Class Note for BIOC 461 at UA
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Date Created: 02/06/15
Chapter 31 Notes Page 1 of 16 Biochemistry 461 Fall 2007 CHAPTER 31 CONTROL OF GENE EXPRESSION LECTURE TOPICS Differential Gene Expression in Procaryotes and Eucaryotes Regulation of Gene Expession o Transcriptional Regulation by DNABinding Proteins Ecoli Lactose Operon O Transcriptional and Postranscriptional Regulation trp Operon Attenuation ferritintransferrin receptor in eucaryotes Helixturnhelix motif of Procaryotic DNA Binding Proteins Eucaryotic Gene Regulation 0 Complexity of Genomes o Elaborate Mechanisms of Regulation 0 Transcription ActivationRepression mediated by ProteinProtein Interactions OVERVIEW PROCARYOTIC GENE EXPRESSION Once again Bacteria g M are most well studied with regard to regulation of gene expression Differences in rates of synthesis of some proteins varies over a 1000fold range in response to changes in quantity and quality of metabolites nutrients environmental challenges etc Regulation of bacterial gene expression occurs most often at the level of transcription Genes are often clustered in operons a ofwhose genes are transcribed coordinately in a single mRNA molecule These genes can be expressed at all times in the cell constitutive genes or they can be under the control of repressor andor activator proteins inducible qenes Page 2 of 16 Many regulatory proteins which bind to DNA have a helixturnhelixmotif in common Examples of operons whose transcriptional regulation is well knovm are the lactose and tmptophan operons of M Gene expression is also regulated at the translational level Exs trp operon in E coli ferritin and transferrin receptor in eucaryotes OVERVIEW EUCARYOTIC CHROMOSOMES AND GENE EXPRESSION Eucaryotic chromosomes are larger have higher degrees of structural order and a more complex composition than procaryotic chromosomes The human genome 4x10a bp 1 meter is 1000 times the size ofthat of iii DNA 4x105bp 14 mm Replication and gene expression is more complex than the procaryotic model Table 311 DNA coman of scvcral prokaryotic and eukaryolic genomes Number of Tm DNA Numbquot of 0 5quot 5 quot rm pairs myth mm chromosomes E coli 4 x 1039 14 1 Veasl ts urevisini 14 x 10 46 15 Fruit lly D molanayism 17 x 1039 56 4 Human 33 x 10 530 23 New The values given are For haploid genmax Eu caryotic chromosomal DNA is wound around histones in complexes called nucleosomes The entire chromosome also contains many other proteins in a complex matrix called chromatin A small fraction 1 2 of eucaryotic DNA is genes which code for proteins There is an abundance of repetitive noncoding sequences which comprise a signi cant 39action of eucarotic genomes Eucaryotic gene expression is regulated primarily at the level of transc ption Transcriptionally active regions of chromosomes are extra sensitive to DNase digestion and have reduced levels of cytosines which have been methylated Expression of genes in these chromosomal regions is regulated by transcriptional factors Page 3 of la Translational regulation occurs In iron metahohsm genes Some developmentallycontrolled genes In insems ano mammals possess a sequence when ine homeo hox Wmen eooesior a an amino acid long DNAV hmdlng ponpeptlde oomam homeo domain Wmen may pe lnvoIved In reguIatlng expression oiinese genes DIFFERENTIAL GENE EXPRESSION IN PRDCARYDTES AND EUCARYDTES Some proearyoile genes are differentlaHy expressed In response lo different environmentaI varlames temperature noinenisr eie Many genes are expressed atdlfferenttlmes ano In different Incatlons irssoes organs In a eoeanoiie orgamsm oonng it s Ilfespan Taple 31 l man 31 Highly expressed proteinencoding genes of the pancreas and liver as percentage nf lolal mRNA paol Rank Pancreas ii Liver 0 i Pmcarl mxypeptidase A1 Albumin 35 2 Pancreatic n ypsmogen 2 Apolipopmtem I 23 3 Chymotr pslnngan Apollpoproiein l 2 s 4 Fanci eauc ll ypsln l Apolipoproiein 7 ii 21 s Elnslzlse 1m A39I39Pasc m 1 s i Pmreasu F Cyrm hmmsoxida 1 I 7 Pancreath lipase Cytochrome oxidasc 2 i l a Procarhoxypepudase B 017 i rAnuh39ypsln 1 o e Pamrem amyIdse I 7 Cytochrome oxida 1 0 9 lo Bile sail annnilaled lipase l l Apolrppprorein E 09 Page 4 of 16 REGULATION OF PROCARYOTIC GENE EXPESSION TRANSCRIPTIONAL REGULATION BY DNABINDING PROTEINS Ecoli Lactose Operon 0 To use lactose as a sole carbon source E Q synthesizes galactosidase thousandscell only when cells are grown on lactose The enzyme is inducible by conversion in the cell of lactose to allolactose a product ofa reaction catalyzed by the 10 or so Bgalactosidase molecules present in the cell prior to induction p868 and Fig311 CH20H CHZOH H U CHyOH CH20H H0 0 O 2 HO O OH 0 OH O OH 5 OH OH BCalactosidase OH HO OH OH OH OH OH Lactose Galactose Glucose Lactose removed 13 galactosidase induction Lactose or IPTG in lab BGalactosidase ug Ii vl39 ull I IIJ U n If quot0 HI H LIlt Jl L N S quotU 00 il l quotUH J H39fi lddbsldds Spontaneoug Br dimerizalion UH Cl 3 Ho IfJ anduxldntion I tail 5539gtDibmmu4l 4iMavnindigo To measure galactosidase activity in the lab Xgal cleavage gives galactose and a blue colored product that cna be quantitated Fig311 Page 5 of 16 The Egalactosidase gene is in an ogeron which contains three structural genes 2 y a two control sites called the promoter p and operator 0 and a regulatory gene i Since there is more than one structural gene cistron the lactose operon is called a polycistronic operon Fig313 Control A ReEUIamr sites Structural genes gene Af B Lactose operon The regulatory gene produces a repressor protein that normally no lactose in medium binds to the ogerator preventing RNA polymerase from transcribing genes 2 y and a An inducerallolactose or IPTG forms a repressorinducer complex that cannot bind to the operator permitting transcription Fig318 a li W 39 39 quot RN Repressor bound to operator site 1 prevents transcription of Z Y and a 00 3 Wu P a imRNA i lacmRNAl l 00 time g lSGalactosidase Permease Transacelylase N Repressorinducer complex does not bind DNA E n ngtngtlt quot1 w wvV qg lbl Page 6 of 16 The lac repressor binds to an inverted repeat operator that is nearly a palindromic sequence with dyad symmetry Fig 369 y Protected by repressor gtl lac repressor DNA interactions Figs315 6 Operator DNA domain y Carboxyl terminal domain lac repressor u hellx An activator CAP protein binds cyclic AMP levels increase when glucose in medium is low and this cAMPCAP complex bind upstream of the promoter site and stimulates transcription 50X more than without CAP This overcomes the lads lack of a strong promoter consensus sequence Fig3110 Fig 3613 CAPRNA polymerase binding sites are adjacent RNA polymeraserepressor binding sites overlap Repressor sterically interferes with RNA polymerase binding while CAP facilitates binding of RNA polymerase A CAP dimer with cAMP binds to DNA in the major groove and bends the DNA by 94 degrees Fig3111 Consensus 35 region gtnmi lt 10 region tam rgtgt i Start site for transcription Page 7 of 16 HELIXTURNHELIX MOTIF OF GENE REGULATING PROCARYOTIC DNA BINDING PROTEINS 0 Three dimensional models of cro lambda repressor and CAP show that these polypeptides share an ahelixBturnahelix motif which is involved in specific proteinDNA interactions These proteins occur as dimers which interact with specific DNA sequences via binding of an orhelical polypeptide domain with the major groove of a symmetricallyoriented recognition site spanning one turn of a B DNA helix Fig 3612729 lac repressor CAP trp repressor gamma 0 Bstrand DNA interactions are basis for recognition in methionine repressor Fig3113 recall also eucaryotic TA TA box binding protein Page 8 of 16 PROCARYOTIC TRANSCRIPTIONAL AND POSTRANSCRIPTIONAL REGULATION TRYPTOPHAN OPERON REGULATION A39I39I39ENUATION n s Transcription m translation interact to regulate the tryptophan operon The tryptophan operon has several structural genes for proteins involved in tryptophan synthesis There is an operator site where tryptophan repressor complexed with tryptophan a corepressor binds inhibiting transcription Figs sis3132 Aleader L sequence is 539 to an attenuator sequence Fig 3634 The leader codes for a polypeptide Fig 3635 whose synthesis occurs at high cellular tryptophan levels and whose synthesis is inhibited at low tmptophan levels The leader polypeptide has two tryptophan codons Fig3134 Antmum rm y M llo Phc Val Lei lysraly mg m Set r liit a rrwnmmAw chuacwmmu UmuGANliC KALLrl llrillrlirltlllllllllln iiilr rrrr ii mil 0 L r a A r F uuurrrrr mu r or u When cellulartryptophan concentration is mgh leader translation is enhanced and transcription is inhibited by the attenuator39s secondary structure which looks like that of a terminator Le a GCrich region with A twofold symmetry WW 2 followed by Us in the quot mRNA which makes a stemloop structure Fig3134 Fig 3636 Ribosome Terminate transcription When tryptophan levels are low leader translation is slow the tryp mRNA does not assume a terminatorlike appearance and the tryptophan operon is transcribed Fig3135 Fig 3636 RNA polymerase B w Alternative 2 SUUCTUIE No termination WNJ N TI Q Ogeron Attenuation Mechanism High Trp W fast translation 34 stemloop stops transc Ribosome Formation of this stem and loop resulls in the terminauon oi Vansmption Leader region is completely translated A High Irypmphan level Transcripl ion Ribosome is stalled 1 LOW TIP W 3 Hp odons 3 slow translation 4 23 stemloop allows transcription a Low tryptophan level Page 9 of 16 TRANSLATIDNAL REGULATION IN EUCARYDTES Proteins svnthesis from ferritin and transferrin receptor mRNAs Figs Bl 38 39 G A U C G A U C A 39 U Ironresponse C 39 5 element u G u A C C c C K C G U G C A U G C V in I39 n G C 5quot Cod g ego J IRE iron response element binding protein blocks translation of ferritin mRNA Ironresponse elements Coding region 5 3 I Transferrin receptor mRNA has several IRES at 339end IREbinding protein located on these lRE s stabilizes mRNA and does not inhibit translation Page 10 of 16 EUCARYOTIC GENE REGULATION COMPLEXITY OF GENOMES EUCARYOTIC CHROMOSOMES 0 SIZE Large genomes 1 meter long in human genome linear molecules Figs311415 Table 5 371 IG 722 I raquot4 a JV Lquot a a 4 1 in Direction of electrophoresis a g a fit 39 100 nm I COMPOSITION Contain five types of basic proteins called histones which have lots 25 of Arg and Lys residues and are 11 to 21 Kd in mass The histones are frequently modified by acetylation methylation Phosphorylation etc These modifications may relate to DNA packaging or availability for replication ortranscription Table 372 Histone amino acid sequences and structures are highly conserved especially histones H3 and H4 in all eucaryotes suggesting that the role of histones was established early during eucaryotic evolution H2 3 4 structures Fig3117 STRUCTURE HZA H23 H3 H4 kg W d 6quot a i 9 5 n Page 11 of 16 o Nucleosomes Fig 375 8 are repeating units of chromatin which consist of core particles a histone octamer around which is wrapped 140 base pairs of DNA connected by linker DNA 20 to 50 base pairs The core particles contain two molecules each of histones H2A HZB H3 and H4 and one molecule of histone H1 binds to the outside of the core particle The DNA wound around the core particle forms a 134 turn lefthanded superhelix Fig3116 Fig 338 Nucleosomes condense DNA into a smaller space packing ratio 71 Further DNA packing must occur in cells since metaphase chromosomes have a packing ratio of 10001 Fig3118 Ami niHermi n al A a C Linker DNA Histones 2A 25 3 4 Histnne 1 39 Linker DNA Page 12 of 16 A protein scaffold of nonhistone proteins provides a higher order structure and higher packing ratio than that of just nucleosomes These proteins include topoisomerase II which suggests that changes in supercoiling are important in dynamic changes of in chromosomal DNA structure and function during the cell cycle Fig 3711 and Lehninger Fig 2320 ismaaepima DNA rm CM h r 2 x 10 mm One mil 30 rnsetlesl One mwite a loopsi 0mian swimbran hull w F o quotN l mu ibcr 1 713931154 w Q FI massr t fax 1mg welcomes Page 13 of 16 REPETITIVE VS SINGLE COPY DNA Renaturation analysis revealed that eucaryotic chromosomal DNA has a large fraction of several types sequences satellites Alu sequences that are repeated up to a million times Results also showed that there was only a small fraction of unique proteincoding sequences Table 376 Table 376 Classes of eukaryotic DNA Proteincoding genes Singlecopy genes Duplicated genes RNAcoding genes Most are tandemly duplicated Pseudogenes Repetitive DNA Simple sequence DNA as in satellite DNA Dispersed repetitive DNA includes mobile genetic elements Spacer DNA of unknown function Ribosomal RNA genes are tandemly repeated several hundred times and can be ampli ed to 2X106 copies during development of Xenopus oocytes to up to 75 of total cellular DNA Amplification provides enough genes to rapidly transcribe rRNA genes to get 1012 ribosomesoocyte Histone genes are clustered repeated lack introns and have mRNA which is not polyadenylated Many major and minor ce proteins are coded by single copy genes For major cellular proteins transcription rates mRNA halflife and use of mRNA for multiple rounds of translation can give up to 109 protein molecules from just one gene copy silk fibroin the human genome is 1000Xthe size of the E M chromosome but only 12 codes for proteins Thus the actual unigue human genome size is only about 50X that of E M Page 14 of 16 REGULATION OF EUCARYOTIC GENE EXPRESSION Elaborate Mechanisms Regions of chromosomes which are transcriptionally active are less condensed form puffs in Drosophila Fig 3729 are undermethylated and are hypersensitive to DNase I mostly at the 539ends of genes These characteristics are tissuespecific and developmentally regulated Methylation of cytosines Fig 3730 in chromosomal DNA is associated with low transcriptional activity in general Azacytosine prevents methylation so transcrption is more active Not all possible regulatory sites on chromosomes in a given celltissue type may be activated at the same time For instance yeast GAL4 protein activates genes for proteins required in galactose utilization as a carbon source But of 4000 possible specific binding sequences only 10 actually have GAL4 bound and only 10 genes must be activated for galactose utilization Page 15 of 16 Disruption of chromatin structure occurs when transcription activator proteins bind to enhancer sequences Many enhancer sequence elements are known The glucocorticoid enhancer is one example Ch28 Notes p13 The muscle creatine kinase gene is another in which 3 copies of one enhancer sequence occurs and two other enhancer sequences exist At least 3 different regulatory proteins are required to bind to all three of these different enhancers Often binding of these proteins disturbs the chromatin structure to expose the gene DNA to allow transcription Figs312021 Start site TATA CAGCTG CAGCTG Enhancer CAGCTG region l TTATAATTAA CCATGTAAGG TRANSCRIPTION ACTIVATIONREPRESSION MEDIATED BY PROTEINPROTEIN INTERACTIONS TRANSCRIPTIONAL ACTIVATORS We already know about transcription factors and enhancer sequences see Chapter 28 Transcriptional activators bind to speci c activator sequences enhancers and these proteins have in common at least two conserved functional domains one which binds to DNA and one which activates transcription Fig3132 Fig3731 In addition other ACt39VatIOn conserved domains may be present doma39n depending on the specific activator Binding domain DNA Page 160f 16 O The family of nuclear hormone binding receptors active as dimers of identical subunits has an additional ligandhormone binding domain The DNA binding domains of nuclear hormone receptor proteins possess globular structural domains in which four cysteines are tetrahedrally coordinated with a divalent zinc ion Two of these zinc clusters are present on each subunit and they stabilize the structure of of the dimer Each subunit of the dimeric steroid receptor has an Oihelix which recognizes and binds to the major groove of the DNA sequence of the steroid response element Fig3122 Fig 3735 5 NAGAACANNNTGTTCTN 339 339 NTCTTGTNNNACAAGAN 5 Glucoconicoid response element GRE 539 NAGGTCANNNTGACCTN 3 3 NTCCAGTNNNACTGGAN 539 Estrogen response element ERE Fig3122 DNA binding domain Fig 3736 ER PR GR VDR RAR TR Ligand binding domain
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