open reading frame is a hallmark property of a protein-coding sequence F) The end of translation is marked by one of three stop codons (UAG, UGA, UAA). G) mrnas have a 3 untranslated region. In some mrnas this region contains a sequence that controls the rate at which the mrna is degraded by the cell. Different mrnas may have widely different half-lives in the cell. 1) Degradation is a highly controlled enzymatic process. 2) Some mrnas last for hours while others may be degraded in seconds. 2

3 3) mrna degradation is important for controlling the amounts of individual proteins that are present in the cell. 4) In some cases, specific mrnas may be targeted for degradation via the binding of specific micrornas to sequences in the 3 UTR (to be discussed in translation lectures) 5) mrna degradation is important for eliminating damaged or nonfunctional mrnas. H) A signal for directing the addition of poly A. The consensus sequence of this signal is AAUAAA (consensus sequence means that some variation in the sequence is allowed). I) About 200 A residues at the 3' end. This is referred to as a poly A tail. 1) The poly A is added post-transcriptionally by poly A polymerase. 2) The poly A sequence is not encoded by the gene (there is no poly T sequence) 5' untranslated region 3' untranslated region Coding region UGA UGA 5' CAP AGG UAA (open reading frame) 3' POLY A AUG UAG UAG 7 Me GpppA AAUAAA A 200 initiator terminator Initiation of translation Termination of translation (poladenylation signal) IV) Structure of mrna-encoding genes A) Transcriptional control elements; 1) Sequences to which the general trascriptional machinery (see below) binds are immediately upstream from the start of transcription. An important component of most (but not all) mrna-encoding genes is the "TATA box" (discussed below) at about -30bp (minus 30 bp). 2) Upstream Activating Sequences and/or Upstream Repressing Sequences are frequently found upstream of the gene, within a few kilo-bp of the start site. These sequences may, however, be located long distances from the start of transcription or even within the gene or downstream from the gene. 3) Control regions that affect more than one gene (Locus Control Regions) may be very far from the start of transcription. 4) Insulators are DNA sequences that define the ends of a DNA region that is affected by enhancers, silencers and/or locus control regions. B) Exons 1) Exons are the sequences that will appear in the final mrna product. 2) The first exon starts at the start of transcription (The 5' untranslated region is part of the first exon). 3) The last exon ends at the site of polyadenylation (The 3' untranslated region is part of the last exon). 3

4 C) Introns 1) Introns are the sequences between exons. They are removed during mrna splicing. 2) Typically, introns are much longer that exons (for most genes about 80-90% of the RNA is lost during splicing). V) Overview of mrna production. A) RNA polymerase II initiates transcription at the beginning of the first exon. The initiating residue of the primary transcript is the first residue following the 7-methy G of the cap, and is present in the final mrna product. The growing RNA chain very transiently has a triphosphate at its 5 terminus. B) Very soon after initiation, a capping system condenses a GTP with the pppa at the 5 terminus of the primary transcript yielding GpppApXpYp... at the 5' end. This is soon methylated to give the mature cap structure. The 3 end of the primary transcript continues to be elongated by RNA polymerase II. C) As RNA is being transcribed, RNA splicing occurs. RNA splicing cuts the intronic sequences from the unspliced precursor, and covalently rejoins the exonic sequences to form the mature mrna. The splicing mechanism recognizes short conserved RNA sequences at and near the exon:intron boundaries. D) The primary transcript is extended beyond the polyadenylation signal. The polyadenylation signal directs an endonuclease to cut the primary transcript about 30 residues downstream from the signal. The resulting 3 residue is the receptor for polyadenylation. 4

5 E) After, the endonuclease has cut the RNA chain, an enzyme, poly A polymerase, adds about 200 A residues. F) The mature mrna consists of the 5 cap, the exons, and the 3 poly A tail. The intron sequences and the sequences beyond the poly A site are degraded. G) mrna is transported to the cytoplasm, where it binds ribosomes and is translated to produce a protein product. The RNA is transported through nuclear pore complexes that are embedded in the nuclear envelope. For some genes, expression is regulated at the level of RNA transport to the cytoplasm (to be covered in Cellular Basis of Medicine). VI) Control of Pol II-directed transcription. A) Promoter structure. 1. A promoter is a set of DNA sequences that regulate the transcription of a gene. 2. Promoters are usually located around and upstream from the nucleotide where transcription starts (figure 8.5). 3. The promoter sequence defines which of the two DNA strands will serve as the template for mrna production, defines the location where RNA synthesis will start, determines which regulatory stimuli will affect transcription of a particular gene, and determines the amount of mrna that will be produced from the gene in response to specific stimuli. 4. The core promoter consists of sequences that span from about 80 base pairs upstream from the start of transcription to about 20 base pairs downstream. These sequences, which usually include TATAAA (TATA box), specify where a preinitiation complex composed of general transcription factors assembles prior to the start of transcription. 5. Enhancer/repressor sequences are usually located immediately upstream from the core promoter but may be anywhere within several kilo-base pairs of the core promoter (even within the coding region of the gene or down stream from the gene). Enhancer/repressor sequences bind tissue-specific transcription factors that stimulate or repress transcription. B) General Transcription Factors 1) These factors work at the promoters of many genes, and form the general transcriptional machinery that assembles at the core promoter (see figure). Factors in this class are TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. The nomenclature TFII indicates that the factor works together with RNA polymerase II. Some genes bind only some of these factors. (a) One subunit of TFIID is a TATA Binding Protein (TBP). TBP binds to the TATA box and bends the DNA. A TATA box is present in most, but not all genes. (b) TFIID recruits several transcription factors, including TFIIA, TFIIB and TFIIE. (c) The assemble transcription factors recruit RNA polymerase II to the promoter. (d) TFIIH joins the growing complex and performs several critical roles in the early steps of initiation. (i) It contains a DNA helicase that promotes the separation of the DNA strands at the initiation site. 5

6 (ii) It possesses a protein kinase activity that phosphorylates the C-Terminal Domain of RNA polymerase II (see below) (iii)it couples transcription with DNA repair and with cell cycle regulation. A. Upstream Downstream Direction of transcription -50 bp +50 bp Gene (double stranded DNA) Core promoter region Transcription initiation site (+1bp) B. RNA polymerase II D TATA B F Direction of transcription H Core promotor region of a typical mrna gene (A) and assembly of general transcription factors at the core promoter (B). Only some of the transcription factors are shown. C) Specialized Transcription Factors. In addition to the core promoter, which is the binding site for the general transcriptional machinery, the regulatory region of a gene contains binding sites for specialized transcription factors. These specialized factors determine the circumstances under which the gene is active. 1) Transcription factors have at least two domains: A DNA binding domain that recognizes specific DNA sequences and a transcription activating (or repressing) domain that functions to regulate the level of initiation. 2) Trans activating or repressing domains generally function by binding coactivators or co-repressors. These may: (a) Interact directly with a component of the general transcriptional apparatus to stimulate or inhibit the formation of the initiation complex. (b) Modify the structure of chromatin (see below). 3) Examples of Specialized Transcription Factors (a) CREB (Cyclic AMP Response Element Binding Protein): Binds as a homodimer to cyclic AMP response elements in DNA. As is discussed below, the activity of CREB is stimulated by cyclic AMP. This leads to the expression of a large variety of genes in response to extra-cellular signals that stimulate cyclic AMP production. (b) AP-1: A family of closely related transcription factors. One member of this family is the Fos-Jun complex, which consists of one molecule of the protein Fos bound to a molecule of the protein Jun. The expression of the Fos and Jun proteins is regulated by a wide range of signals that are communicated to the cell across the plasma membrane. These include signals provided by growth factors and neurotransmitters. 6

7 (c) Nuclear receptors: This is a family of proteins that includes the steriod receptors (such as the receptor for estrogen). The binding of a specific hormone to a specific receptor (e.g. binding of estrogen to the estrogen receptor) will stimulate transcription of a large number of specific genes. 4) The following figure shows a hypothetical promoter that might be activated under a number of different conditions. It would be activated by: (a) growth factors which induce the synthesis of Fos and Jun proteins (b) neuropeptides that activate G-protein coupled receptors, elevating camp levels. (c) synaptic activity, which induces Ca 2+ fluxes in neurons. D) Chromatin structure and gene expression 1) Assembly of the large initiation complex at the promoter requires a loosely folded chromatin structure. Factors that promote this loosely folded structure stimulate transcription, those that induce a tightly folded chromatin structure are inhibitory. 2) Histone acetylation, on the N-termini of the core histones (2a, 2b, 3, and 4), is particularly important. The acetylation loosens the chromatin structure and promotes transcriptional activity in the region of chromatin that has been modified. 3) Co-activators contain histone acetyl-transferases (HATs) that acetylate the tails of histones in nucleosomes that are close to where the coactivator binds. Histone acetylation decreases the degree of chromatin structure by decreasing nucleosome-nucleosome interactions. Co-activators also recruit chromatin remodeling complexes that use energy from ATP hydrolysis to move the nucleosomes away from the promoter. 4) Co-repressors deacetylate histone tails and thereby increase chromatin structure. Most transcriptional co-repressors contain a histone deacetylase (HDAC) activity as a part of the co-repressor complex. 7

8 5) DNA methylation at CpG sequences represses transcription. One mechanism through which this works is through a methyl-cpg binding protein that associates with a histone deacetylase. 6) Histone methylation is also an important regulator of chromatin structure as discussed in the Nucleic Acids lectures. 7) Non-histone chromatin proteins contribute to the formation of specific chromatin structures. VII) mrna elongation. A. When the pre-initiation complex is fully assembled, RNA polymerase II switches from initiation mode to elongation mode. The switch between modes is triggered by phosphorylation of the C-terminal domain (CTD) of the largest RNA polymerase II subunit. B. The CTD consists of 52 repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro- Ser. The CTD extends a great distance from the body of the enzyme and interacts with different sets of proteins as translation progresses from initiation to termination. C. The CTD repeats contain two serines, serine-2 and serine-5. Phosphroylations of these serines act as switches that switch RNA polymerase II between initiation mode, early elongation mode and late elongation mode. These modes affect transcription initiation, elongation, RNA processing and the chromatin structure of the gene being transcribed. 1) In the unphosphorylated state, the CTD interacts with general transcription factors, thereby stabilizing the pre-initiation complex. 2) TFIIH contains a kinase that phosphorylates serine 5 in each CTD repeat When phosphorylated, the CTD no longer interacts with the general transcription factors. 3) Enzyme complexes that catalyze the RNA processing events of RNA capping, RNA splicing, RNA 3 end formation and RNA transport out of the nucleus are all associated directly or indirectly with phosphorylated forms of the CTD. This physical association assures that all of the individual steps of RNA formation that take place in the nucleus are tightly coupled. D. The phosphorylated CTD interacts with histone methylases that maintain the promoter in an open chromatin conformation and the rest of the gene in more compact chromatin conformation. Histone H3 lysine 4 methylation occurs near the promoter. Histone H3 lysine 36 methylation occurs over the rest of the gene. VIII) Termination A. The 3 -end forming complex that recognized the plolyadenylation signal is associated with the RNA polymerase II CTD. It cleaves the newly formed RNA a few nucleotides past the polyadenylation signal. After the mrna has been released from the polymerase, RNA synthesis continues for a hundred to several thousand nucleotides. After cleavage, a 5 3 exonuclease binds to the RNA that is still being synthesized. This exonuclease is needed to dissociate the RNA polymerase from the template and thereby stop transcription of the gene. One hypothesis for how transcription is terminated by the exonuclease is a torpedo model. According to this model, the 5 3 exonuclease, acting in analogy to a torpedo, digests the RNA, 8

9 moving toward the slowly moving RNA polymerase II. When the exonuclease catches up to the polymerase, the polymerase is knocked off the DNA template. IX) RNA Splicing A) The splicing enzymes cleave the RNA molecule at the junctions between exonic (retained) and intronic (removed) sequences and rejoining the exonic sequences to generate a mature spliced mrna. B) Splicing must take place with perfect accuracy: addition or deletion of a single residue within a coding sequence will shift the reading frame and the resulting mrna will encode a defective or altered protein product. Usually, as a matter of chance, the new reading frame will contain a stop codon. Thus, inaccurate splicing typically results in nonsense-mediated-decay of the mrna (see below). C) Sequences near exon-intron boundaries confer cleavage specificity. Mutations in these sequences can lead to: 1) The formation of an alternative splice site and a different protein product. 2) The formation of an alternative splice site, resulting in a frame shift and no protein product. 3) The loss of splicing at the affected site, resulting in a frame shift and no protein product. 4) Reduced efficiency of splicing and the production of less protein product (a fraction of the transcripts being properly spliced and others improperly spliced as in 1-3 above) 5) Steps in splicing required to remove an intron which lies, for example, between exon I and exon II are: a) Cleavage of the mrna precursor at the exon I-intron boundary. b) Linkage of the 5 residue of the intron to the 2 -OH of a nucleotide near the intron-exon II boundary to form a lariat shaped molecule. c) Cleavage of the intron-exon II junction and rejoining of exon I and exon II. d) Degradation of the intron. e) The enzymology of splicing involves snrnps (small nuclear ribonucleoproteins; small nuclear RNAs complexed with specific proteins), which play essential roles in the process. D) Alternative splicing 1) Alternative patterns of splicing can allow a single DNA sequence to encode more than one protein. Frequently, different isoforms of a protein are produced by alternative RNA splicing patterns 2) A given RNA precursor may be spliced differently in different cell types or under different growth conditions in a single cell type. 9

10 Exon 1 Splicing factors, including U1 snrnp and U2 snrnp, U1 recognize the splice junctions. snrnp U2 snrnp G GU Intron A AG G Exon 2 Intron U1 snrnp G U2 snrnp HO- A snrnp complexes, including U1 and U2, bring the intron 3' A nucleotide and the splice donor close together G The 2' OH of the A nucleotide that will become the branch point attacks the phosphodiester of the splice donor Intron The 3' OH attacks the phosphodiester of the splice acceptor. Splicing proteins hold the splice donor and acceptor close together. G-OH G A 2' - 5' phosphodiester is formed. The A nucleotide has both a 2' - 5' phosphodiester and a 3' - 5' phosphodiester. Intron Completed splice O-P-O-A O-P-O-A G G Intron (to be digested) Mechanism of RNA splicing. Splicing factors, that are associated with the carboxy terminal domain of RNA polymerase assemble the many components required for splice site recognition and the formation of the bonds. Two of these, U1 snrnp and U2 snrnp are shown here. A color version is available on the molecules to cells web site. 10

11 X) Splice site mutations A) Mutations that affect splice junctions may lead to errors in the joining of two exons. Usually this leads to a frame shift in the resulting mrna and hence a premature termination codon. The premature stop in translation results in decay of the mrna via the nonsense mediated decay pathway. B) Mutations that affect splice junctions may lead to alternative patterns of splicing (as opposed to no splicing of the affected intron). C) Splice site mutations may lead to a lowered efficiency of splicing which will result in reduced amounts of the correct mrna and reduced production of the affected protein (as opposed to no protein at all). XI) Nonsense Mediated Decay A) Messenger RNAs that contain premature stop codons would, if not destroyed by the cell, lead to the production of partially formed proteins that could be detrimental. 1. They may not fold correctly, leading to accumulation of amyloid (aggregates of denatured protein). 2. They may lack important control functions; the loss of control of a key growthregulating protein could lead to the formation of a tumor. B) Nonsense mediated decay works as follows: 1. Following splicing, a protein complex (EJC; see the figure) remains at each exon junction. 2. These EJCs are removed by the first ribosome to pass along the mrna. 3. If the ribosome stops before it passes an EJC, the EJC remains on the mrna and targets the mrna for rapid digestion. Normal Normal stop codon Mutant mrna: Premature stop codon (due to a mutation) EJC EJC EJC EJC Exon Junction Exon Junction Translation Translation 1 2 Ribosome some Ribo EJC Passage of the first ribosome removes EJC Protein Translation Ribosome is halted at the premature stop EJC is not removed. This targets the mrna for rapid degrsdation mrna digestion No Protein 11

12 XII) Cellular responses to extracellular signals. A) CREB, cyclic-amp and Calcium 2. CREB is a leucine zipper type transcription factor involved in the regulation of many cell processes. In different cell types it may activate different sets of genes. One example is in neural cells where it plays a role in learning and memory. 3. CREB homodimerizes through a leucine zipper and binds to CRE (cyclic AMP response element) sequences in DNA (CREB is an acronym for CRE Binding protein). 4. CREB is phosphorylated on serine 133 by protein kinase A, which is activated by the second messenger camp. 5. Once phosphorylated on serine 133, CREB binds CBP (CREB binding protein), which in turn attracts co-activators that have histone acetylase activity (HAT), HAT acetylates nearby histones, altering the local chromatin structure. In addition, the phosphorylated CREB and CBP interact directly with the general transcriptional machinery, stabilizing its interaction with the promotor. Hormone receptor Inactive protein Kinase A camp Active protein Kinase A P CREB CREB ATP ADP Histone acetylase activities of the co-activator p300 and of CBP acetylate histone tails.in nucleosomes (N) p300 Interactions of p300 and CBP with TFIID and TFIIB help stabilize the pre-initiation complex. CBP CBP Chromatin assumes a more open structure; chromatin remodeling complexes remove nucleosomes from the core promoter, the core promoter becomes available for general transcription factor binding Ac P i CREB P i Ac Ac Ac N CRE N N N TFIID TFIIB gene 12

13 5) CREB serine 133 is also phosphorylated by Cam kinase, which is activated by Ca 2+. a) Ca 2+ enters cells as a result of neuronal stimulation. b) Ca 2+ binds to calmodulin, a small peptide, and Ca 2+ -calmodulin binds to Cam kinase, activating its ability to phosphorylate proteins on serine and threonine. Ca +2 Ca +2 CaM Kinase Calmodulin Calmodulin Ca +2 CREB CaM Kinase ATP ADP CREB P 6) Overall, these pathways enable the second messenger, camp or fluxes of Ca 2+ to activate specific sets of genes. B) Nuclear Receptors 1) Nuclear receptors are a class of transcription factors that are present in the nucleus. Some members of the class are also present in the cytoplasm, and some of these are regulated by controlling their entry into the nucleus. 2) Examples of nuclear receptors are a) Steroid hormone receptor (e.g. estrogen receptor; glucocorticoid receptor mineraolcorticod receptor) b) Thyroid hormone receptor c) Receptors that regulate bile salt and fatty acid metabolism (LXR, FXR, PPAR) 3) Binding of an activating ligand causes a change in the conformation of the transcription-activating domain of the receptor. This allows the binding of coactivator proteins that in turn bind histone acetylases and factors that stabilize the assembly of the core transcription complex at the TATA box. 4) In the absence of an activating ligand, many of the nuclear receptors bind corepressors in place of co-activators. The co-repressors recruit histone deacetylases and destabilize interactions required for the assembly of the core transcription complex at the core promoter 13

14 P300 acetylates histones in the region of the promotor Chromatin structure is loosened Binding of transcription factors is favored. A coactivator protein (e.g. p160) binds to the estrogen receptor. Other proteins such as p300 bind to p160 Direct interactions with TFIID and other TFII complexes stabilize the preinitiation complex P300 P160 P300 E E ER ER ERE TBP TATA TFIID +1 Transcriptional activation of estrogen-sensitive genes results from recruitment of activating proteins (co-activators) to the estrogen response element, a DNA sequence found in the promoters of estrogen responsive genes. When estrogen enters the target cell, it binds to the estrogen receptor, causing a conformational change. This enables the binding of the co-activator protein p160. Other proteins, such as p300, bind to p160 and affect transcription through direct interactions with the basal transcription complex (represented here by TFIID) and by altering chromatin structure to a less compact form. This increases access to DNA sequences enabling other transcription factors to bind which further stimulate formation of the basal transcriptional complex. C) Growth factors 3) Growth factors are polypeptides that bind to receptors on the cell membrane and activate the process of cell division. 4) Growth factors bind to receptors that span the cell membrane. d) The growth factor.binds to a domain of the receptor that lies outside the cell. e) This activates the receptor via the cytoplasmic domain, which lies inside the cell. f) Binding of a growth factor to its receptor in many cases causes two receptor molecules to come together in the plasma membrane, forming a dimer. The cytoplasmic domains of the two receptor molecules are thereby juxtaposed to one another. When juxtaposed, each receptor tyrosine kinase domain of the dimer phosphorylates the other domain. 5) The phosphotyrosine residues that are generated bind "SH2" domains found in a second set of proteins (including GRB2) which reside in the cytoplasm near the receptors. 6) GRB2 activates of a guanine nucleotide exchange factor, for example SOS. 7) SOS interacts with the G protein Ras, exchanging GDP for GTP. This places Ras in an active state. 8) Active Ras stimulates a protein kinase (the Raf kinase) which phosphorylates proteins on serine or threonine residues. The substrates for Raf include other protein kinases, which may in turn phosphorylate and active yet other protein kinases. 14

15 9) Since these kinases are activated by mitogens (the growth factors), this pathway is called the Mitogen Activated Protein kinase cascade, or MAP kinase cascade. 10) Specific kinases that are activated in the MAP kinase cascade can move from the cytoplasm to the nucleus and phosphorylate transcription factors, activating these factors and thereby controling transcription. 11) An example of this cascade is the activation of Fos gene expression, which is rapidly induced by a wide range of agents, including growth factors. 12) Fos, together with Jun proteins activate the transcription of a large number of other genes. Different sets of genes may be activated in different cell types. Induction of the Fos and Jun family of transcription factors is one of the earliest nuclear responses to external stimuli. Members of the Fos and Jun families (e.g. FosB, Fra1, JunB, JunD) are induced with different kinetics or are expressed at different levels. This suggests that there may be a continuously changing population of Fos- Jun related heterodimers following stimulation, each of which may have different effects on gene regulation. These proteins set in motion the long term changes in gene transcription required for the cell s response to the incoming signal. Fos and Jun have been implicated in diverse processes including nurturing of young and memory. 15

16 XIII) Synthesis of rrna by RNA polymerase I (poli) A) The rrna genes are located in specialized regions near the tips of the short arms of the five acrocentric chromosomes (13, 14, 15, 21, and 22) (acrocentric means the centromere is near the end of the chromosome). 1) Each of the above regions contains many copies of rrna genes in tandem arrays. 2) Each of the above regions also contains a "nucleolar organizer region" (NOR). 3) The ten NORs aggregate to form nucleoli (one to ten nucleoli per nucleus), the regions of the nucleus where the assembly of ribosomes begins. Ribosome synthesis requires: (a) Synthesis of rrna precursors (b) Processing of the precursors to yield rrnas (c) Import of some ribosomal proteins from the cytoplasm (d) Partial assembly of the proteins and rrnas (e) Export of the immature ribosomes to the cytoplasm for final assembly B) rrna is synthesized by RNA polymerase I, which is localized in the nucleolar regions of the nucleus C) Specific RNA poli transcription factors regulate polymerase I activity. Conditions that promote cellular growth stimulate these transcription factors via phosphorylation. Phosphorylation is due to several kinases including the Map Kinases and mtor (discussed in protein synthesis lectures). D) rrna synthesis and cancer Greatly enlarged nucleoli are one of the most striking features of cancer cells, a trait commonly used by pathologists. The nucleolar hypertrophy results from a greatly increased production of rrna and ribosome biosynthesis. In normal cells, the maximal rate of protein synthesis, and hence cell growth is limited by the availability of rrna. Tumor cells often acquire mutations that increase rrna synthesis so as to maximize the rate of cell growth. These mutations commonly result in a permanently stimulated MAP kinase pathway, which then maximally stimulates, via phosphorylation, RNA polymerase I (and pol III) transcription factors. rrna is synthesized as a precursor rrna which must be processed. The processing involves several cleavages of the precursor to yield three of the four ribosomal RNAs.. Ribosomal RNA Production RNA polymerase I rrna gene (DNA) ppp OH Precursor RNA RNA processing p p p Mature rrnas 18S 5.8S 28S (S refers to Svedberg units) 16

17 E) The other rrna is 5S, which is synthesized in other areas of the nucleus by RNA polymerase III. XIV) Synthesis of small RNAs by RNA polymerase III (pol III) A) These RNAs include the trnas, 5S rrna and several other short RNAs that are present in the cell. Initiation of synthesis by RNA polymerase III is governed by an internal promoter, which consists of sequences within the transcription unit. To initiate RNA synthesis by pol III, several proteins referred to as transcription factors form a complex with the internal promotor. These transcription factors then recruit the pol III and position it with respect to the position where transcription starts. In the absence of the transcription factor complex, pol III has negligible affinity for the gene. The internal promoter sequences have two functions: they bind transcription factor proteins that are required for gene activity, and they serve as part of the template for transcription of the RNA. The amount of RNA that is produced is determined by several factors including 1) Amount of the transcription factors in the cell 2) Affinity of the transcription factors for the promotor sequences 3) Affinity of pol III for the transcription factors These factors include SP1 which binds to GC-rich sequences (the GC box and CTF (also known as C/EBP) which binds to the sequence CAAT (CAAT box). 17