Outline. Gene Expression. One Gene One Enzyme Hypothesis. 1. Central Dogma DNA RNA Protein

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1 ene Expression: RN and Synthesis ene Expression RN synthesis synthesis enetic ode Outline Splicing enes Introns and Exons omparison of ene Expression in Prokaryotes and Eukaryotes ene Expression 1. entral Dogma 2. Evidence for role of DN enes influence enzymes enes specify order of amino acids Fig. 14.3(E rt) Egg UV light destroys nucleus, or it is removed with micropipette. adpole or frog embryo Differentiated cells are isolated from tadpole or frog embryo. Nucleus ontains a Full Set of enetic Material Briggs & King (1952) No growth 1 2 Embryo 3 Differentiated cell nucleus is inserted into enucleate egg. Embryo Nucleus is adpole removed in micropipette. bnormal embryo Occasionally, an adult frog develops. One ene One Enzyme Hypothesis Beadle & atum s Experiments - rginine No growth Results Fig (E rt) enes Specify Sequences of mino cids Normal hemoglobin β chain (Sanger 1953) Valine Histidine cine hreonine Proline lutamic acid lutamic acid Wild-type Mutagenize Neurospora with X-rays + rginine rowth Valine Histidine cine hreonine Proline Sickle cell anemia hemoglobin β chain (Ingram 1956) ENE = Unit of Heredity Valine lutamic acid lutamate Ornithine itrulline rginosuccinate rginine rg Enzymes rg enes E F H rg E rg F rg rg H ENE = Sequence of nucleotides that determines the amino acid sequence of a protein

2 entral Dogma of Molecular Biology DN he enetic ode DN is read in sets of 3 nucleotides for each amino acid. ranscription RN ranslation Different amino acids he enetic ode DN is read in sets of 3 nucleotides for each amino acid. RN Synthesis Occurs on a DN emplate DN U U Same amino acids Newly synthesized RN RN-DN hybrid helix messenger RN makes protein U U U codon codon codon codon enetic ode (messenger RN codons) enetic ode haracteristics 1. riplet ode 2. Degenerate (redundant) 3. Start & Stop odons 4. Universal

3 ene Expression RN Synthesis ranscription = RN synthesis ranscription proceeds through: Initiation: DN unwinds at Promoter site. RN polymerase binds to promoter site. ranscription factors guide RN polymerase. Elongation: ranscription bubble forms. Single-stranded DN used to form new RN using complementary RN nucleotides added in a direction. ermination: RN polymerase stops transcription. DN terminator sequence stops RN synthesis. ene Expression RN Synthesis Initiation of transcription 1. DN Strands DN template strand: strand of DN double helix used to make RN DN coding strand: strand of DN complementary to the template strand 2. Enzyme in RN synthesis RN polymerase: enzyme that synthesizes RN from the DN template 3. Promoter Initial site on DN to attach RN polymerase Fig. 15.8a(E rt) ranscription: DN Promoter Sites are Start sites Initiation DN RN polymerase σ Promoter site oding strand Bacterial DN ranscription in Eukaryotes requires ranscription factors ranscription factor ore promoter Eukaryotic DN Eukaryote Initiation omplex ranscription factors emplate strand 35 sequence 10 sequence Prokaryote RN Polymerase RN polymerase mrn Initiation omplex RN Synthesis: Elongation & ranscription Bubble Elongation: RN nucleotides added RN Synthesis: ermination ermination ranscription stops at termination sequence DN emplate strand Newly synthesized RN U U DN oding strand RN-DN hybrid helix RN polymerase - Region Four or more U ribonucleotides

4 Elements of ranscription DN components RN Nucleotide Structure Upstream (-) Downstream (+) ore Promoter DN recognition & RN polymerase binding site Start Site First odon ranscription Unit odons ranslated erminator Signal to End ranscription Initiation Elongation ermination Nitrogen Base 1. RN sugar is RIBOSE 2. Uracil is unique to RN 3. Polymer is single-stranded omparison of ranscription in Prokaryotes & Eukaryotes Prokaryotes Eukaryotes RN polymerase One hree Promoter site + + Initiation or One Many transcription factors (RN polymerase holoenzyme) (bind to multiple sites on DN) mrn ranscription unit & amino acid sequence ermination of transcription olinear - hairpin & weak U- pairing Not colinear Introns & Exons Require cutting & splicing Not well-defined mrn transcript Not processed Processed cap & poly- tail added Mature RN transcript oupled to translation ransferred to cytoplasm through nuclear pores ene Expression ypes of RN ene expression requires multiple types of RN messenger RN (mrn) encodes proteins ribosomal RN (rrn) a structural component of the ribosome transfer RN (trn) carries amino acids to the ribosome 22 aminoacyl-trn synthetase ransfer RN Structure & Function trn red & yellow P (green) Enzyme (blue) aminoacyltrn synthetase nticodon loop t-rn cceptor arm amino acid attaches here nticodon loop ene Expression - ranslation ranslation = protein synthesis Steps: Initiation mrn, trn, and ribosome come together Elongation trns bring amino acids to the ribosome for incorporation into the elongating polypeptide ermination ribosome encounters a stop codon and releases polypeptide 24

5 ene Expression ranslation Ribosome Structure ene Expression Initiation of ranslation Initiator trn mrn Initiation Elongation ermination Ribosome large Ribosome small functional Ribosome Fig. 15.2b(E rt) Ribosome Ribosome Structure & Function Peptide Bond Formation t RN binding sites Initiation Elongation ermination E = Exit site P = Peptide site = mino acid site MINO Large ribosomal Small ribosomal E P Ribosome functions: (1) decode the mrn (2) form peptide bonds mrn binding site RBOXYL E NIODON ODON P t RN t RN Fig d(E rt) Synthesis: Initiation Initiation Elongation ermination Fig a(E rt) Synthesis: Elongation E site = EXI P site = Peptide Site = mino acid trn site P site (occupied) trn Elongation factor NIODON ODON U U E site site U U U U mrn

6 Fig b(E rt) Fig c(E rt) Synthesis: Elongation Synthesis: ranslocation Peptide Bond Forms U U U U U U U U Fig d(E rt) Synthesis: ranslocation Fig e(E rt) Synthesis: Elongation & ranslocation 5 P site ytoplasm E site site trn U U U U trns bring their amino acids in at the site on the ribosome. Peptide bonds form between amino acids at the P site, and trns exit the ribosome from the E site. Fig b(E rt) Synthesis: ermination ene Expression Synthesis Val Ser la rp Polypeptide chain released ranslation consists of Initiation mrn, trn, and ribosome come together trn Elongation trns bring amino acids to the ribosome for incorporation into the elongating polypeptide. Release Factor ermination ribosome encounters a stop codon and releases polypeptide U U SOP ODON 36

7 RN & Synthesis in Prokaryotes Eukaryotic pre-mrn Splicing In eukaryotes, the final mrn transcript is not colinear with DN. In eukaryotes, the primary transcript must be modified by: addition of a cap addition of a poly- tail removal of non-coding sequences (introns) Fig a(E rt) Exon coding region mrn is processed in Eukaryotic ells Intron noncoding region Eukaryotic RN may be spliced in more than one way lternative splicing may generate two or more types of mrn from the same transcript Exons DN Primary RN transcript cap ranscription Introns are cut out and coding regions are spliced together poly- tail DN 1 o RN transcript mrn or Mature mrn transcript opyright 2005 Pearson Education, Inc. Publishing as Benjamin ummings Fig (E rt) RN & Synthesis - Prokaryotes and Eukaryotes Prokaryote chromosome Eukaryotic hromosome ranscription ranslation mrn DN Primary Intron ranscription RN transcript Processing mature mrn ap Poly- tail ranslation END ENE EXPRESSION END ene Expression