Molecular Biology of the Cell Designer Genes

8/10/2019 Molecular Biology of the Cell Designer Genes

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Basic Molecular Genetic Mechanisms

Structure of Nucleic AcidsTranscription of Protein-Coding Genes and Formation of mRNADecoding of mRNA

Stepwise Synthesis of Proteins on RibosomesDNA ReplicationDNA Repair and RecombinationVirusesMolecular Genetic Technique Analysis of Mutations to Identify and Study GenesCloning and Characterization(PCR, ECP)Using Cloned Fragments to Study Gene Expression

Locating and IDing Human Disease GenesInactivating Eukaryotic GenesGenes, Genomics, and ChromosomesEukaryotic Gene StructureChromosomal Organization of Genes and Noncoding DNATransposable DNA ElementsOrganelle DNAsGenomicsStructural Organization of Eukaryotic ChromosomesMorphology and Functional Elements of Eukaryotic ChromosomesTranscriptional Control of Gene ExpressionControl of Gene Expression in BacteriaOverview of Eukaryotic Gene ControlRNA Polymerase II Promoters and General Transcription FactorsRegulatory Sequences and Proteins


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Molecular Mechanisms of Transcription, Repression, and ActivationRegulation of Transcription-Factor ActivityEpigenetic Regulation of TranscriptionPost-Transcriptional Gene ControlProcessing of Eukaryotic Pre-mRNARegulation of Pre-mRNA ProcessingTransport of mRNA Across the Nuclear EnvelopeCytoplasmic Mechanisms of Post-transcriptional Gene ControlProcessing of tRNA & rRNA

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Basic Molecular Genetic MechanismsStructure of Nucleic AcidsDNA, RNA both polymers composed nucleotidesRNA more diverse functions (eg. as catalyst)All nucleotides are made of an organic base+5 Carbon Sugar+ phosphate group

Purine =A&G=fused double bonds Pyrimidine = C+T+U=Single bonds5 end has phosphate/hydroxyl group on 5 carbon of end sugar, 3 end has hydroxylon 3 carbon of terminal sugar (sequences read 5 --> 3)bond between nucleotides is a phosphodiester bond (2 phosphoester bonds)Chargaffs LawDNA usually right-handed double helix, normally less compact B form (when wateris removed in lab, it turns into A form)


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DNA has hydrogen in 2 position, making it more chemically stable than RNA withhydroxyl in 2denatured at temperature Tm, which varies depending on G-C concentration(because they have more stability with 3 hydrogen bonds) and ion concentration(direct relationship) and extreme pHmay renaturate , a feature used in hybridization of sequencestopoisomerase I can relieve torsional stress from a broken DNA fragmentRNA falls apart into bases in an alkaline solutionRNA secondary structure: hairpin, stem-loopRNA tertiary structure: Psuedoknot , can be ribozyme Transcription of Protein-Coding Genes and Formation of mRNAgene is DNA sequence that specifies synthesis of one polypeptide or functionalRNA sequencemiRNAs =microRNA fragments that regulate tRNA activityRNA polymerase translates in 5-3RNA polymerase binds to promoter, seperates the DNA in a 12-14 bp area calledthe transcription bubble , continues along at rate of 1000 bp/minute until stop site,where it releases the mRNA strand. In prokaryotes only, translation on the 5 endcan beginbefore transcription for the sequence has finishedIn prokaryotes, an operon containing multiple genes is transcribed from a singlepromoter, in eukaryotes each protein-encoding gene has its own promoter

RNA polymerase has 2 large subunits ( beta, betaprime) , and 2 smaller alphasubunits, as well as one omega subunit for structural stabilityexons vs introns (exons usually in multicellular eukaryotes only), operons arefunctional gene sequences with mutiple proteinsIn eukaryotes, RNA synthesis creates pre- mRNAs , which are transformed in RNAprocessing to mRNA: a 5-cap is created to prevent degradation , poly(A)polymerase adds Poly(A) Tail (100-250bp) at 3 end to help guide the mRNAthrough the cell, and RNA splicing (taking out the introns)

mRNA still retains untranslated regions at the endsalternative splicing uses introns to creates multiple proteins from a single gene(Fibronectin is an example)Decoding of mRNAtranslation code degenerate because multiple codons can specify the same aminoacidAUG is start codon,UGA,UAG, and UAA are stop codons


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sequence from start to stop is reading frame occasionally can frame-shift to read multiple proteins from one RNA seuqencemitochondria, ciliated protozoans, and Acetabularia (a single-cell plant) havevariations in the amino acid code (read stop codons as amino acids)tRNAs link to amino acids with help of aminoacyl-tRNA synthetase (whose 20variations recognise 1 amino acid and all its cognates , or codon triplets coding forit)The reaction uses ATP to affix an amino acid to the 2 or 3 hydroxyl at the end ofthe acceptor stem in a high-energy bond that is termed activated, whose energy isused to create the peptide bonds between amino acidsThe correct tRNA is recognized by the enzyme by the structure of the anticodonloop & acceptor stem, + the absence of bases in tRNAthe enzyme also proofreads, so the total error is 1 in 50000 codons (in E. coli)the D loop and the T CG loops interact with the ribosome to maintain the stabilityof the tRNA-mRNA temporary bondthe third position of a codon is the wobble position , which can pair with multipletypes of tRNA anticodons, this means that some tRNAs can recognise multiplecodon triplets that code for the same amino acid, so many cells contain fewer thatnthe 61 tRNAs that would otherwise be necessarymany tRNAs have inosine (I) in place of adenine in the wobble position, which canrecognise A, C, or U mRNA base pairs (ex, CUA, CUC CUU and UUA, which all code

for Leu, are all recognized by GAI anticodon)

Stepwise Synthesis of Proteins on Ribosomesribosomes most common RNA, work at 3-5 amino acids per secondup to 3 hours to make largest proteins (eg titin)ribosomes are made of 3 RNA molecules in prokaryotes(pro), 4 RNA in eukaryotes(euk)Large subunit contains 1 molecule large rRNA(23S pro, 28S euk), one molecule of 5S

(svedburg/sedimentation units ) rRNA, and 1 molecule 5.8S in vertebrates (total50S pro, 60S euk)Small subunit contains 1 molecule small rRNA(16S pro, 18S euk)total ribosome is 70S in bacteria (30S small subunit, 50S large subunit), 80S ineukaryotes (60S small subunit, 40S large subunit)plants and yeasts can be larger


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specific tRNA called tRNA iMet binds to the P site on the small subunit to initiatetranslation (when it reads the AUG codon)eukaryotic translation initiation factors(eIFs) mediate initiation:1.a ribosome finished with translation binds with eIFs 1, 1A, and 3 to form a 43Spreinitiation complex2.eIF2 binds GTP to tRNA iMet 3. a eIF4 complex binds to mRNA to activate it (5 end and Poly(A) tail)4. the preinitiation complex binds with the eIF4/mRNA complex j5. the eIF4 works as a helicase to undo the RNA secondary structure and feed itinto the ribosome6. The AUG start codon is recognized by the ribosome,causing GTP to be hydrolyzedto GDP (as a sort of proofreading switch to confirm that translation has started),creating a 48S initiation complex7. The small subunit joins the bottom of the large subunit, over the RNA8.When this occurs correctly, the remaining GTP is hydrolyzed to GDP as aproofreader switch, the eIFs are released, and the full 80S ribosome is createdSome mRNAs have an internal ribosome entry site (INES), which forms an RNAcomplex that interacts with eIFs to bind to the 40S subunit , which then is bound tothe mRNA and the 60S subunit.Elongation Factors (EFs) are used to guide the translocation of ribosomes overthe mRNA sequence

the pepidyltransferase reaction is catalyzed by the large rRNA itself (not aprotein), and GTP hydrolysis is used again to signal thisA, P ,E sitesto terminate the translation, Release Factors (RFs) bound directly to the A site(eRF1) recognise the stop codon and signal to eRF3-GTP to cleave the completedprotein from the ribosome.the protein ABCE1 uses ATP to release the mRNA and RFs to go to anotherribosome, and the IFs come marching in again to form the 43S preinitiation complex

DNA Replication

first learned about with SV40 virusesthese viruses use only 1 viral protein ( large T-antigen) to reproduce, the rest comefrom inside the cell
https://docs.google.com/document/d/176BMfc5Q4L6_5q8MGDqYp2Rq63xRQ4K5P1NFx-M14pY/edit#bookmark=id.bld9zpc8bd8


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DNA Repair and RecombinationDNA polymerase in eukaryotes produce 1 mutation in 10 4 nucleotides, inprokaryotes 10 9 proofreading from exonuclease activity of polymerase (base pair returned toexonuclease site( a polymer domain) unless forms base pair with template dna)the bond between a purine and deoxyribose is prone to hydrolysis!mitochondria and peroxisomes create hydroxyl radicals and superoxide, which alsodamage dnapoint mutations are a change in 1 base pairnonsense mutations are new stop codons in the wrong placemissense mutations are a change in the amino acid sequencesilent mutations dont change the sequence (changes between codon cognates)deamination is one of the more frequent mutations: C-->U (or C--> T in humans)excision-repair systems excise and repair damaged dna by referencing thetemplatein the case of a G-T mismatch, the repair system knows to automatically replace Twith C(because it must have been caused by deamination)this all happens before replication or it wouldnt be recognizeddepurination is the loss of a guanine or adenine base from the hydrolysis of its

bond, which forms abasic sites, which generate mutationsmismatch excision repair corrects mismatches after replicationnucleotide excision repair fixes chemically warped bases that distort the DNAshape locally, including thymine-thymine dimers which is when the carbons in thymine bond,warping the dna (caused by UV light)the excision repair uses helicase, polymerase, ligase, as normalx, gamma radiation and anticancer drugs lead to double strand breaks that often

rejoin incorrectlynonhomologous end-joining (NHEJ) is the predominant way to repair thesebreaks, although they lose a few base pairs at the joining zonessometimes, ends from 2 different chromosomes are joined together, leading to acancerous geneshomologous recombination was found to be very important when a strongcorrelation between mutations in its genes and cancer was discovered


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used as important repair mechanismto find homologous strands, it takes DNA from separate molecules in geneticrecombination , an important source of genetic diversityreplication fork collapse, caused by a nick in the phosphodiester backbone, can befatal to the cell (destroys the genetic data)its repaired by: strand invasion of the DNA by a complementary strandbranch migration is when the cell uses ATP to extend the hybrid zone away fromthe breakbranch= where the target dna crosses from the whole DNA strand to its brokencomplimenta holliday structure is then formed, as the broken off strand base pairs with thebottom, whole strand, crossing over the hybrid strandthe strands are broken at the crosspoint and the breaks are ligated (this makes it sothe 2 original strands of dna, as well as the transcribed dna, are all preservedVirusesthere are RNA and DNA viruses: RNA replicates in cytoplasm, DNA replicates innucleusviruses can encode between 4 and 200 proteinsthe infectious particle is a virion the host range is the set of cells that viruses infect (usually pretty narrow (onephyla max)

phagevesicular stomatitis virus has a wide host range (insects and many mammals)polio and many other viruses only infect specific cell types (intestine for polio)HIV infects lymphocytes and glial cellsthe capsid is the protein coat around the nucleic acid of a virusits made with a small number of distinct genes to minimize the nucleic acid needeto encode for itcapsid+nucleic acid= nucleocapsid

helical nucleocapsids are rodlike tubes with the nucleic acid in a helical groove (extobacco mosaic virus)icosahedral nucleocapsids are hedrons made of 20 sides (each an equilateraltriangle)some use grooves between capsid subunits to interact with host cells, some usefibers extending from the surfacemany bacteriophages have a icosaderal head and a rod tail


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many viruses have an envelope of a phospholipid bilayer + a few glycoproteinsplaque assays can find how many viral particles are in a sampleits done by culturing a sample of viral particles on host cells and then counting thenumber of lesions ( plaques) that developlytic cycle:adsorption: binding of capsids to the cell membranepenetration, replication, assembly, releasetemperate phages establish nonlytic association that doesnt kill the cellprophage= integrated viral dnathis is lysogenymust switch to lytic cycle at some point to get out of the cell again--this isinductionretroviruses, reverse transcriptasecancer-causing genes in retroviruses

Molecular Genetic TechniquesAnalysis of Mutations to Identify and Study Genesmutation analysis reveals genes required for the process, the order that genes actin the process,a nd whether encoded proteins interact with each other

allele wild type= non-mutated, standard genephenotype/genotypemutagenconditional mutations used to isolate mutants, most common typetemperature-sensitive mutations which can be isolated in bacteria and lowereukaryotes but not in warm-blooded eukaryotes.THese are mutations that function

(ie produce proteins) at one temperature, but denature at another (when a normalprotein would be stable throughout that temperature range)nonpermissive temperature is when the phenotype is observed ( permissive isopposite)mutations are used to find the order of protein function (with mutations defectiveat a certain point in the process)


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genetic suppression occurs when a mutation in the structure of one protein ismatched by a mutation in a interacting protein, such that the functioning of theprocess involving the proteins is unimpeded iff both mutations occu (this helpsdetermine if 2 proteins interact)synthetic lethality is the opposite (ie proteins fail to function iff both mutationsoccur)mutations can be used to map genes by tracking instances of crossing over (whenyou get 0 or both mutations in the recombinant chromosome), as lessrecombinations occur if the genes are closer together (discovered by A. Sturtevant)1 genetic map unit is the distance between 2 positions that results in a 1/100recombination rateloci are unlinked if the recombinant rate=parental rateCloning and Characterization(PCR, ECP)recombinant DNA dna from different sourcesvector DNA: dna inside the cell combined with a DNA fragment (the one you wantto replicate) (commonly E. coli)dna is cut by using restriction enzymes, which find restriction sites ( usuallypalindromic) on enzymesthe bacteria that produce restriction enzymes also produce modification enzymes ,which adds methyl groups to native DNA to prevent restriction enzymes(RE) fromcutting at that point

sticky ends are produced with staggered cuts in the double helix ( leaving singlestrands), blunt/flush ends are produced when the enzyme cuts across both strandsat the same placex RE will always cut y sequence at a predictable set of locations, spaced 4^n basesapart (n=length of restriction site)ligase can easily join 2 complementary sticky ends with covalent phosphodiesterbonds, ligase from bacteriophage T4 can inefficiently join blunt endsSmaI & AluI REs produce blunt ends

plasmids are rings (1-3 kb) of extrachromosomal dna found in lower eukaryotes, E.coli ones are often used as cloning vectorsplasmids have a replication origin (ORI), a marker that can be selected (eg drugresistance) and a place to insert DNAonce a host cell starts replicating a plasmid at the ORI, it will continue replicatingthe rest if the plasmid including inserted DNA


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in transformation , ~1/10000 E coli cells mixed with modified plasmids will take upa plasmid (these are then selected for with the marker and left to reproduce)fragments from 3-10000 bp can be inserted into vectorsvector versatility is increased with polylinkers , which are synthetic vectors withseveral different restriction sites, meaning that they can be used with DNAsequences cut with multiple REs.Bacterial Artificial Chromosomes(BACs) are used to clone long (millions bp)sequences, one type uses a ORI called the F factor DNA libraries are collections of DNA molecules each cloned into vectors; thecloned set of all sequences in a genome is a genomic library because large genomes contain too many introns, complementary DNA (cDNA)libraries, which store DNA copies of mRNAs, are used for higher eukaryotespoly(A) tails are used to recognize mRNA in the cell (using thymidylate)the mRNA is then synthesized into cDNA with reverse transcriptase (thx HIV)This is then methylated to prevent cleavage, ligated to an EcoRI linker with T4Ligase, and attached to an E coli vectordifferences in transcription rates mean that #occurrences of a gene in a cDNAlibrary is variable (libraries thus contain millions of individual recombinant clones)libraries are screened with oligonucleotide probes ( 20 bp) that bind to a selectedclone, and finding genes based on encoded proteinsprobes use hybridization: denature the library replica and add the (fluorescent)

probe, then renature it and wash away the excess probe, scan sample forfluorescent objects (ie the hybrid dna)gel electrophoresis:for sequences 10-2000 bp, use acrylamide gels, 2000-20 kb need agarose gelssubcloning is rearranging parts of genes (eg change out a promoter)PCR es lo que esdenature and then add in synthetic oligonucleotides in excess100 bp DNA fragments can be sequenced with PCR using fluorescent DNA

polymeraseUsing Cloned Fragments to Study Gene Expressionsouthern blotting is used to find a specific gene fragment: 1st use gelelectrophoresis to separate the genome by length, then denature the DNA at thedesired length and put in hybridization probes.northern blotting : southern blotting with RNAin situ hybridization is used to preserve the relative location of mRNA


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DNA microarrays are thousands of DNA sequences attached to a slide1500 sequences/cm^2transfection clones genes into animal cellselectroporation is the application of electric shock to open the pores of a cell toDNAtransient transfection is when a viral vector is used to put in a lot of plasmidsquickly, but these arent distributed to all daughter cells (thus transient)stable transfection uses mammalian enzymes with a selectable marker, thosewhich integrate into the genome are then selected forretroviral expression systems: lentivirus systems can be used to speed uptransfectioncan flag proteins with GFP (green fluorescent protein) or an antibody called anepitope

Inactivating Eukaryotic Genes

the genome of the yeast S. cerevisiae can be easily changeddisruption constructs are ersatz sequences of DNA spliced into the genome(created with PCR), to see if the gene it replaces is vital to cell functionthis method found that 4500/6000 yeast genes not essentialcan also use a conditional promoter: eg GAL1 promoter in yeast only permitsreplication when galactose is present: allows researchers to control replication ofthat genegene knockout

RNA interference (RNAi) uses double-strand rna to block expression ofcomplementary rna (see chapter 8 )

Genes, Genomics, and ChromosomesEukaryotic Gene Structure


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bacterial mRNAs are sometimes polycistronic ( a cistron is a sequence encodingone polypeptide), but most eukaryotic mRNAs are monocistronic: each mRNAmolecule encodes 1 proteinas a result, bacterial translation can proceed from multiple points on the mRNAthus, bacterial transcription units are distinct from genes (transcription units aresingle operons)simple transcription units are processed to create an mRNA for one 1 protein90% of human transcription units are complex ( it can be processed in more than 1way, all results monocistronic)mutations in control units affect all of it, mutations in exons affect only that mRNAthe various proteins encoded from different expressions of a gene are calledisoformsthere are solitary and duplicated genesduplicated genes create a gene family, and are similar but non-identical forms of agene located close together on the transcription sequence, created by unequalcrossing-over, they separately evolve in a beneficial mannerpsuedogenes: gene duplications that became non-functionalheavily used genes (like that to produce rRNAs) have multiple identical copiesother genes code:small nuclear RNAs, which function in RNA splicingsmall nucleolar RNAs: help rRNA processing in nucleolus

micro RNAs (miRNAs) regulate the translation and stability of mRNAs

Chromosomal Organization of Genes and Noncoding DNANo direct correlation between gene length and complexity--amoeba dubia has 200times more DNA/cell than humansselective pressure on commonly used genes to reduce intron size1/3 of DNA is transcribed (but 95% of this is introns), and the rest is between genes,repeated DNA sequencesmicrobes have less introns because the cost of gene synthesis is proportionallygreater to themrepetitious dna: multiple copies of DNA sequencessimple-sequence DNA: is 6% of the genome and is identical copies of varioussequences. When 1-13 bp, it is called microsatellites created by backward slippage during replication


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interspersed repeats are much longer ( more info )microsatellites cause many genetic diseases (by creating coding sequences thatcode for bad proteins)extended repeats can also occur in non-coding sequences, where they can formlong RNA hairpins that sequester the proteins that are supposed to regulatesplicing14-100 bp regions are minisatellitestoday, satellites are used for dna fingerprinting, by using PCR to find amplifytandem repeats, which gives different results for every person (other than identicaltwins)

Transposable DNA ElementsInterspersed repeats are also known as moderately repeated DNAthey also can move around the genome in a process called transposition , they

seem to only exist to maintain themselves (thus also called transposons)retrotransposons copy to rna then to dna, DNA transposons cut out their sequenceand move somewhere else.retroviruses may have evolved from these transposonscan aid evolution by transposing sequences around them too (mutation)transposons are copied (rarely so they dont disrupt essential genes) bytransposaseretrotrans

they have an inverted repeat on either end, and then a direct repeat on eiteherend--wild-type has it once, but the IS has it on both endsactivator elements are highly correlated with reversible mutationsdissociation (Ds) elements are correlated with mutations that dont reversethemselves, except in the presence of the 1st classactivator elements are IS elementsdissociation elements are IS elements with damaged transposase--transfer only inpresence of 1st mutation (ie transposase)retrotransposons can be divided into those with long terminal repeats(LTR)LTR retrotransposons make up 8% of human DNAbecause they code for all retroviral proteins, and have LTRs like integratedretroviruses, theyre called retrovirus-like elementsendogenous retroviruses are the most common, lotsof isolated LTRs most common mammal transposons are nonviral retrotransposons long interspersed elements (LINE) are 6 kbp long


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three types of LINES, L1-L3: only L1 still transposesin total 21% of DNAit has direct repeat, a region with a lot of A&T, then ORF (open reading frame) 1,encoding for a rna-binding protein, then ORF2 which encodes a long region withreverse transcriptase and DNA endonucleases.Short INterspersed Elements (SINE) are 13% of DNA100-400bpstill have A-T rich sequence at ends like LINEshort because they dont encode protein, the rely on the reverse transcriptase fromLINESmany SINEs are Alu elements evolved from a RNA in the signal recognitioncomplex, which target polypeptides to the ER1 in 8 individuals have non-LTR retrotranspositions occurring, 60% SINE (90% Alu),40% LINE L1retrotranspositions can come from processed mRNA, too, creating psuedo-genestranspositions crucial source of mutations--duplications can evolve separately toperform mutually beneficial effects.recombinations between mobile elements in separate genes () can generate totallynew genes--called exon shufflingin DNA transposons happens when when an exon is flanked by 2 transposons andthe whole thing gets transposed

in retrotransposons it happens when the LINE poly(A) signal is too weak, andtranscription continues through another exon (and the whole thing isretrotransposed)transposons are used for gene therapy (to insert a gene) (sleeping beautytransposon)

Organelle DNAsmitochondrial dna (mtDNA) inherited cytoplasmically--as the sperm haslesscytoplasm, most human mitochondria hail from the motherthe mitochondria has its own rRNA, but most of the proteins it needs are importedfrom the cytosol (the mitochondria usually produces only a few subproteins thatare assembled into multimers with imported parts from rIKEA.in animals and protozoa there are few introns (mitochondria need to reproducefrequently and quickly), but in plants there are many (as a result plant mtDNAs can


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Transcriptional Control of Gene ExpressionControl of Gene Expression in Bacteriasigma factors are necessary to activate prokaryotic genes--protein that enablesbinding between RNA polymerase and promoters

lac and trpphosphorylation and small-molecule ligands can regulate promotion and repressionsigma factor in a complex with RNA polymerase can be activated by enhancers farfrom the start site2-component regulatory sequences have 1 sensor protein that transfers agamma-phosphate (in ATP) to the response regulator proteinattenuation (with Trp) :the ribosome follows right behind RNA polymerase to translate directly after

transcription--speed varies directly with on concentrations of tRNA Trp isection 3 of the translated RNA can bind with either section 2 or 4--if section 2 is inthe ribosome (because the ribosome has moved quickly) 3-4 will link, forming ahairpin and stopping transcription, otherwise 2-3 will link and transcriptioncontinues (because the polymerase has continued past)attenuation also occurs through riboswitches --tertiary RNA formations that canbind small molecules and create a termination hairpin when present at sufficientconcentration

Overview of Eukaryotic Gene Controlgene control more permanent, serves whole organismcan analyze gene control regions with reporter genes such as luciferase and GFP3 RNA polymerases in eukaryotes#1 transcribes pre-rRNA #2 transcribes all protein-coding genes & most RNA splicers & siRNAs

#3 transcribes tRNA 5S rRNA and random small RNAs like the signal-recognitionparticle and a RNA splicerall 3 can be distinguished by their net charges (in an ionic solution)plants also have RNApolys 4&5, synthesize siRNAsRNA poly has similar structure throughout all organismsRNA poly 2 has a carboxyl-terminal domain (CTD) , 7 amino acid sequencerepeated min. 10 times--critical for viabilityCTD becomes phosphorylated during transcription


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RNA Polymerase II Promoters and General Transcription Factorstranscription start sites can be found by identifying the DNA sequence under the 5cap in the corresponding mRNATATA boxes are promoters similar to those in E. Coli, ~30 bp upstream of the startsite.

also initiators, much closer to transcription start sitevery few CG sequences because deamination of C turns it to T, so most CGsequences are in:islands of CpG promoters , which initiate transcription on both sides, the nonsenseside stops being transcribed .5-1 kb from the start sitegeneral transcription factors are proteins required for RNApoly to transcribemost placesthey separate dna strands to get the RNApoly to the sequence and form the

preinitiation complex, which is formed by:TFIIB (gen. transcription factor) binds to a TATA box, and bends the DNA to allowtranscription, lets the PolyII bindTFIIH works as a helicase, then most GTFs release and transcription beginsTAF subunits can be ~30 bp downstream, in a downstream promoterelement(DPE)

Regulatory Sequences and Proteinspromoter-proximal elements are upstream elements who must be located closeto the promoter70% of eukaryote genes are promoted by CpG islandsyeast has upstream activating sequences (UASs) which work like enhancers ineukaryotesdna binding-domains of eukaryotic transcription chan have zic finger,

homeodomain, basic helix-loop-helixenhanceosomes are the multiprotein complexes of activators bound to enhancers

Molecular Mechanisms of Transcriptional Repression andActivation


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histone tails can be modified to change relative condensation ofchromatin--changes ability to transcribeactivators/repressors interact with a large protein complex (the mediator) this regulates transcription preinitiation complexesheterochromatin is more condensed, and thus less active, than euchromatin activation and repression domains

Regulation of Transcription-Factor Activitynuclear receptors can regulate transcription factorsresponse elements are where nucleotides bind nuclear receptorsheterodimeric nuclear receptors in the nucleus repress transcription when boundto cognate sites (by directing histone deacetylation), but when the hormone ligandbinds, it starts the preinitiation complexhomodimeric steroid hormone receptors are normally in cytoplasm (trapped by

inhibitor proteins), but when liganded they activate transcriptionexample is heat shock genes: when there is a heat shock, the heat-shocktranscription factor activates, stimulates the polymerase, and brings in morepolymerase to get the reaction done more quickly--before the shock thepolymerase is paused mid transcription for faster responsepolymerase can transcribe at different rates at different times.

Epigenetic Regulation of TranscriptionMethylation and acetylation of histones is the primary manner of epigeneticregulationmethylation of CpG sequences in CpG island promoters in mammals creates bindingsites for methyl-binding proteins that associate with histone deacetylase--inducestranscriptional repressorspolycomb complexes maintain gene repressionOther Eukaryotic Transcription Systems:Pol 1 transcription (in the nucleoleus) is highly regulated to correspond with cellgrowth: ribosomes need to be created when new cells are created...the complex NoRC relocates the Pol1 transcription start site into a nucleosome(then methylates it)Pol3 is unique because it has internal promoter regions-- A box and B box for tRNAC box for 5S rRNAstable RNAs coding sequences have upstream promotersmitochondrial RNA polymerase is encoded in nuclear DNA,mtDNA promoter sequences are A-base rich


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chloroplasts have 2 rna polymerases-- plastid polymerase with multiple subunitslike bacteria (has core encoded in chloroplast still), and a bacteriophage-likepolymerase that encodes some of the bacterial polymerase subunitstranscription regulated by sigma factors responding to light and metabolic stress

Post-Transcriptional Gene Control (look and see if this has itall)

Processing of Eukaryotic Pre-mRNA5 cap and poly(A) tail are used to shield the mRNA from the enzymes breakingdown introns: 5 exoribonucleases digest unprotected rna

mRNAs are always part of heterogeneous ribonucleoprotein (hnRNP) complexes 5 cap made of methylated riboses and guaninecapping enzyme is catalyzed by the phosphorylating of the CTD of Pol II--thisdistinguishes mRNA from r&tRNAepibecause 1 CTD can bind to multiple proteins at the same time, transcription can beefficiently coupled with splicing, + CTD coordinates transcription with processingand ensures that the processing machinery is in the right place for transcription tobegintranscription starts very slowly because of the NELF (negative elongation factor) ,NELF disassociates when the 5cap is put onhnRNP proteins prevent RNA secondary structure formation, aid in splicing andtransport3 end of introns have a lot of PRNA recognition motif (RRM) is the most common RNA-binding domain, highly

conserved across speciesin short transcription units, splicing occurs after cleavage from the template, inlonger ones splicing can start before the 3 end is cappedsplice sites can be found by comparing the template DNA to mRNA cDNAtransesterification reactions switch phosphodiester bonds to splice out introns,usually by pairing a G to an A (introns start with GU and end with AC)(called thebranch point A) in the intron, and cutting the loop out of the mRNArarely the intron starts with AU and ends with AC , this uses 4 rare snRNAs


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splicing uses snRNAs (U1-6, because theyre rich in U) and other proteins assembledon a pre-mRNA to form a spliceosome complex, size= of a ribosomeexon-junction complex then formed, with a RNA export factor (REF) to guide thecomplete mRNA out of the nucleus and enzymes to quality-control and break downbad splicingsome protozoans, and C . elegans, use trans-splicing, where they synthesizetogether pre-mRNAs to form the final mRNAthe exact location of splice sites is determined by SR proteins interacting withexonic splicing enhancers to form a cross-exon recognition complex15% of diseases, including spinal muscular atrophy is caused by poor exondefinition leading to mis-splicingsome introns are self-splicing: group 1(ss) introns are in protozoans, coding fornuclear rRNAs, and group II are organelle dna encoding for all RNAsnRNAs may have evolved from self-splicing RNAs, which could have acceleratedevolution by allowing for creative splicing and facilitating exon shuffling (becausethis frees up intron sequences to be anything without threatening cell viabilityAAUAAA acts as a poly(A) signaler upstream, then G/U rich sequence downstreamsignaling proteins bind to AUUAAA to create the cleavage/polyadenylationcomplex, complex only starts cleavage when poly(A) polymerase (PAP) bonds tothe complex so that the 3 end is capped before degradation startsPAP starts adenylation slowly, but poly(A) binding protein comes in to speed it up

and guide the mRNA through the cytoplasmexonucleases linked in an exosome degrade introns5 cap protected by nuclear cap-binding complex

Regulation of Pre-mRNA Processingproteins previously encoded can regulate gene splicingin drosophila, Sxl prevents splicing, Tra promotes splicingRNA binding sites for protein splicing repressors are called exonic splicingsilencers


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RNA editing post-transcription in organelle dna: short sequences encodedelsewhere are applied to change the exon sequence at certain sites--potential foruse in drugs

pTransport of mRNA Across the Nuclear Envelopenuclear pore complexes are symmetrical structures with copies of nucleoporinproteins, FC-NPCs are semi permeable pores --random coils of amino acids andFG-repeats limit diffusionproteins


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Molecular Biology of the Cell Designer Genes

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