1 RNA synthesis, regulation of gene expression Ó Department of Biochemistry 2013 (E.T.) 2 Synthesis of RNA from a DNA template Only one strand of double helix DNA is transcribed – template strand. The second strand is coding strand ( its sequence is identical with primary transcript only U is replaced by T) Transcription 3 5´ 3´ G G A T C 3´ 5´ C C T A G 5´ 3´ G G A U C Coding strand Template strand DNA RNA RNA transcript is synthetized in the 5´® 3´ direction Primary transcript Terminology 4 For transcription are necessary: dsDNA RNA-polymerase ATP, GTP, CTP, UTP Mg2+ ions 5 Replication x Transcription Characteristic replication transcription Enzymes: Location: Initiation: Procedure: Control: nucleotides: DNA-polymerases On chromosome at S phase RNA primer is necesssary Both strands are copied Proofreading and DNA repair dATP, dGTP, dCTP, dTTP RNA-polymerases Selected fragment of DNA no primer is necessary Only one strand is copied polymerase do not include proofreading ATP, GTP, UTP, CTP 6 Transcription is carried out by DNA-dependent RNA polymerases (transcriptases) Prokaryotes: •only one polymerase composed of 5 subunits plus sigma factor. • it transcripts all forms of RNA Eukaryotes 4 different RNA polymerases RNA pol I – synthesis of r RNA (in nucleolus) RNA pol II – synthesis of mRNA (nucleus) RNA pol III – synthesis of tRNA, 5S RNA (nucleus) RNA pol IV - synthesis of mitochondrial RNA The mechanism of the action is the same, they recognize various promoters 7 Amanitine (bacterial toxine from Amanita phalloides) - cyclic octapeptide with unussual amino acids Inhibitor of eukaryotic RNA polymerase (mainly of II-type) Strukt_vzorec_a-amanitin 8 Three phases of transcription • initiation • elongation • termination • 9 Mechanism of RNA polymerase function •Synthesis of RNA occurs in 5´→3´ direction •Nucleotides ATP,GTP,CTP,UTP are necessary • Each nucleotide pairs with the complementary nucleotide on the DNA template • Polymerase catalyzes formation of phosphodiester bond between 3´-OH end of ribose on growing RNA-strand and a-phosphate bonded to 5´OH of ribose new nucleotide • the energy for polymeration is provided by cleavage of NTP and release of diphosphate •In contradistinction to DNA polymerases, RNA polymerases don´t exhibit any nuclease (proof-reading) activity so that they cannot correct mismatches. • • + PPi OH OH 10 Terminology of transcription Boxes (elements): small sequences in the promoter region Cis-acting sequences: lying on the same molecule of DNA that is transscribed, near the gene Trans-acting factors : proteins that bind to these DNA sequences and facilitate or prevent binding of DNA polymerase (genes for their synthesis are lying on different chromozomes) Promoter – specific sequence on DNA template about 40 nucleotides long lying in upstream position to the initiation site Transcription unit - sequence of nucleotides in DNA that codes for a single RNA molecule, along with the sequences necessary for its transcription; normally contains a promoter, an RNA-coding sequence, and a terminator Primary transcript - RNA product synthesized in direction u 5´®3´ 11 Initiation of transcription •RNA polymerase (RNAP) binds to specific nucleotide sequences – promoters • stable complexes with template DNA strand and RNA at the promoter region are formed The promoter contains characteristic consensus sequences (common conserved regions that are found in certain area of all genes) 12 Promoter in DNA of prokaryotes In position ~ -10 TATAAT box (Pribnow box ) In position ~ -35 another sequence TTGACA These sequences are recognized by s -factor of prokaryotic RNA polymerase Sequences in prokaryotic promoter -35 Pribn.box ~ 15 b Start of transcription ~ 10b DNA 13 Transcription in prokaryotes Iniciation: Binding of RNA-polymerase to promotor region of DNA by sigma subunit Local unwinding of DNA caused by RNA polymerase Pairing of ribonucleotide bases with template strand and formation of several phosphodiester bonds between nucleotides promoter Beginning of the newly synthesized RNA Movement of RNA polymerase RNA polymerase is inhibited by antibiotics rifampicine 14 Transcription in prokaryotes Elongation: Release of sigma subunit from RNA polymerase RNA polymerase moves along the template strand, unwinding of double helix Formation of covalent ester bonds among the nucleotides Newly formed RNA is released Termination signal – transcription is finished. Newly synthesized RNA separates from DNA template. Transcription rate 20-50 nukleotides/s Action of topoisomerases 15 Termination Termination signals usually contain a palindromic (selfcomplementary) GC-rich region and an AT-rich region. Thus the mRNA transcripts of this DNA palindrome can pair to form a hairpin structure with a stem and loop followed by a sequence of more uracil base – RNA transcripts end within or just after them 16 Transcription in eukaryotes It is far more complicated than transcription in prokaryotes DNA is temporary released from chromatine structure Most active are relaxed parts of chromatin – euchromatin Relaxation of chromatin is mediated by acetylation of lysine residue at the amino terminus of histone proteins by the action of histone acetyltransferase 17 Promoter u eukaryotes (RNA polymerase II) Transcription factors mediate the binding of RNA polymerase and the initiation of transcription. Many transcription factors are involved that bind to different regions of DNA positive strand template strand 5´- 3´- GGCAATC ATATAA promoter ~ –100 –1 +1 –25 start of transcription TATA box (Hogness box) coding region CAAT box in basal gene expression specifies the frequency of initiation directs TF II D and RNA pol II to the correct site Boxes in the promotor of eukaryotes: TATA (analogic to Pribnow sequence) Sometimes present CAAT 18 Transcription factors in eukaryotes Basal transcription factors transcription factors bound onto the promoter Are necessary for transcription of all genes Gene specific transcription factors bind to regulatory DNA sequences distant from promoters. 19 Basal transcription factors in eukaryotes • They must be attached to RNA polymerase before the transcription starts and are at the same time associated with promoter sequences •They are necessary for recognition of promoter and facilitate the binding of RNA polymerase • Polymerase II and transcription factors bound onto the promoter form a complex called the basal transcription apparatus. It regulates basal gene expression • At least six basal transcription factors in eukaryotes Basal TF = are necesary for transcription of all genes 20 Basal transcription factors TFIID – the biggest of basal factors of transcription - 11 subunits One of the subunits is TBP (TATA box binding protein). TBP binds to TATA box, the other subunits reacts also with TBP and RNA polymerase One of factors is an ATP-dependent helicase that separates the DNA duplex for the polymerase II TFIIB TFIIF TFIIE TFIIA TFIIH RNA polymerase TBP TFIID transcription TBP Polymerase II then slides to the start of transcription and initiate transcription http://www.youtube.com/watch?v=WsofH466lqk 21 Gene specific regulatory proteins (factors) Specific transcription factors - proteins that bind to specific regulatory DNA sequences (enhancers, silencers, HRE) lying on the same chromosome, distant from promoters (very often in large distance). They act as activators or repressors of the given gene transcription. Specific transcription factors interact with mediator proteins (coactivators, corepressors) that are in contact with basal transcription factors. A typical gene coding synthesis of a protein in eukaryotes has many binding sites for specific transcription factors in the template DNA strand 22 Regulation of a typical eukaryotic gene by a specific transcription factor TF IID Pol II CTD ~ 2 000 bp mediator proteins TF IID Pol II CTD regulatory sequence ~ 2 000 bp promoter specific transcription factor basal transcription apparatus (Pol II and basal factors) CTD-carboxyterminal domain upstream Interaction with mediator proteins 23 Examples of specific transcription factors Specific transcription factors are proteins whose specific effect is often activated by cellular signal pathways: Examples of specific transcription factors and their activation a) Intracelular receptors activated by binding of hydrophobic hormones b) Membrane receptors of hormons producing second messenger after the binding of hormon. One of the effects of second messenger is activation of proteinkinase that activates transcription factor by phosphorylation c) Ras signal pathway activated by binding various growth factors on membrane receptor is terminated by phosphorylation of transcription factors (proteins Fos, Jun, Myc a dalších) d) SREBP (sterol regulatory element binding protein) activated at low sterol concentration in the cell e) Binding of cytokine to membrane receptors activates JAK-STAT signal pathway. Phosphorylated STAT protein functions as transcription factor. 24 a) Intracellular receptors of hormons are specific transcription factors • receptors of steroidal (thyroidal) hormones are present in cytoplasma or nucleus in inactive form. They bind inhibitory protein in inactive form (e.g. heat shock protein). • hormon permeates across the plasmatic membrane to the cell and is specifically bonded to the receptor in cytoplasma or nucleus • inhibitory protein is separated, the complex hormon-receptor is formed, the conformation of receptor protein is changed •the complex hormon receptor is translocated to the nucleus • the complex hormon-receptor acts as the specifis transcription factor in the nucleus and binds to DNA at hormon response element (HRE) • the complex hormon-receptor attached to DNA reacts at the same time with coaktivatr (mediator protein) that is in contact with basal transcription complex. Thereby is transcription of a gene stimulated or inhibited. 25 Example: Initiation of transcription by cortisol • hydrophobic molecule difuses into a cell Inactive form of a glucocorticod receptor is present in cytoplasma and is associated with dimer of hsp 90 protein (chaperon) and other proteins Binding of cortisol to its receptor in cytoplasma causes a conformational change in the receptor Hsp proteins are separated from the receptor active complex dimerizes and is translocated into the nucleu by nuclear pores GR cortisol CBG • cortisol in plasma is transfered by CBG (corticosteroid-binding globulin) Hsp - protein 26 Dimeric complex cortisol-receptor binds to dsDNA at the specific GRE sequence (glucocorticoid response element), it means on HRE (hormone response element) specific for glucokortikoids. DNA binding domain GR receptor DNA GRE Cortisol binding domain Binding sites of the glucocorticoid receptor Initiation of transcription by cortisol - continuation 27 GR dimer – intracellular glucocorticoid receptor (dimer) GRE – glucocorticoid response element GREB protein – GRE binding protein (a specific transcription factor) TF IID Pol II CTD > 1 000 bp mediator proteins enhancer coactivator GREB protein GRE cortisol-GR dimer complex promoter basal transcription apparatus Active complex cortisol-receptor binds onto DNA at the specific sequence GRE (glucocorticoid response element, one of the HRE – hormone response elements). The coactivator and specific hormone response element-binding proteins (HREB-proteins) are also attached. This complex supports initiation of transcription on the promoter by means of mediator proteins. Initiation of transcription by cortisol - continuation 28 Intracelular receptors for hydrophobic hormons •Members ofthe nuclear receptor superfamily (there is known more than 150 proteins). •Present in cytoplasma or nucleus •Main group are steroidal-thyreoidal receptors Examples: Androstane receptor AR Estrogene receptor ER Progesterone receptor PR Glucocorticoid receptor GR Mineralocorticoid receptor MR 29 Transcription factors are attached to DNA usually in the major groove. Transcription factors that bind onto regulatory DNA sequences comprise mostly one of the typical structural motifs: helix-turn (or loop)-helix, zinc-finger, and leucine zipper. Only the small part of protein molecule (called DNA-binding domain) is responsible for the interaction with DNA. It is usually represented by two adjacent a-helical segments. NRS helix-turn-helix zinc finger leucine zipper Zinc finger, e.g., occurs in DNA binding domains of steroid-hormone receptors. NRS (nucleotide recognition signal) is a part of a-helix containing amino acid sequence that is able to recognize specific regulatory sequence of nucleotides in the major groove DNA. 30 Zn N S N S Zinc finger NRS E.g.binding domains of steroidal hormone receptors. Zn2+ is chelated by four ligands ether by histidine(N) or by cysteine (S) Zn2+ maintains the tertiary structure of the domain NRS (nucleotide recognition signal) is a part of a-helix containing the sequence of amino acids that serves for recognition of specific sequence in major groove DNA 31 Initiation of transcription - summarization •Transcription is initiated only after all transcription factors are attached •The completed assembly of transcription factors and RNA polymerase bind to the promoter, forming a transcription initiation complex. •RNA polymerase is attached to the transcription factors and DNA in promoter region •It melts 10-15 nucleotide base pairs around the transcription start site, allowing for ribonucleotides to bind to the template strand. •After the first bond is synthesized, the RNA polymerase must clear the promoter, most of transcription factors are separated • • 32 Elongation phase As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy. unwinding rewinding elongation site nascent transcript 5´ 3´ RNA-DNA hybrid template strand capping enzyme (CE) methyltransferase (MT) Both those enzymes modify the 5´-end of the nascent transcript to 5´-m7Gppp-cap CTD is phosphorylated 33 Capping of mRNA 5´ 5´ H N N O N H 2 N N O O H O H C H 2 C H 3 + O P P P C H 2 O P O C H 2 O O C H 3 base base 5´-5´ phosphate linkage 7-methylguanosine is attached by 5´-5´phosphate bond to the 5´-terminal end of the mRNA Complex of proteins is then bonded to the cap that prevents RNA from the action of 5´-exonukleases and enable the transport of RNA through the nuclear pores Čapka je také nutná pro zahájení translace 5´-terminal 34 Termination in eukaryotes no perspicuous termination signal has been found. Transcripts produced by DNA polymerase II are released from the transcription apparatus after the polyadenylation signal AAUAAA and the GU- or U-rich sequence that is able to bind cleavage stimulation factor (CStF) had been transcribed. The terminal sequences of the transcripts are decomposed in the course of 3´-polyadenylation (not encoded by template DNA). 35 Eukaryotic transcription and translation are separated in space and time Prokaryotes Eukaryotes exons introns nucleus cytosol translation translation transcription DNA transcription nuclear export splicing pre-mRNA mRNA processing 36 Prokaryotes - transcription occurs in the cytoplasm. Translation of the mRNA into proteins also occurs in the cytoplasm. DNA is much more accessible to RNA polymerase than DNA in eukaryotes. RNA polymerase interacts directly with prokaryotic DNA. mRNA produced as a result of transcription is not modified in prokaryotic cells. Eukaryotes - transcription occurs in the cell's nucleus. mRNA then moves to the cytoplasm for translation. Eukaryotic DNA is wrapped around histones to form nucleosomes. Eukaryotic DNA is packed to form chromatin. Other proteins mediate the interation between RNA polymerase and DNA in eukaryotes. Eukaryotic cells modify mRNA by RNA splicing, 5' end capping, and addition of a polyA tail. Differences between prokaryotes and eukaryotes 37 Processing of primary transcripts •Primary transcript is precise copy of precursor template DNA with exception of T®U •Primary transcripts of tRNA and rRNA are posttranscriptionaly modified by nucleases in both prokaryotes and eukaryotes •Prokaryotic mRNA is practically identical with primary transcript (is used for translation before the synthesis finishes) •Eukaryotic mRNA is significantly modified 38 Processing of eukaryotic mRNA The primary transcript is hnRNA It is a transcript of the structural gene at which the coding sequences (exons) alter with non-coding sequences (introns or interventing sequences). Exon 1 Exon 2 Exon 3 Exon 4 Intron 1 Intron 2 Intron 3 Non-coding sequences must be removed during processing 39 • Chemical modification (capping at 5´ terminal) – prevents mRNA against 5´-endonucleases and it is also the marker recognized in proteosynthesis. • Splicing (removal of introns) • Polyadenylation (addition of polyA on 3´ terminal) – it prevents against the 3´exonucleases Procesing of hnRNA in nucleus 40 Splicing of hnRNA Splicing is ensured by the action of small nuclear complexes – splicesomes Splicesomes contain five small RNA rich in uracil (U1, U2,U4,U5 and U6) Small RNAs are associated with proteins and form snRNPs (small nuclear ribonucleoprotein particles). Nucleotide sequence AGGU determine the splice sites.These sequentions are recognized by snRNPs. 5´-----AGGU-------AGGU------3´ exon exon intron 5' splice site 3' splice site 41 Exon 1 Exon 2 GU A AG U1 U2 U4 U5 U6 GU A U6 U5 U4 GU A U6 U5 U4 Exon 1 Exon 2 Splicing laso 5' splice site branch site 3' splice site http://www.youtube.com/watch?v=4X8eK15R8yY 42 Mechanisms of RNA-splicing 5´ 3´ pGU OH A AGp Exon 1 Exon 2 G p U A OH AGp Exon 1 Exon 2 mRNA G p U A spliced intron AG Adenine nucleotide in the branch site 2´-OH group of adenine nucleotide attacches phosphate in the position 5´of guanine nucleotide at the place of splicing and forms a lariat. The chain on the 3´end of exone 1 is interrupted, 3´-OH become free and attaches the 5´-end of exon 2 43 Alternative splicing Alternative splicing – various groups of exon originating from one gene form various molecules of mRNA that provide various proteins Exon 1 Exon 4 Exon 3 Exon 2 Primary transcript Alternative splicing Exon 1 Exon 2 Exon 3 Exon 1 Exon 2 Exon 4 Protein A Protein B 44 Gen for a-tropomyosine Alternative splicing of m RNA transcription, splicing exons introns mRNA – striated muscle mRNA – smooth muscle mRNA - fibroblasts In many cases, the splicing process can create a range of unique proteins by varying the exon composition of the same messenger RNA. Alternative splicing can occur in many ways. Exons can be extended or skipped, or introns can be retained. 45 Splice site mutations Mutation at splice sites can lead to improper splicing and production of abberant proteins E.g. b - thalasemia: b-subunit of hemoglobin is not formed in sufficient amount It results from point mutation in b-globin gene where the G®A mutation occurs This creates a new splice acceptor site nineteen nucleotides upstream from the normal splice acceptor A faulty beta-globin protein is made, leading to severe anemia. 46 46 5SRNA pre45SRNA + proteins Syntéza eukarytic rRNA 5S 45S 41s 5sRNA 5S 5S 18S 28S 5,8 S 20S 32S nucleolus nucleus ribonukleoproteins Smaller ribosomal subunit Larger ribosomal subunit 47 Processing of 45 S eukaryotic rRNA 45 S 41 S 20 S 32 S 18 S 28 S 5,8 S 48 Synthesis of eukaryotic rRNA Nucleolus: 45S RNA is synthetized in form of preRNA Complexation with proteins – formation of ribonucleoproteins Methylation and shortening 5S RNA is synthetized in nucleoplasma, it migrates into the nucleolus and is attached to ribonucleoproteins Transport of shortened RNAs to nucleoplasma and through nuclear pores to cytoplasma. Formation of ribosomes. 49 Synthesis of eukaryotic tRNA Synthesis in a form of pre t-RNA in the nucleus Removal of nucleotide sequences on 5´, 3´terminal and nucleotide intron in the anticodon loop Modification of bases: methylation of uracil to thymin dehydrogenation of uracil formation of pseudouridine ( C-C bond between uracil and ribose) deamination of adenosine to inosine Addition of CCA sequence on 3´end Migration to cytoplasma 50 Examples of tRNA processing: methylation uridine ribosyl thymine 5 pseudouridine (j) transformation of the linkage to ribosyl Modification of some bases processing transcript of intron mature tRNA precursor tRNA anticodon a leader sequence 3´-terminal UU replaced by amino acid attachment site CCA-3´-OH 51 Regulation of gene expression Gene expression – formation of proteins or RNA products Generally only small fraction of genes in a cell are expressed at any time Gene expression is regulated differently in prokaryotes and eukaryotes 52 Circular DNA mRNA •only one DNA in the cell •DNA is not complexed with histones •nucleus is not separated from cytoplasma •transkripts of genes do not include introns •translation and transcription occur simultaneously The main features of gene expression in prokaryotes 53 Regulation of gene expression in prokaryotes Regulation is less complex than in the multicellular eukaryotes Gene expression is regulated mainly by controlling the initiation of gene transcription. The most extensive studied is bacterium E.coli. Its genom includes 4x106 base pairs ®E.coli should be able of making several thousands of proteins Under normal conditions they synthesize only about 600-800 different proteins. Many genes are inactive and only those genes are expressed that generate the proteins required for growth in that particular enviroment 54 Operons theory Structural gens of bacterias are grouped into units called operons promotor 1 3 2 Structural genes operon AUG AUG UAA AUG UGA UAG 5´ Prot 1 Prot 1 Prot 1 DNA 3´ mRNA 55 Operon • It includes structural genes for proteins that are metabolically related •Genes in operon are ussually coordinately expressed (they are either all „turned on“, or all „turned off“) •The product of transcription is single polycistronic mRNA •Transcription is regulated by single promotor which is located in the operon at the 5´-end, upstream from the structural genes 56 Regulation of RNA polymerase binding by repressors – negative control Regulatory gene DNA mRNA repressor A B C operator structural genes repressor je encoded by regulatory gene Its product, the repressor protein, difuses to the promoter and binds in the region of the operon called operator Operator is located within the promoter or near of its 3´-end Repressor blocks the binding of RNA-polymerase to the promotor Synhesis of mRNA does not occur > 57 Repressor is controlled by two mechanisms Induction Corepression Inductor is a small molecul that binds to repressor, changes its conformation and triggers its release from the operator transcription can start Inductors: small molecules of nutrients or their metabolites repressor is not active until corepressor is bonded to it. The complex repressor-corepressor binds to operator preventing binding of RNA polymerase transcription stops Corepressors: small molecules of nutrients or their metabolites > 58 Example of induction Induction of lac operon in E.coli by lactose Enzymes for metabolizing glucose by glycolysis are produced constitutively If the milk sugar lactose is available, the cell adapt and begin to produce three additional enzymes required for lactose metabolism These enzymes are encoded by lac operon A metabolite of lactose (allolactose -isomer of lactose that is formed spontaneously) serves as an inducer, binds to the repressor and inactivates it. RNA polymerase can bind to promotor and transcribe the structural genes of the lac operon (b-galactosidase, permease and transacetylase) Glucose can prevent activation of the lac operon (see fig.80) 59 Example of corepression Corepression of trp operon (synthesis of tryptophane at E.coli) genes for enzymes of tryptophane synthesis (5 enzymes) are located in trp operon Tryptophan ise corepresssor, it binds toinactive repressor, binds its conformation. The complex tryptophan-repressor inhibits transcription of operon. http://www.biology.ualberta.ca/facilities/multimedia/uploads/genetics/trpoperon2.swf 60 Stimulation of RNA polymerase binding - positive control Regulatory proteins binds to promoter and stimulate the binding of RNA-polymerase Regulatory protein is activated on the base of presence/or absence of small molecule of nutrient of its metabolite in the cell Catabolite repression > 61 Example of positive control Transcription of lac operon at E.coli transcription of lac operon is affected by allolactose only when the glucose is absent Decrease of glucose level results in increase of cAMP (it is not known why) cAMP binds to its receptor in the cell (cAMP-receptor protein → CRP) Complex cAMP-CRP binds to the regulatory site of lac operon, stimulates the binding of RNA polymerase to promotor and transcription of genes for metabolism of lactose → cells metabolize lactose, only when it does not have adequate supply of glucose 62 Atenuation of transcription •Some operons are regulated by process that interrups (attenuates) transcription after it has been initiated •The cause is the change of secondary structure of mRNA •Attenuator („retarder“ ) is a sequence included in operon •Processes of translation occurs at the same time with transcription •The rate of transcription affects the formation of hair-pin loops on mRNA http://www.biology.ualberta.ca/facilities/multimedia/uploads/genetics/trpoperon2.swf 63 Atenuation of transcription – trp operon E.coli 1 2 3 4 1 2 3 4 1 2 Low conc. of Trp High conc. of Trp STOP Ribosom is attenuated Quickly moving ribosome Sequence 1 contains codones for Trp 5´ mRNA 64 • mRNA is transcripted from the trp operon, ribosomes bind to it and rapidly begin to translate the transcript. • RNA polymerase is followed by moving ribosome • near the 5´end of the transcript there are number of codons for trp. • initially, high levels of trp in the cell result in high levels of trp-tRNAtrp and rapid translocation of the transcript • rapid translocation generates a hairpin loop in the mRNAthat serves as a termination signal for RNA polymerase and transcription terminates. • when trp levels are low, levels of trp-tRNAtrp are low and ribosome stall at codons for trp. A different hairpin loop forms in mRNA that does not terminate transcription •RNA polymerase can complete the transcription •The level of transcription is regulated by the amount of trp in the cell Atenuation 65 Regulation of protein synthesis in eukaryotes The main features of gene expression in eukaryotes (differences from prokaryote): • DNA is organized in nucleosomes of chromatin • gene must be in an active structure to be exxpressed in a cell • operons are not present • genes encoding metabolically related proteins are located on different chromosomes • each gen has its promotor • transcription and translation are separated • 66 Regulation of eukaryotic cells gene expression occurs at multiple level •A) DNA and the chromosome including chromosome remodeling and gene rearrangement •B) transcription, primarily through transcription factors affecting binding of RNA polymerase •C) processing of transcripts •D) initiation of translation and stability of mRNA 67 Regulation of availability of genes for transcription Chromatin in the nucleus: Condensed (heterochromatin ) – genes are inactive Euchromatin- genes produce mRNA In cells of differentiated tissues only the genes that have some role in the cell are active Long-term changes in the activity of genes occur during development as chromatin goes from a diffuse to a condensed state and vice versa. 68 A) Examples of regulation on the chromosom structure level ØChromatin remodeling ØDNA methylation Ø gene rearangement Ø gene amplification Ø gene deletion 69 ØChromatin remodeling - change of chromatin state that results in activation of transcription ® displacement of of nucleosome from chromatin so that the transcription can start Remodeling mechanisms: • an ATP driven unwinding of certain section of DNA from nuclosome core • covalent modification of the histone tails through acetylation or deacetylation (acetylation of e -amino group in side chain of lysin on N-terminals of histones H2A,H2B,H3 a H4). 70 ATP ATP driven unwinding of certain section of DNA from nuclosome core 71 Histon-acetyltransferase catalyzes acetylation of histones. This reaction removes a positive charge from the e--amino group of the lysine, thereby reducing the electrostatic interactions between the histones and the negatively charged DNA® this results in an easier unwinding. This makes RNA polymerase and transcription factors easier to access the promoter region. Therefore, in most cases, histone acetylation enhances transcription while histone deacetylation represses transcription. Significance of histon-acetyltransferase and histon deacetylase + H+ 72 ØDNA methylation Methylation of cytosine residues in DNA by SAM ® 5-methylcytosine SAM + DNA ® DNA-CH3 + S-adenosylhomocystein Methylations are located in GC-rich sequences (GC-islands) that are often near or in the promoter region of gene (postsyntetic modifiation of DNA – enzym methylase) Genes that are methylated are less readily transcribed Example: globin genes are more extensively methylated in nonerythroid cells than in cells in which these genes are expressed (erytroblasts and retikulocytes) 73 DNA methylation Initiation of transcription Transcription is inhibited by methylation 74 ØGene rearrangement Segments of DNA can move from one location to another in the genome, associating with each other in various ways Example : rearrangement of genes in cells that produce antibodies (imunoglobulins) (see Imunology) 75 ØGene amplification Certain region of a chromosome undergo repeated cycles of DNA replication The newly synthesized DNA is excised and forms small, unstable chromosomes called double minutes. They intgrate into other chromosomes thereby amplifying the gene in the process. It is not usual physiological means of regulating of gene expression in normal cells, it occurs in response to certain stimuli Normally gene amplification occurs through errors during DNA replication and cell division – then cells containing amplified genes may have a grow advantage, if the enviromental conditions are appropriate. Example: patients treated by methotrexate (nhibitor of dihydrofolate reductase) can develop drug resistance. The cause is that some rapidly deviding cancer cells amplify the gene for dihydrofolate reductase, producing hundreds of copies in the genome. These cells generate large amount of dihydrofolate reductase and normal doses of methotrexate are not longer adequate. 76 B) Regulation at the level of transcription Basal regulation of transcription (common for all genes) Regulation by the components of „basal transcription complex“ (RNA polymerase binding the TATA box, TATA binding proteins and further basal transcription factors binding on promoter or RNA-polymerase) Genes regulated only in this way: constitutively expressed genes Specific regulation of gene expression: Gene specific transcription factors bind to the specific regulatory sequences. 77 Regulation of transcription factors Down(up)-regulation of transcription factors formation (see SRBP in synthesis of cholesterol) Modulation by binding of stimulatory and inhibitory ligands (CREBP) Mutual cooperation of transcription factors Phosphorylation/ dephosphorylation of transcription factors regulated growth actors, cytokines, peptide hormones atd. 78 C) Postranscriptional regulation Alternative splicing Alternative splicing and variation of the site of polyadenylation cause that one gene can produce various proteins (see the lecture 13) RNA editing In some instances, RNA is „edited“ after transcription. Primary transcript and the sequence of gene are the same, but bases are altered or nucleotides are added or deleted after the transcript is synthesized. 79 79 RNA editing synthesis of apoB in hepatocytes and enterocytes CAA Gen apoB transcript CAA hepatocyte enterocyte CAA mRNA UAA Stop! translation Liver apoB -4563 AA Intestinal apoB – 2152 AK 80 Synthesis apoB in hepatocytes and enterocytes (apo B is a component of chylomicrons and VLDL) Gen apoB produces protein containing 4563 amino acids in liver The same gene produces apoB containing only 2152 amino acids in enterocytes Conversion of C(cytosin) to U (uracil) by deamination of mRNA transcript generates the stop-codon in intestinal mRNA. Thus the protein formed in the enterocyte has only 48% of lenght in comparision with apoB in liver 81 D) Regulation at the level of translation Regulation ussualy involves the initiation of protein synthesis by eIFs (eukaryotic initiation factors)