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From Gene to Protein. Campbell and Reece Chapter 17. Gene Expression. process by which DNA directs the synthesis of proteins or RNA synthesis of proteins transcription translation. How Gene to Protein Figured Out. Evidence from study of metabolic disorders:

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from gene to protein

From Gene to Protein

Campbell and Reece

Chapter 17

gene expression
Gene Expression
  • process by which DNA directs the synthesis of proteins or RNA
  • synthesis of proteins
    • transcription
    • translation
how gene to protein figured out
How Gene to Protein Figured Out
  • Evidence from study of metabolic disorders:
  • 1902: British physician 1st to suggest genes responsible for phenotype thru enzymes that catalyze specific chemrx in the cell
inborn errors of metabolism
Inborn Errors of Metabolism
  • Garrod hypothesized that symptoms of an inherited disease are due to a gene that leads to inability to make a certain enzyme
  • 1 of 1st to realize Mendel’s principle’s of heredity applied to more than pea plants
alkaptonuria
Alkaptonuria
  • Signs & Symptoms:
    • urine turns black when alkapton (chemical in urine) reacts with air
    • missing enzyme in pathway that degrades phenylalanine (a.a.)
beadle tatum experiment
Beadle & Tatum Experiment
  • worked with a bread mold Neurosporacrassa
  • bombarded it with radiation (already known to cause genetic changes)
  • then checked for survivors who had different nutritional needs from wild-type mold
beadle tatum experiment1
Beadle & Tatum Experiment
  • individually put yeast in different mediums (agar with different nutrients)
  • identified mutants that could not survive on minimal nutrients placed them in complete growth medium (minimal med. + all 20 a.a. + few vitamins & minerals)
1 gene 1 polypeptide
1-Gene-1-Polypeptide
  • Beadle & Tatum’s results supported their hypothesis
  • 1958: Nobel prize
1 gene 1 polypeptide1
1-Gene-1-Polypeptide
  • revised over time:
    • not all proteins are enzymes
    • some proteins have >1 polypeptide
    • now: 1- gene-1-protein hypothesis
    • not 100%: some eukaryotic genes can each code for a set of closely related polypeptides via alternative splicing
transcription short version
Transcription: short version
  • the synthesis of RNA using information in DNA
  • mRNA made using complimentary base pairing
translation short version
Translation: short version
  • synthesis of a polypeptide using the information in mRNA
  • “translates” message in mRNA  a.a.
the genetic code
The Genetic Code
  • 4 nucleotide bases to code for 20 a.a.
  • triplet code: 3 consecutive bases code for 1 of the a.a./ stop
template strand
Template Strand
  • during transcription:
    • DNA helix unwound
    • 1 strand only transcribed (could be either side depending on the gene)
slide21
mRNA
  • uracil added as compliment to adenine
  • ribose as its 5-carbon sugar
  • single stranded
codons
Codons
  • nucleotide triplets of DNA or mRNA that specifies a particular amino acid or termination signal
  • basic unit of the genetic code
  • written in 5’  3’ direction (in DNA 3 bases read in 3’  5’ direction)
cracking the code
Cracking the Code
  • early 1960’s
  • Nirenberg: synthesized mRNA using only uracil (UUUUUUU…)
    • added it to test tube with all 20 a.a., ribosomes
    • translated into polypeptide made up of phenyalanine
    • now knew UUU = Phe
    • did same for AAA= Lys, CCC = Pro, GGG = Gly
cracking the code1
Cracking the Code
  • all 64 a.a. deciphered by mid-1960’s
  • 3 codons code for “stop” marking end of translation
  • AUG functions as “start” & Met
    • Met may or may not be clipped off later
genetic code is redundant
Genetic Code is Redundant
  • >1 triplet codes for each of the a.a. but any 1 triplet codes for only 1 a.a
  • redundant triplets usually only differ in the 3rd base
reading frame
Reading Frame
  • translating the code in correct groupings
  • example:

Did the red dog eat the bug?

Idt her eddogeatthebug?

evolution of genetic code
Evolution of Genetic Code
  • code is nearly universal:
    • bacteria  complex multicellular organisms CAU = His
    • insert genes into other species & get same result (human insulin gene in bacteria)
  • exceptions: certain unicellular eukaryotes & in organelle genes of some species
rna polymerase
RNA Polymerase
  • unwinds 2 strands of DNA
  • binds nucleotides together as build mRNA
    • only in 5’  3’ direction (like DNA polymerase)
3 stages of transcription
3 Stages of Transcription
  • Initiation
  • Elongation
  • Termination
initiation
Initiation
  • After RNA polymerase binds to promoter, ¤ DNA strands unwind
    • polymerase begins RNA synthesis @ start pt. on template strand
initiation1
Initiation
  • promoter: usually includes w/in it the transcription start point (a nucleotide where transcription begins) & extends several dozen or more nucleotide pairs upstream from start pt.
  • RNAP can assemble nucleotides only in 5’  3’ direction (just like DNA polymerase)
  • unlike DNAP, RNAP does not require a primer
start point
Start Point
  • nucleotide where RNA synthesis actually begins
  • RNAP binds in precise location & orientation on the promoter  where determines where transcription starts & which of the 2 strands will be transcribed
rna polymerase1
RNA Polymerase
  • Bacteria:
    • 1 single RNAP used to make all types RNA
  • Eukaryotic Cells:
    • @ least 3 types RNA polymerase
    • II used for RNA synthesis
    • I and III used to transcribe RNA not used for protein synthesis
rna polymerase2
RNA Polymerase
  • Prokaryotes :
    • RNAP recognizes & binds to the promoter by itself
  • Eukayotes:
    • collection of proteins , transcription factors, mediate the binding of RNAP & initiation of transcription
transcription factors
Transcription Factors
  • must 1st attach to promoter b/4 RNAP II can bind to it
  • RNAP II + transcription factors =

Transcription Initiation Complex

  • TATA box: DNA sequence in eukaryotic promoters crucial in forming the transcription initiation complex
elongation
Elongation
  • RNAP moves downstrean, unwinding the DNA & elongating the RNA transcript 5’  3’
  • ~ 10 – 20 nucleotides exposed
  • in wake of transcription the 2 DNA strands spontaneously rewind
  • length of DNA transcribed = transcription unit
termination
Termination
  • mechanism differs between prokaryotes & eukaryotes
  • Bacteria: transcription proceeds thru terminator sequence in the DNA  the transcribed RNA functions as the terminator sequence  causing RNAP to detach
  • prokaryotes have no further modification
termination in eukaryotes
Termination in Eukaryotes
  • RNAP II transcribes a portion of DNA called the polyadenylation signal (AAUAAA) in the pre-mRNA
  • ~10 – 35 nucleotides downstream from that sequence proteins ass’c with transcription cut the pre-mRNA free from the polymerase
  • pre-mRNA then  modified
rna processing
RNA Processing
  • in eukaryotes only
  • both ends of primary transcript altered
  • certain interior sections cut out & remaining parts spliced back together
mrna ends
mRNA Ends
  • 5’ end receives a 5’cap: modified G is added after ~ 20 – 40 nucleotides in mRNA
  • 3’ end modified: enzyme adds 50 -250 A’s to the AAUAAA forming a poly-A tail
functions of modified ends of mrna
Functions of Modified Ends of mRNA
  • facilitate exit of mRNA from nucleus
  • protect mRNA from degradation of hydrolytic enzymes
  • help ribosomes attach to the 5’ end
rna splicing
RNA Splicing
  • cut-and-paste job removing segments of RNA that were transcribed
  • average size transcript: 27,000 nucleotides
  • average size protein: 1,200 nucleotides (400 a.a.)
introns
Introns
  • noncoding, intervening sequence w/in primary transcript that is removed from the transcript during RNA processing; also refers to the region of DNA from which this sequence was transcribed
exons
Exons
  • sequence w/in primary transcript that remains in the RNA after RNA processing; also refers to the region of DNA from which this sequence was transcribed
rna splicing1
RNA Splicing
  • signal: short nucleotide sequence @ each end of an intron
  • particle called “snurp” recognizes splice sites
    • small nuclear ribonucleoproteins (snRNP’s)
    • in nucleus
    • made of RNA + protein
    • small nuclear RNA ~150 nucleotides
spliceosome
Spliceosome
  • combination of several different snRNP’s (almost size of ribosome)
  • interact with certain sites along intron releasing intron rapidly degraded
  • then joins ends of exons together
ribozymes
Ribozymes
  • RNA molecules that function like enzymes in some organisms
  • intron RNA can act like ribozyme & catalyze its own excision
ribozymes1
Ribozymes
  • 3 properties of RNA enables some RNA molecules to function as enzymes:
  • single-stranded: 1 sequence can interact w/another using base pairing
  • some of bases contain functional groups (like a.a) that could participate in catalysis
  • ability to form H-bonds adds specificity
importance of introns
Importance of Introns
  • still having debate about importance of introns & RNA splicing in evolution
  • they both have adaptive benefits
  • do not know functions of most introns
importance of introns1
Importance of Introns
  • single gene can encode >1 kind of polypeptide
  • know many genes that make 2 or more different polypeptides depending on what was removed as introns during gene splicing
  • called: alternative RNA splicing
alternative rna splicing
Alternative RNA Splicing
  • Drosophila sex differences due to how RNA transcript is spliced
  • Human Genome Project: 1 of reasons humans get by with same # genes as a nematode
translation closer look
Translation: Closer Look
  • tRNA: transfers a.a. from cytoplasmic pool of a.a to ribosome where it’s a.a. is added to polypeptide chain
  • cell keeps supply of all 20 a.a. on hand
    • degradation of other molecules
    • synthesizes them using building blocks in cytoplasm
slide63
tRNA
  • brings specific a.a to ribosome
  • 1 end has a.a./ other end has anticodon which H-bonds with codon on ribosome
  • tRNA translates the codes into the corresponding a.a.
  • tRNA is transcribed from DNA templates & used repeatedly
  • tRNA made of ~80 nucleotides long with some regions folded back on self due to base pairing
trna structure1
tRNA Structure
  • 3’ end: a.a. attached
  • opposite end: anticodon
accurate translation
Accurate Translation
  • requires 2 instances of molecular recognition:
  • tRNA that binds to particular a.a.
    • correct match made by group enzymes called aminoacyl-tRNAsynthetases: their active site fits only 1 of the 20 a.a.
  • pairing of tRNAanticodon with mRNA codon
trna wobble
tRNA Wobble
  • ~ 45 different ones (not 61 like genetic code would suggest)
    • possible because pairing the 3rd base of codon & 3rd base of anticodon: relaxed base pair rules
      • U can pair with A or G in 3’ end of codon (3rd position)
      • called a “wobble”
ribosomes
Ribosomes
  • subunits made in nucleolus
    • rRNA transcribed & added to proteins imported from cytoplasm
    • ribosomal subunits  cytoplasm, join only when translating mRNA
    • subunits ~1/3 protein & 2/3 rRNA
      • bacteria: 3 molecules rRNA
      • eukaryotes: 4 molecules rRNA
ribosome structure
Ribosome Structure
  • eukaryotic ribosomes slightly larger than prokaryotic ones
  • pharmaceutical products (antibiotics) designed to inactivate bacterial ribosomes that have no effect on ours
    • Tetracyclines
    • Streptomycin
ribosome structure1
Ribosome Structure
  • 4 binding sites: (1st for mRNA, others for tRNA)
  • mRNA binding site
  • P site: peptidyl-tRNA holds the tRNA carrying the growing polypeptide chain
  • A site: aminoacyl-tRNA holds tRNA carrying next a.a to be added
  • E site: exit, where discharged tRNAs leave ribosome
ribosome
Ribosome
  • holds tRNA & mRNA in close proximity & catalyzes the formation of new peptide bond holding the 2 a.a together adding to carboxyl end of last a.a. in growing polypeptide chain
  • peptide chain passes thru exit tunnel in large subunit as it grows longer
translation
Translation
  • 3 Stages:
  • Initiation
  • Elongation
  • Termination
initiation2
Initiation
  • small ribosomal subunit attaches to mRNA
  • downstream from this attachment is the start codon AUG
  • tRNA with UAC (Met) binds to it
  • large ribosomal subunit attaches (1GTP)
  • initiation factors (proteins) required to bring it all together
elongation2
Elongation
  • Codon recognition
    • anticodon of incoming tRNA w/c’ base
    • 1 GTP increases accuracy & efficiency
  • Peptide bond formation
    • part of rRNA catalyzes reaction
    • amino end of newest a.a + carboxyl end of peptide chain transferring pep. chain to tRNA @ A site
  • Translocation
    • ribosome moves so tRNA @ A site  P site
    • 1 GTP
termination1
Termination
  • ribosome reaches stop codon the A site accepts a “release factor” (shaped like tRNA but does not have aminoacyl part)
  • promotes release of bond between P site, mRNA, & last tRNA
  • 2 ribosomal subunits & ass’c proteins come apart
animation time
Animation Time!
  • http://bcs.whfreeman.com/thelifewire/content/chp12/1202003.html
  • http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis
try at home interactive
Try at home: interactive
  • http://www.wiley.com/college/boyer/0470003790/animations/translation/translation.htm
polyribosomes
Polyribosomes
  • 1 ribosome can make polypeptide of average size: 1 min
  • typically many ribosomes are translating a single mRNA @ given time
  • 1st ribosome gets far enough past start codon 2nd ribosome can get started
  • allow cell to make many copies of polypeptide very quickly
primary structure
Primary Structure
  • as polypeptide chain grows longer from ribosome it will spontaneously start to fold & coil as result of a.a side chain interactions
  • genes determine 1’ structure which then determines 2’, 3’ and 4’ structures
chaperonins
Chaperonins
  • proteins that help with the folding
post translational modifications
Post-Translational Modifications
  • additional steps that may be required b/4 protein can do its job
    • attachment of sugars, lipids, phosphate groups to a.a
    • enzymatic removal of 1 or more a.a. from leading end (amino end)
targeting polypeptides specific locations in cell
Targeting Polypeptides  Specific Locations in Cell
  • free ribosomes make proteins used in cytoplasm
  • bound ribosomes (RER) attached to cytosolic side while polypeptide being released into endomembrane system
  • both have identical small & large subunits
signal peptide
Signal Peptide
  • growing polypeptide cues ribosome to attach to ER
  • polypeptides of proteins destined for endomembrane system have signal peptide: sequence of ~20 a.a. at or near leading end (N-terminus) is recognized by a protein-RNA complex called signal-recognition particle or SRP
slide94
SRP
  • escorts ribosome to receptor protein on ER membrane
  • receptor part of multiprotein translocation complex
  • ribosome continues to make polypeptide which enters ER thru protein pore
  • signal protein usually removed by enzyme
proteins organelles
Proteins  Organelles
  • use other signal peptides for protein destined for chloroplast, mitochondria, or interior of nucleus
  • in these, proteins made in cytosol then  to organelle
  • signal proteins target or “address” proteins for secretion or to cellular locations
    • used by prokaryotes too
mutations
Mutations
  • ultimate source of new genes
    • large scale mutations
      • chromosomal rearrangements: chap. 15
    • small scale mutations
      • 1 or a few nucleotide bases changed
try @ home
Try @ Home
  • http://www.bodrum-hotels.com/gene-mutations/gene-mutations-and-proteins-worksheet.html
point mutations
Point Mutations
  • changes in single nucleotide pair
  • if occurs in gamete or cell that  gamete will be passed on to offspring
  • if mutation has adverse effect on phenotype is called a genetic disorder or hereditary disease
  • if mutation causes organism to die before fully developed it is said to be lethal
  • if mutation results in no change in phenotype is said to be silent
familial cardiomyopathy
Familial Cardiomyopathy
  • point mutation
  • dominant
  • possible cause of sudden death of young athletes
substitutions
Substitutions
  • replacement of 1 nucleotide pair by another pair: a few will improve activity of protein it is coding for but most will be detrimental
  • some silent due to redundancy of genetic code
  • if changes 1 a.a. for another called missense mutation
    • if substituted a.a. similar to real one no effect
    • some substitutions will have major consequences
nucleotide pair substitution
Nucleotide-Pair Substitution
  • Silent
  • Missense:
    • most substitutions in this category
  • Nonsense: substitution changes from 1 a.a.  stop codon
    • resulting polypeptide is shorter
    • nearly all  nonfunctional proteins
insertions deletions
Insertions & Deletions
  • (+) or (-) of nucleotide pairs in a gene
  • disastrous effects
  • may alter reading frame triplet codon shifts on mRNA
  • called frameshift mutation
    • whenever insertion or deletion not in a multiple of 3
    • if not causes major missense
mutagens
Mutagens
  • any chemical or physical agent that interacts with DNA & can cause a mutation
  • 1920’s: Muller used x-rays to make mutant Drosophila & he discovered it does same in humans
  • mutagenic radiation includes:
    • UV radiation cause thymine dimers in DNA
chemical mutagens
Chemical Mutagens
  • nucleotide analogs
    • similar to normal DNA nucleotides
    • insert self into DNA
chemical mutagens1
Chemical Mutagens
  • some cause chemical changes in bases that changes their pairing properties
differences
Differences
  • some in gene expression among eubacteria, archaea, and eukaryotes
  • if no nucleus: translation can begin b/4 transcription is over
  • Archaea show similarities to Eubacteria and eukaryotes in processes of gene expression
what is a gene
What is a Gene?
  • region of DNA whose final functional product is either a polypeptide or an ENA molecule
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