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Chapter 14 From Gene to Protein Metabolism Teaches Us About Genes Metabolic defects studying metabolic diseases suggested that genes specified proteins A alkaptonuria (black urine from alkapton a.k.a. homogentisic acid ) PKU (phenylketonuria) Genes each disease is caused by non-functional enzyme B C create phenotype… D E 1 Gene – 1 Enzyme Hypothesis Beadle & Tatum Compared mutants of bread mold, Neurospora fungus created mutations by X-ray treatments X-rays break DNA inactivate a gene wild type grows on “minimal” media sugars + required precursor nutrient to synthesize essential amino acids mutants require added amino acids each type of mutant lacks a certain enzyme needed to produce a certain amino acid non-functional enzyme = broken gene 1941 | 1958 Beadle & Tatum George Beadle Edward Tatum Beadle & Tatum’s Neurospora Experiment So… What is a Gene? One gene – one enzyme One gene – one protein each protein has its own gene but many proteins are composed of several polypeptides One gene – one polypeptide all proteins are coded by genes but not all proteins are enzymes but many genes only code for RNA One gene – one product but many genes code for more than one product … Where does that leave us?! Defining a Gene… “Defining a gene is problematic because… one gene can code for several protein products, some genes code only for RNA, two genes can overlap, and there are many other complications.” – Elizabeth Pennisi, Science 2003 gene RNA polypeptide 1 gene polypeptide 2 polypeptide 3 It’s hard to hunt for wabbits, if you don’t know what a wabbit looks like. The “Central Dogma” How do we move information from DNA to proteins? transcription DNA replication translation RNA protein For simplicity sake, let’s go back to genes that code for proteins… From nucleus to cytoplasm… Where are the genes? Where are proteins synthesized? genes are on chromosomes in nucleus proteins made in cytoplasm by ribosomes How does the information get from nucleus to cytoplasm? messenger RNA nucleus RNA ribose sugar N-bases uracil instead of thymine U : A C : G single stranded mRNA, rRNA, tRNA, siRNA…. transcription DNA RNA Transcription Transcribed DNA strand = template strand Synthesis of complementary RNA strand untranscribed DNA strand = coding strand transcription bubble Enzyme that facilitates the building of RNA: RNA polymerase Transcription Initiation RNA polymerase binds to promoter sequence on DNA Role of promoter 1. Where to start reading = starting point 2. Which strand to read = template strand 3. Direction on DNA = always reads DNA 3'5' Transcription Elongation RNA polymerase unwinds DNA ~20 base pairs at a time reads DNA 3’5’ builds RNA 5’3’ (the enzyme governs the synthesis!) No proofreading 1 error/105 bases many copies short life not worth it! Transcription RNA Transcription Termination RNA polymerase stops at termination sequence mRNA leaves nucleus through pores RNA GC hairpin turn Prokaryote vs. Eukaryote Genetics Differences between prokaryotes & eukaryotes time & physical separation between processes RNA processing Transcription in Eukaryotes only 1 prokaryotic enzyme 3 eukaryotic RNA polymerase enzymes RNA polymerase I RNA polymerase I I transcribes genes into mRNA RNA polymerase I I I only transcribes rRNA genes only transcribes rRNA genes each has a specific promoter sequence it recognizes Eukaryotic Post-transcriptional Processing Primary transcript eukaryotic mRNA needs work after transcription Protect mRNA from RNA-ase enzymes in cytoplasm add 5' G cap mRNA 5' cap add polyA tail 5' G PPP CH 3' A 3 Edit out introns intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequence primary mRNA transcript mature mRNA transcript pre-mRNA spliced mRNA Primary Transcript Processing mRNA protecting RNA from RNase in cytoplasm add 5’ cap add polyA tail remove introns AUG UGA Protecting RNA 5’ cap added G trinucleoside (G-P-P-P) protects mRNA from RNase (hydrolytic enzymes) 3’ poly-A tail added 50-250 A’s protects mRNA from RNase (hydrolytic enzymes) helps export of RNA from nucleus UTR UTR Dicing & Splicing mRNA Pre-mRNA mRNA edit out introns splice together exons intervening sequences expressed sequences “AVERAGE” “gene” = 8000b pre-mRNA = 8000b mature mRNA = 1200b protein = 400aa lotsa “JUNK”! In higher eukaryotes 90% or more of gene can be intron no one knows why…yet there’s a Nobel prize waiting… Splicing Enzymes snRNPs small nuclear RNA RNA + proteins Spliceosome several snRNPs recognize splice site sequence cut & paste RNA as ribozyme some mRNA can splice itself RNA as enzyme 1982 | 1989 Ribozyme RNA as enzyme Sidney Altman Yale Thomas Cech U of Colorado Splicing Details No room for mistakes! editing & splicing must be exactly accurate a single base added or lost throws off the reading frame AUGCGGCUAUGGGUCCGAUAAGGGCCAU AUGCGGUCCGAUAAGGGCCAU AUG|CGG|UCC|GAU|AAG|GGC|CAU Met | Arg | Ser | Asp | Lys | Gly | His AUGCGGCUAUGGGUCCGAUAAGGGCCAU AUGCGGGUCCGAUAAGGGCCAU AUG|CGG|GUC|CGA|UAA|GGG|CCA|U Met | Arg | Val | Arg |STOP| Alternative Splicing Alternative mRNAs produced from same gene when is an intron not an intron… different segments treated as exons Hard to define a gene! Discovery of Split Genes Richard Roberts NE BioLabs Philip Sharp MIT 1977 | 1993 adenovirus common cold Structure of Antibodies Y Y Y Y s s s light chain B cell membrane s s s s s s s s s s s s s s s s s s s s s s Y s Y s s s Y s s Y s variable region s Y s s Y s Y Y antigen-binding site light chain heavy chains light chains antigen-binding site heavy chains antigen-binding site How do vertebrates produce millions of antibody proteins, if they only have a few hundred genes coding for those proteins? By DNA rearrangement & somatic mutation vertebrates can produce millions of B & T cells antibody mRNA DNA of differentiated B cell C chromosome of undifferentiated B cell rearrangement of DNA V D C J B cell The Transcriptional Unit (gene?) enhancer 1000+b 20-30b 3' RNA TATA polymerase translation start TAC translation stop exons transcriptional unit 5' DNA ACT DNA UTR promoter UTR introns transcription start transcription stop 5' pre-mRNA 5' GTP mature mRNA 3' 3' AAAAAAAA Prokaryote vs. Eukaryote Genetics Prokaryotes DNA in cytoplasm circular chromosome naked DNA no introns Eukaryotes DNA in nucleus linear chromosomes DNA wound on histone proteins introns vs. exons intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequence From Gene to Protein aa aa aa transcription DNA translation aa aa mRNA protein aa aa aa mRNA leaves nucleus through nuclear pores nucleus ribosome cytoplasm proteins synthesized by ribosomes using instructions on mRNA Translation in Prokaryotes Transcription & translation are simultaneous in bacteria DNA is in cytoplasm no mRNA editing needed How Does DNA Code for Proteins DNA TACGCACATTTACGTACGCGG mRNA AUGCGUGUAAAUGCAUGCGCC ? protein Met Arg Val Asn Ala Cys Ala How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)? Cracking the Code 1960 | 1968 Nirenberg & Matthaei determined 1st codon–amino acid match UUU coded for phenylalanine created artificial poly(U) mRNA added mRNA to test tube of ribosomes, tRNA & amino acids mRNA synthesized single amino acid polypeptide chain phe–phe–phe–phe–phe–phe Heinrich Matthaei Marshall Nirenberg Translation Codons blocks of 3 nucleotides decoded into the sequence of amino acids mRNA Codes for Proteins in Triplets TACGCACATTTACGTACGCGG DNA codon mRNA AUGCGUGUAAAUGCAUGCGCC ? protein Met Arg Val Asn Ala Cys Ala mRNA codes for proteins in triplets… CODONS! The Code! For ALL life! strongest support for a common origin for all life Code is redundant several codons for each amino acid Why is this a good thing? Start codon AUG methionine Stop codons UGA, UAA, UAG How are Codons Matched to Amino Acids? 3' DNA 5' TACGCACATTTACGTACGCGG 5' mRNA 3' AUGCGUGUAAAUGCAUGCGCC UAC GCA 3' 5' tRNA amino acid Met Arg codon CAU Val anti-codon aa aa aa cytoplasm transcription translation aa aa aa aa aa protein aa aa aa nucleus tRNA Structure “Clover leaf” structure anticodon on “clover leaf” end amino acid attached on 3' end Loading tRNA Aminoacyl tRNA synthetase enzyme which bonds amino acid to tRNA endergonic reaction ATP AMP energy stored in tRNA-amino acid bond unstable so it can release amino acid at ribosome Ribosomes Facilitate coupling of tRNA anticodon to mRNA codon organelle or enzyme? Structure ribosomal RNA (rRNA) & proteins 2 subunits large small Ribosomes P site (peptidyl-tRNA site) A site (aminoacyl-tRNA site) holds tRNA carrying growing polypeptide chain holds tRNA carrying next amino acid to be added to chain E site (exit site) empty tRNA leaves ribosome from exit site Building a Polypeptide Initiation brings together mRNA, ribosome subunits, proteins & initiator tRNA Elongation Termination Elongation: Growing a Polypeptide Termination: Release Polypeptide Release factor “release protein” bonds to A site bonds water molecule to polypeptide chain Now what happens to the polypeptide? Protein Targeting Signal peptide address label Destinations: ex. start of a secretory pathway secretion nucleus mitochondria chloroplasts cell membrane cytoplasm RNA polymerase DNA Can you tell the eukaryotic story? amino acids exon intron pre-mRNA tRNA 5' cap mature mRNA aminoacyl tRNA synthetase polyA tail large subunit 3' polypeptide ribosome 5' tRNA small subunit E P A Putting it all together… Don’t forget the YouTube videos!