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Chapter 17: Gene Expression Gene Expression DNA houses all genetic information Proteins are bridge between genotype & phenotype Two parts o Transcription – DNA to RNA o Translation – RNA to Protein Archibald Garrod (1909) Inborn Errors of Metabolism o Genetic disorders caused by errors in metabolic pathways Caused by faulty enzymes o Mendel’s discrete factors cause faulty enzyme o One gene – One enzyme Beadle & Tatum (1928) Exposed Neurospora (bread mold) to x-rays creating mutants o Wild type grow on minimal medium (Inorganic salts, sucrose, biotin) o Mutant’s deficiency found by growing on minimal plus one amino acid (no growth = biosynthesis) Found 3 mutants in arginine pathway o Each had different faulty enzyme in pathway o Supported Garrod’s Hypothesis Evolving Hypothesis One Gene – One Protein o Not all proteins are enzymes Keratin, insulin One Gene – One Polypeptide o Not all proteins a single polypeptide Hemoglobin – 2 different subunits (only one subunit bad in Sickle Cell) One Gene – One polypeptide or RNA o All RNAs come from genes too Transcription Overview DNA too large to function in cytoplasm for translation mRNA (messenger RNA) transcribed as the go between RNA differences o Ribose sugar (-vs- deoxyribose) o Uracil (instead of Thymine) o Single-stranded & one gene RNA Polymerase o Opens DNA o Builds RNA strand on 3’ 5’ template (like leading strand) o RNA Pol only adds to 3’ end, so RNA built in 5’ 3’ direction Base pairs = G-C & A-U The Genetic Code o Must code for 20 amino acids o Codon If each base a ‘letter’, how long word? 1 letter = 4 words 2 letters = 16 words 3 letters = 64 words 61 codons for AAs, 3 for ‘stop’ o Uniform Across Species Transcription Process Prokaryotes have one RNA Polymerase that transcribes all RNA Eukaryotes have three RNA Polymerases in nucleus o RNA Pol II synthesizes mRNA Polymerases bind ‘upstream’ of transcription unit (gene to be read) Initiation o Promoter o Specific DNA nucleotide sequence marking where transcription begins o Upstream of transcription unit (start point) o TATA box in eukaryotes o Transcription factors (protein) bind to promoter region, allow RNA Pol II to bind o Transcription Initiation Complex formed Elongation o RNA Pol II untwists 10-20 nucleotides at a time o Builds 40 nucleotides per second o Bases always added to 3’ end o A-U & G-C o Multiple RNA Pol II can transcribe gene simultaneously Termination o Prokaryotes – end at terminator sequence o Eukaryotes o RNA Pol hits polyadenylation sequence (AAUAAA) o 10-35 nucleotides downstream, pre-mRNA cut & released o RNA Pol II keeps going for a bit RNA Processing o pre-mRNA altered before leaving o 5’ cap on 5’ end when transcription starts Modified guanine added Helps protect from hydrolytic enzymes ‘Attach here’ signal for ribosome o Poly-A Tail on 3’ end right after released 50-250 adenines added Facilitates export from nucleus o RNA Splicing Introns (interrupt transcript) – long regions of noncoding RNA segments Exons (expressed transcript) – RNA that will be expressed by translation Spliceosome – cut introns, splice exons Large protein plus… snRNP (aka ‘Snurps’) Small nuclear ribonucleoproteins 150 nucleotides (snRNA) + protein Recognize intron & signal cutting Splicing Importance Some can control gene activity Can regulate export to cytoplasm Alternative RNA splicing o Changing introns/exons during splicing can yield different proteins o One gene = multiple products o About 20k genes & 175-200k proteins Different exons often different domains Prokaryote mRNA translated immediately Translation Overview Once mRNA codon produced, must be turned into amino acids (polypeptide) Ribosomal complex (rRNA, mRNA, tRNA) build polypetides o Free Ribosomes in cytosol o Bound Ribosomes on rough ER o Why different locations? tRNA (Transfer RNA) o 80 nucleotide RNA strand Complimentary regions allow it to H-bond & fold over on itself o Anti-codon – one end has 3 nucleotide segment to match mRNA codon (61) o 3’ end extends off other end & will bind an amino acid o 45-50 human tRNA (vs. 61 AA codons) due to ‘wobble’ o Aminoacyl-tRNA Synthetase Enzyme that catalyzes attachment of amino acid to tRNA 20 different synthetases Each can recognize all tRNAs for its amino acid Costs 1 ATP to attach amino acid Aminoacyl-tRNA or charged tRNA rRNA (Ribosomal RNA) o Synthesized in nucleolus o Component of ribosomal subunit Performs most of activity Protein is 1/3rd mass & largely structural o Eukaryotic subunits Small (40S) = 1 rRNA + 33 proteins Large (60S) = 3 rRNA + 50 proteins Ribosome o 80S complex of small/large subunits o mRNA binding site o 3 tRNA binding sites A site – for aminoacyl-tRNA or charged tRNA to bind P site – for peptidyl-tRNA Has growing polypeptide attached E site – tRNA exits Translation Process o 3 stages – Initiation, Elongation, & Termination All need protein factors to help o Initiation & Elongation require GTP energy (like ATP) Guanosine Triphosphate o Initiation Each step aided by initiation factors (IF) Small subunit binds tRNA-Methionine Then finds 5’ cap & binds in UTR upstream of start codon (AUG) mRNA scanned for first AUG, once found, codon & anticodon bind, creating P site IFs released & GTP energy attaches large subunit o Elongation Codon Recognition Anticodon of charged tRNA matches mRNA codon for next AA Elongation factor aids in H bonding b/w tRNA & mRNA costing 2 GTP Always read from 5’ 3’ First codons transcribed first Peptide Bond Formation rRNA catalyzes bonding b/w polypeptide in P site & amino acid in A site Growing polypeptide to A site tRNA Translocation Ribosome moves down 1 codon (1 GTP) o A site moves to P site w/ growing polypeptide o P site moves to E site & exits o A site open again o Termination Begins when stop codon enters A site 3 that don’t code for AAs Releasing factor protein binds to stop codon in A site Catalyzes hydrolysis of bond b/w polypeptide chain & tRNA Both released from ribosome 2 GTP break down rest of complex o Polyribosomes Multiple ribosomes perform translation simultaneously o Polypeptide Completion Protein folding can start during translation Spontaneously occurs sometimes Others helped by chaperonins Posttranslational Modification Additions of other molecules (lipid, sugar, phosphate, etc.) Parts cleaved off or can be split into two polypeptides (e.g. insulin) 2+ polypeptides joined (quaternary fold) o Polypeptide Targeting All ribosomes (identical) start in cytosol Developing protein can signal ribosome to bind to Rough ER Signal peptide (series of AAs) read by a signal recognition particle (SRP) SRP moves ribosome to a receptor on Rough ER o Peptide moves into ER as its made Other SRPs can signal final destination Gene Mutations Replication or mutagen 3 General categories o Substitution – base changed to another o Deletion – base lost in DNA o Addition – base added to DNA Point Mutations o aka Base-pair mutation o Alters one base o In protein, is called… Silent – no AA change Missense – one AA change (good, bad, or no change) Nonsense – change created a stop codon ending translation Frameshift Mutations o Results from insertion or deletion 1-2 changes shifts reading frame (very bad) All downstream codons affected (AAs all wrong) Can cause missense or nonsense 3 changes will add/remove 1 AA