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Chapter 17. From Gene to Protein
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Metabolism teaches us about genes
Metabolic defects studying metabolic diseases suggested that genes specified proteins alkaptonuria (black urine from alkapton) PKU (phenylketonuria) each disease is caused by non-functional enzyme Genes create phenotype A B C D E
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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
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1941 | 1958 Beadle & Tatum George Beadle Edward Tatum
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Beadle & Tatum’s Neurospora experiment
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Where does that leave us?!
So… What is a gene? One gene – one enzyme but not all proteins are enzymes but all proteins are coded by genes One gene – one protein but many proteins are composed of several polypeptides but each polypeptide has its own gene One gene – one polypeptide 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?!
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if you don’t know what a wabbit looks like.
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 It’s hard to hunt for wabbits, if you don’t know what a wabbit looks like. 1990s -- thought humans had 100,000 genes ,000 was considered a good estimate ,000 ,000 is our best estimate polypeptide 1 polypeptide 2 polypeptide 3 gene
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let’s go back to genes that code for proteins…
The “Central Dogma” How do we move information from DNA to proteins? transcription translation DNA RNA protein For simplicity sake, let’s go back to genes that code for proteins… replication
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From nucleus to cytoplasm…
Where are the genes? genes are on chromosomes in nucleus Where are proteins synthesized? proteins made in cytoplasm by ribosomes How does the information get from nucleus to cytoplasm? messenger RNA nucleus
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transcription and translation
RNA ribose sugar N-bases uracil instead of thymine U : A C : G single stranded mRNA, rRNA, tRNA, siRNA…. To get from the chemical language of DNA to the chemical language of proteins requires 2 major stages: transcription and translation transcription DNA RNA
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Transcription Transcribed DNA strand = template strand
untranscribed DNA strand = coding strand Synthesis of complementary RNA strand transcription bubble Enzyme RNA polymerase
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Transcription in Prokaryotes
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'
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Transcription in Prokaryotes
Promoter sequences RNA polymerase molecules bound to bacterial DNA
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Transcription in Prokaryotes
Elongation RNA polymerase unwinds DNA ~20 base pairs at a time reads DNA 3’5’ builds RNA 5’3’ (the energy governs the synthesis!) No proofreading 1 error/105 bases many copies short life not worth it!
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Transcription RNA
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Transcription in Prokaryotes
Termination RNA polymerase stops at termination sequence mRNA leaves nucleus through pores RNA GC hairpin turn
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Transcription in Eukaryotes
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Prokaryote vs. Eukaryote genes
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
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Transcription in Eukaryotes
3 RNA polymerase enzymes RNA polymerase I only transcribes rRNA genes RNA polymerase I I transcribes genes into mRNA RNA polymerase I I I each has a specific promoter sequence it recognizes
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Transcription in Eukaryotes
Initiation complex transcription factors bind to promoter region upstream of gene proteins which bind to DNA & turn on or off transcription TATA box binding site only then does RNA polymerase bind to DNA
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Post-transcriptional processing
Primary transcript eukaryotic mRNA needs work after transcription Protect mRNA from RNase enzymes in cytoplasm add 5' cap add polyA tail Edit out introns A 3' poly-A tail CH3 mRNA 5' 5' cap 3' G P A’s intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequence pre-mRNA primary mRNA transcript mature mRNA transcript spliced mRNA
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Transcription to translation
Differences between prokaryotes & eukaryotes time & physical separation between processes RNA processing
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Translation in Prokaryotes
Transcription & translation are simultaneous in bacteria DNA is in cytoplasm no mRNA editing needed
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DNA mRNA protein From gene to protein transcription translation
aa transcription translation DNA mRNA protein ribosome mRNA leaves nucleus through nuclear pores proteins synthesized by ribosomes using instructions on mRNA nucleus cytoplasm
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How does mRNA code for proteins?
TACGCACATTTACGTACGCGG DNA AUGCGUGUAAAUGCAUGCGCC mRNA ? Met Arg Val Asn Ala Cys Ala protein How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?
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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
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Heinrich Matthaei Marshall Nirenberg
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Translation Codons blocks of 3 nucleotides decoded into the sequence of amino acids
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mRNA codes for proteins in triplets
TACGCACATTTACGTACGCGG DNA AUGCGUGUAAAUGCAUGCGCC mRNA AUGCGUGUAAAUGCAUGCGCC mRNA ? Met Arg Val Asn Ala Cys Ala protein
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The code For ALL life! Code is redundant Why is this a good thing?
strongest support for a common origin for all life Code is redundant several codons for each amino acid Why is this a good thing? Strong evidence for a single origin in evolutionary theory. Start codon AUG methionine Stop codons UGA, UAA, UAG
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How are the codons matched to amino acids?
3' 5' TACGCACATTTACGTACGCGG DNA 5' AUGCGUGUAAAUGCAUGCGCC 3' mRNA codon 3' 5' UAC Met tRNA GCA Arg amino acid CAU Val anti-codon
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cytoplasm transcription translation protein nucleus
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tRNA structure “Clover leaf” structure anticodon on “clover leaf” end
amino acid attached on 3' end
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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 The tRNA-amino acid bond is unstable. This makes it easy for the tRNA to later give up the amino acid to a growing polypeptide chain in a ribosome.
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Ribosomes Facilitate coupling of tRNA anticodon to mRNA codon
organelle or enzyme? Structure ribosomal RNA (rRNA) & proteins 2 subunits large small
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Ribosomes P site (peptidyl-tRNA site) A site (aminoacyl-tRNA site)
holds tRNA carrying growing polypeptide chain A site (aminoacyl-tRNA site) holds tRNA carrying next amino acid to be added to chain E site (exit site) empty tRNA leaves ribosome from exit site
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Building a polypeptide
Initiation brings together mRNA, ribosome subunits, proteins & initiator tRNA Elongation Termination
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Elongation: growing a polypeptide
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Termination: release polypeptide
Release factor “release protein” bonds to A site bonds water molecule to polypeptide chain Now what happens to the polypeptide?
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start of a secretory pathway
Destinations: secretion nucleus mitochondria chloroplasts cell membrane cytoplasm Protein targeting Signal peptide address label start of a secretory pathway
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Can you tell the story? RNA polymerase DNA amino acids tRNA pre-mRNA
exon intron tRNA pre-mRNA 5' cap mature mRNA aminoacyl tRNA synthetase polyA tail 3' large subunit polypeptide ribosome 5' tRNA small subunit E P A
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Put it all together…
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Any Questions??
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Chapter 17. Mutations
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Universal code Code is redundant several codons for each amino acid
“wobble” in the tRNA “wobble” in the aminoacyl-tRNA synthetase enzyme that loads the tRNA Strong evidence for a single origin in evolutionary theory.
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Mutations Point mutations single base change base-pair substitution
silent mutation no amino acid change redundancy in code missense change amino acid nonsense change to stop codon When do mutations affect the next generation?
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Point mutation leads to Sickle cell anemia
What kind of mutation?
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Sickle cell anemia
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Mutations Frameshift shift in the reading frame insertions deletions
changes everything “downstream” insertions adding base(s) deletions losing base(s)
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What’s the value of mutations?
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Chapter 17. RNA Processing
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Transcription -- another look
The process of transcription includes many points of control when to start reading DNA where to start reading DNA where to stop reading DNA editing the mRNA protecting mRNA as it travels through cell
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Primary transcript Processing mRNA
protecting RNA from RNase in cytoplasm add 5’ cap add polyA tail remove introns AUG UGA
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Protecting RNA 5’ cap added 3’ poly-A tail added
G trinucleoside (G-P-P-P) protects mRNA from RNase (hydrolytic enzymes) 3’ poly-A tail added A’s helps export of RNA from nucleus UTR UTR
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Dicing & splicing mRNA Pre-mRNA mRNA edit out introns
intervening sequences splice together exons expressed sequences In higher eukaryotes 90% or more of gene can be intron no one knows why…yet there’s a Nobel prize waiting… “AVERAGE”… “gene” = 8000b pre-mRNA = 8000b mature mRNA = 1200b protein = 400aa lotsa “JUNK”! average size gene (transcription unit) = bases average size primary transcript = 8000 bases average size mature RNA = 1200 bases average size protein = 400 amino acids lots of “junk DNA”
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Discovery of Split genes
1977 | 1993 Discovery of Split genes Richard Roberts Philip Sharp adenovirus NE BioLabs MIT common cold
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Splicing enzymes snRNPs Spliceosome RNA as ribozyme several 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
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Ribozyme 1982 | 1989 RNA as enzyme Sidney Altman Thomas Cech Yale
U of Colorado
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Splicing details No room for mistakes!
editing & splicing must be exactly accurate a single base added or lost throws off the reading frame AUGCGGCTATGGGUCCGAUAAGGGCCAU AUGCGGUCCGAUAAGGGCCAU AUG|CGG|UCC|GAU|AAG|GGC|CAU Met|Arg|Ser|Asp|Lys|Gly|His AUGCGGCTATGGGUCCGAUAAGGGCCAU AUGCGGGUCCGAUAAGGGCCAU AUG|CGG|GUC|CGA|UAA|GGG|CCA|U Met|Arg|Val|Arg|STOP|
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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!
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Domains Modular architecture of many proteins
separate functional & structural regions coded by different exons in same “gene”
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The Transcriptional unit (gene?)
enhancer 1000+b translation start translation stop exons 20-30b transcriptional unit RNA polymerase 3' TAC ACT 5' TATA DNA transcription start UTR introns transcription stop UTR promoter DNA pre-mRNA 5' 3' mature mRNA 5' 3' GTP AAAAAAAA
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Any Questions??
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The Transcriptional unit
enhancer 1000+b exons 20-30b transcriptional unit RNA polymerase 3' TAC ACT 5' TATA DNA introns 5' 3' 5' 3'
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