From Gene Protein Part 1 Transcription
Lecture 16 and 17; May 26th and 31stFrom Gene Protein – Part 1Transcription
From Gene to ProteinReplicationDNACentral DogmaRNATranscriptionPROTEINTranslation
From Gene to Protein: Key Experiment!1940s Beadle and Tatum: One Gene – One Enzyme What is the relationship between a gene anda protein? Combined Biochemistry and Genetics Used Neurospora crassa (bread mold) N. crassa can live on a minimal foodsource Minimal medium (food source) Inorganic salts Glucose VitaminsNeurospora crassa (SEM)Bombarded Neurospora with X-rays and then looked for mutants thatdiffered in nutritional needs!
From Gene to Protein: Key Experiment!1940s Beadle and Tatum: One Gene – One EnzymeX-rays is a mutagen that causes breaks in the phosphodiester bondsof DNA which results in mutations in the DNA.
From Gene to Protein: Key Experiment!1940s Beadle and Tatum: One Gene – One Enzyme Normal (wild type) neurospora can live on minimal growth medium Prototrophs Neurospora mutants need complete growth medium supplemented Auxotrophwith all 20 amino acidsCharacterized their Arginine auxotrophs can’t live without argininesupplementationIdentified 3 classes of mutants: Class I, II, and IIIX-raysClass IClass IIWild TypeClass IIIArginine auxotrophs
From Gene to Protein: Key Experiment!1940s Beadle and Tatum: One Gene – One EnzymeArginine auxotrophs 3 classes of mutants: Class I, II, and IIIArginine Biosynthesis: Ornithine and citrulline are precursorsWild typeMinimalmedium(MM)(control)MM OrnithineMM CitrullineMM Arginine(control)Class IMutantsClass IIMutantsClass IIIMutants
From Gene to Protein: Key Experiment!1940s Beadle and Tatum: One Gene – One EnzymeArginine auxotrophs 3 classes of mutants: Class I, II, and IIIGene AWild typeClass IMutants(mutationin gene neArginineArginineEnzymeAOrnithineGene BEnzymeBCitrullineGene CEnzymeCArginineClass IIMutants(mutationin gene B)Class IIIMutants(mutationin gene C)PrecursorA
From Gene to Protein: Key Experiment!1940s Beadle and Tatum: One Gene – One EnzymeArginine auxotrophs 3 classes of mutants: Class I, II, and IIIPrecursorEnzyme AOrnithineEnzyme BCitrullineEnzyme CArginineKey! Different Genes involved in Arginine biosynthesis!One Gene One Enzyme HypothesisOne Protein
From Gene to ProteinCentral DogmaRNADNATranscriptionPROTEINTranslation TranscriptionIs the synthesis of RNA under the direction of DNAProduces messenger RNA (mRNA) TranslationIs the actual synthesis of a polypeptide, which occurs under thedirection of mRNAOccurs on ribosomes
From Gene to ProteinIn prokaryotes:transcription and translation occur togetherTRANSCRIPTIONIn eukaryotes:transcription occurs in nucleustranslation occur in cytoplasm(RNA transcripts are PTIONTRANSLATIONPolypeptidePre-mRNARNA PROCESSINGmRNARibosomeTRANSLATIONPolypeptide
From Gene to Protein Lyse a cell Remove its nuclei Collect the cytoplasm Protein Synthesis(in vitro)Add a Protease No ProteinsAdd DNase Protein synthesis intactAdd RNase Protein synthesis stoppedMessage RNA was found in the 1950’sDNA RNA PROTEIN
From Gene to ProteinDilemma: How does a RNA direct the synthesis of a protein?Francis Crick: Adaptor HypothesisProposed that there are 20 adaptor molecules (one for each amino acids)Together with a set of proteins that directs the synthesis of proteinstRNA transfer RNAcan base pair (H-bonds) with mRNAattached to a specific amino acidsRibosomes
From Gene to ProteinCracking the Genetic CodeHow does 4 nucleotides code for 20 different amino acids?1 nucleotide (41) only 4 amino acids2 nucleotides (42) only 16 amino acids3 nucleotides (43) 64 different amino acidsA triplet code is the smallest unit that can code for an amino acid!
From Gene to ProteinCracking the Genetic Code1961 Niremberg deciphered the first codon (triplet DNA sequence)In a test tube (in vitro) added:cytoplasmic extractribosomesother components (tRNA and other proteins)amino acidspolypeptideArtificial mRNA:5’-UUUUUUUUU-3’ PHE PHE PHE PRO PRO PRO5’-CCCCCCCCC-3’5’-AAAAAAAAA-3’ LYS LYS LYS
From Gene to Protein Genetic Code Codon: 3 bases long (1 codon 1 amino acid) 64 codons total 61 code for amino acids More than 1 codon can encode the same amino acid 3 STOP codons: UAG, UGA, UAA Signals the ribosome to stop and release the protein!! 1 Start codon: AUG encodes Methionine (MET)(every preotin starts with MET!)
From Gene to Protein Open Reading Frames (ORF)ORF 1ACCGCCGACUUUTHRALAASPPHEORF 2PROPROTHR?ORF 3ARGARGLEU? Every mRNA has 3 ORF Key! Find the start codon and you will be in the right reading frame Ribosomes read from 5’ 3’
Genetic CodeRemember all T’s are replaced with U’s in RNA
From Gene to Protein: Transcription Transcription is the DNA directed synthesis of RNATranscriptional UNIT5’-TATAA-3’(TATAA box Sequence- can direct synthesis from either strand)1st base of mRNAPromoterPTerminatorStopStartTCoding Region5’ UTR (untranslated region)Important for ribosome binding3’ UTR (untranslated region)Important for RNA functionand stabilityNote: Gene can be very big relative to the actual coding region for a given protein
From Gene to Protein: TranscriptionTranscription Basics1. Initiation Promoter: DNA sequence that directs that start of mRNA synthesis RNA polymerase recognizes promoter and unwinds DNA Promoter chooses the orientation of the gene Directs RNA polymerase to the right DNA strand (template) Includes the 1st base of the mRNA2. Elongation RNA Polymerase synthesizes mRNA in the (5’ 3’ direction)3. Termination Termination sequence: stretch of DNA recognized by RNApolymerase, which tells it to stop and release the mRNA
From Gene to Protein: TranscriptionTranscription Basics: RNA synthesis2Pi5’PPi3’OHP-P-P-OBASE (A,G,C,and U)RNAHOOH2’OH Not reactive3’OH reactiveRNA polymerase – driven by PPi 2Pi (14 kCal – exergonic!)Prokaryotes:1 RNA polymerase makes mRNA, tRNA, and rRNAEukaryotes:RNA Pol I rRNA (ribosomal)“ II mRNA (message)“ III tRNA (transfer), and snRNA (small nuclear)
From Gene to Protein: TranscriptionPromoter5 3 n unitStart pointRNA polymerase3 5 DNA1Initiation.5 3 UnwoundDNATemplate strand ofDNAtranscript2Elongation.3 5 RNARewound5 3 RNA5 RNAtranscript5 3 Figure 17.75 3 5 3 3Completed RNAtranscriptTermination.3 3 5
ElongationNon-templatestrand of DNARNA nucleotidesRNApolymeraseA3 TCCAA3 endU5 AUCCATAGGTTDirection of transcription(“downstream”)5 Newly madeRNATemplatestrand of DNA
From Gene to Protein: UUUCCGCCGACUCoding region(triplet code for a.a.)3’UTR
From Gene to Protein: TranscriptionRNA PolymeraseUnwinds 10-20 bp at a timeMoves in the 3’ 5’ directionSynthesizes mRNA 5’ 3’DNA is a template: chooses next base by base-paringDNA T ARNA A UFidelityRNA polymerase can not proof read (check for mistakes)Codon variation helps to solve this problem 1 codon 1 amino acidsError rate: 1/10,000 basesmRNA is not permanent!!
From Gene to Protein: Transcriptionmature mRNA50 to 250 adenine nucleotidesadded to the 3 endA modified guanine nucleotideadded to the 5 end5 GPP5 CapProtein-coding segmentPPolyadenylation signalAAUAAA5 UTRStart codonStop codon3 3 UTRAAA AAAPoly-A tail5’Cap : modified Guanine - helps ribosome (in translation) find the 5’END3’ polyAAA tail - export out of nucleusBoth 5’Cap and 3’ polyAAA tail – helps stabilize RNA in cytoplasm!!
From Gene to Protein: TranscriptionModification of the pre-mRNAEukaryotes:pre-mRNARNA processingmature mRNASPLICINGExons: protein coding region of a geneIntrons: intervening sequence which does not code for a proteinSplicing: cut and religate mRNA in order to remove intronsTRANSCRIPTIONDNARNA PROCESSINGPre-mRNA5 ExonIntronPre-mRNA 5 1053 146Poly-A tailIntrons cut out andexons spliced togetherTRANSLATIONPolypeptidemRNA5 Cap13 UTRPoly-A tail1463 UTR
From Gene to Protein: TranscriptionModification of the pre-mRNAALL take place in nucleus5’Cap, 3’polyAAA tail, splicing of RNA is required to exit nucleusSpliceosome: splices out introns to produce mature mRNAComposed of protein and RNAsnRNA’s (small nuclear ribonucleoproteins)base pair with intro/exon junctioncut and rejoin RNAKEY! Spliceosome has to recognize intron/exon boundaries!!!15% of all Inherited diseases involve splicing defects!!
From Gene to Protein: TranscriptionSpliceosome5 1RNA transcript (pre-mRNA)IntronExon 1ProteinExon 2Other proteinssnRNAsnRNPsSpliceosome25 Spliceosomecomponents35 mRNAExon 1Exon 2Cut-outintron
From Gene to Protein: TranscriptionSplicing in Eukaryotes (Prokaryotes do not modify their RNA!)1. 1 gene can encode 1 proteinpre-mRNA 5’Cap12435AAAAAAAAAAAAAlternative Splicingmature Different tissues, different times in development, etc.2. Bigger genes: more opportunity for recombination and thus diversity!3. Introns can regulate gene expression
From Gene Protein – Part 2Translation
From Gene to Protein: TranslationProkaryotesHow does the ribosome find mRNA?AUGMET (start codon)TPRibosome binding site (RBS)Eukaryotes don’t have this! Use the 5’Cap insteadBoth scan 5’ 3’ direction to find start codon
From Gene to Protein: TranslationTransfer RNA (tRNA)METAntiparallel basepairng w/ itselfHairpin loopsAnti-codon loop that base pairs w/3 AAmino acidCattachment site CA 5 C GG CC GU GU AA UA UU CUAC A C AG*G*G U G U *CC* *U C** G AG CG CU AmRNA codon* GAA*CU*UCAC U CG A GA G **GA G GHydrogenbondsAnticodon3’-UAC-5’5’ .AUG .AAAA 3’
From Gene to Protein: TranslationAttachment of amino acid to tRNA: action of aminoacyl-tRNA synthase61 codons but only 45 tRNAsWobble Theoryrelaxation of basepairing �CUC’3’Leucine
Action of Aminoacyl-tRNA synthase!Aminoacyl-tRNA synthaseBinds ATP and a.a.Adenosine-P-P-P loses 2Piand is attached to a.a.(14 kCal of eneergy)exergonicActivated tRNA-amino acidmolecule is releasedCorrect tRNA is bound and a.a.is transferred to tRNA.Adenosine-monphosphate isreleased
From Gene to Protein: TranslationTranslation RULES-mature mRNA-5’Cap or RBS (prokaryotes)-Ribosome scans 5’ to 3’ looking for AUG (MET) start codon-syntheses of protein in : N-term to C-term-stop codon stops synthesisSense strand (encodes protein)DNA5’ .TC ATG GAC CAT TGA G .3’3’ .AG TAC CTG GTA ACT C .5’Template strand for mRNA (anti-sense)mRNA 5’ .UC AUG GAC CAU UGA G .3’Stop codonN-MET-ASP-HIS-CLinked by peptide bond
From Gene to Protein: TranslationRibosomessmall – 30Slarge – 50SMany proteins plus ribosomal RNA (rRNA)assembled in nucleolusA site: amino acid addition siteP site: Protein site (growing protein located here)E site : exit site for tRNAlargeEP AsmalltRNA base pairs with the mRNA in the A and P site!mRNAbinding site
From Gene to Protein: TranslationStages of TranslationInitiationsmall subunit recognizes mRNA: 5’Cap (EUK) or RBS (PROK)scan 5’to3’ for the AUG start codonInitiator tRNAMET base pair with start codon on mRNAlarge subunit joinsother protein factors needed: “initiation factors”GTP as energy sourcetRNAMET now in the P siteElongationgrowing peptide starts in the P siteCodon recognitionthe right tRNAa.a. brought into A siteelongation factors (GTP used) neededFormation of peptide BONDProtein now in the A siteP site has empty tRNATranslocationRibosome shifts 5’ to 3’
From Gene to Protein: TranslationInitiation
From Gene to Protein: TranslationElongation
From Gene to Protein: TranslationStages of Translation – Con’tTerminationProtein in the P siteA site has a stop codonRELEASE factor recruitedbreaks bond between peptide and tRNAstop (hydrolysis)protein is freed from ribosometRNA exits from the E siteRibosome disassembles into large and small subunitsReleasefactorFreepolypeptide5 3 3 5 5 Stop codon(UAG, UAA, or UGA)3
From Gene to Protein: TranslationPolysomesmultiple ribosomes translating protein on the same mRNAall go 5’ to 3’ directiona way for 1 mRNA to make many proteins
From Gene to Protein: TranslationRibosomes on the Rough ER: translation of a secreted or integralmembrane protein!initiate translation the same1st 20 amino acids that exits ribosome is a signal peptiderecognized by signal-recognition particle (SRP)SRP docks ribosome onto the ER “translocation complex”SRP released and growing protein deposited into ER by ribosomeIn RER – signal peptide cleaved by proteaseElongation continuesFolding and glycosylation in cle(SRP) SRPreceptorCYTOSOL proteinERLUMENFigure braneProtein
From Gene to Protein: Transcription pre-mRNA mature mRNA RNA processing SPLICING Exons: protein coding region of a gene Introns: intervening sequence which does not code for a protein Splicing: cut and religate mRNA in order to remove introns TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA
One Gene-One Enzyme Hypothesis (Beadle & Tatum) The function of a gene is to dictate the production of a specific enzyme One Gene—One Enzyme but not all proteins are enzymes those proteins are coded by genes too One Gene—One Protein but many proteins are composed of several polypeptides, each of which has its own gene One Gene—One Polypeptide
44, 45, 61, and 62. The gene 43 protein is a DNA polymerase (1); the gene 32 protein is a DNA helix-destabilizing (2); and the gene 41 protein and the gene 61 protein act together to make short RNA primers (3, 4). Of special rele- vance to this communication, the protein products of genes 44
protein:ligand, K eq [protein:ligand] [protein][ligand] (1) can be restated as, K eq 1 [ligand] p 1 p 0 (2) where p 0 is the fraction of free protein and p 1, the fraction of protein binding the ligand. Assuming low protein concentration, one can imagine an isolated protein in a solution of Nindistinguishable ligands. Under these premises .
The N protein is (circle one) a repressor. an activator. (c) In further study, you discover that transcription of the N gene is controlled by protein A (protein A is the product of gene A shown in the diagram), and theose binds to the N protein. You examine the transcription of genes N, X, Y, and Z in cells where gene A is
Process Development 1. Identification of target protein w/therapeutic value 2. Identification of target gene 3. Isolation of the target gene 4. Insertion of the target gene into a host cell (such as E.coli) & express protein 5. Purification: Separation of the target protein from the host cell protein
biological significance of protein complexation with RNA has been well recognized, the specific mecha-nism of protein–RNA interaction is not fully understood [10]. Measurement of sequence–specific DNA– protein and RNA–protein interactions is a key experimental procedure in molecular biology of gene regulation.
21], protein-protein interactions [22, 23], and as antigens for vaccine studies [24]. 2.1.1.1.2 Maltose-binding protein Maltose-binding protein (MBP) is a 42 kDa protein encoded by the malE gene of Escherichia coliK12 [25]. MBP fusion proteins have been utilized for single-step purification by affinity to cross-linked amylose [26].
Araling Panlipunan Grade 10 . Alternative Delivery Mode . Ikalawang Markahan- Modyul 3: Mga Dahilan at Epekto ng Migrasyon . Unang Edisyon, 2020 . Isinasaad ng Batas Republika 8293, Seksiyon 176na “Hindi maaaring magkaroon ng karapatang- sipi sa anomang akda ang Pamahalaan ng Pilipinas. Gayon pa man, kailangan muna ang pahintulot ng ahensiya o tanggapan ng pamahalaan na naghanda ng akda kung .
