Download Chapter 17: Gene Expression Gene Expression DNA houses all

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