Chapter 17: From Gene to Protein (Protein Synthesis)

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Chapter 17 From Gene to Protein(Protein
Synthesis)
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Essential Knowledge

• 3.a.1 DNA, and in some cases RNA, is the
  primary source of heritable information
  (17.1-17.4).
• 3.c.1 Changes in genotype can result in changes
  in phenotype (17.5).

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Question?

• How does DNA control a cell? (or identify a
  phenotype)
• By controlling protein synthesis (otherwise known
  as gene expression)
• Proteins are the link between genotype and
  phenotype

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1909 - Archibald Garrod

• Suggested genes control enzymes that catalyze
  chemical processes in cells
• Inherited Diseases - inborn errors of
  metabolism where a person cant make an enzyme
• Symptoms reflect persons inability to make
  proteins/enzymes

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Example

• Alkaptonuria - where urine turns black after
  exposure to air
• Lacks - an enzyme to metabolize/break down
  alkapton

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George Beadle and Edward Tatum

• Worked with Neurospora and proved the link
  between genes and enzymes
• Grew Neurospora on agar
• Varied the nutrients in the agar
• Looked for mutants that failed to grow on minimum
  agar

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Conclusion

• Mutations were abnormal genes
• Each gene dictated the synthesis/production of
  one enzyme
• One Gene - One Enzyme Hypothesis

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Current Hypothesis

• One Gene - One Polypeptide Hypothesis.
• Why change? Not all proteins are enzymes
• We now know proteins may have 4th degree
  structure.

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Central Dogma

• DNA
• Transcription
• RNA
• Translation
• Polypeptide chain
• (will become protein)

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Explanation

• DNA the genetic code or genotype
• RNA - the message or instructions
• Polypeptide - the end product for the phenotype

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Why is there an RNA intermediate?

• Evolutionary adaptation
• Check-point in process
• Provides protection for DNA code
• More copies can be made simultaneously

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Genetic Code

• Sequence of DNA bases that describe which amino
  acid to place in what order in a polypeptide
  chain
• The genetic code gives ONLY the primary protein
  structure
• All other protein structures result from chemical
  interactions amongst primary protein structure

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Genetic Code

• Is based on triplets of bases (called codons)
• Has redundancy some AA's have more than 1
  code/3-base codon
• Proof - make artificial RNA and see what AAs are
  used in protein synthesis (early 1960s)

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Codon

• A 3-nucleotide word in the Genetic Code
• 64 possible codons known

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• Codon
• Amino
• acid

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Codon Dictionary

• Start- AUG (Met)
• Stop- UAA UAG UGA
• 60 codons for the other 19 AAs

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Code Redundancy

• Third base in a codon shows "wobble effect
• First two bases are the most important in reading
  the code and giving the correct AA
• The third base often doesnt matter
• This allows for mistakes during DNA replication

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Reading Frame

• The reading of the code is every three bases
• Ex the red cat ate the rat
• Ex ATT GAT TAC ATT
• The words (codons) only make sense if read in
  this grouping of three (in correct letter order)

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Code Evolution

• The genetic code is nearly universal
• Ex CCG proline (all life)
• Reason
• Code must have evolved early
• Life on earth must share a common ancestor

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Protein Synthesis Intro

• Intro movie

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Protein Synthesis Intro

• Step 1 Transcription
• DNA ? mRNA
• Step 2 Translation
• mRNA ? tRNA ? Am. Acid ? Polypep. chain
• Polypeptide chain then becomes protein

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Transcription

• Process of making RNA from a DNA template
• RNA type mRNA (messenger)
• Intermediate type
• Takes place in nucleus (in eukaryotes)

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Transcription Steps

• 1. RNA Polymerase Binding
• 2. Initiation
• 3. Elongation
• 4. Termination

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RNA Polymerase

• Enzyme for building RNA from RNA nucleotides
• Prokaryotes - 1 type
• Eukaroyotes- 3 types
• Splits two DNA strands apart
• Hooks RNA nucleotides together (as they pair with
  DNA)

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1st Step RNA Polymerase Binding

• Requires that the enzyme find the proper place
  on the DNA to attach and start transcription
• Different from DNA polymerase
• Doesnt require an RNA primer

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RNA Polymerase Binding Needs

• Promoter Regions (on the DNA)
• Special sequences of DNA nucleotides that tell
  cell where transcription begins
• Transcription Factors
• Proteins

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Promoters

• Regions of DNA where RNA Polymerases can bind
• About 100 nucleotides long. Include initiation
  site and recognition areas for RNA Polymerase
• Also decide which DNA strand to use

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TATA Box

• ONLY in eukaryotes
• Short segment of T,A,T,A
• Located 25 nucleotides upstream from the
  initiation site
• Recognition site for transcription factors to
  bind to the DNA

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Transcription Factors

• Proteins that bind to DNA before RNA Polymerase
• Recognizes TATA box, attaches, and flags the
  spot for RNA Polymerase
• RNA poly wont attach unless these are present

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Transcription Initiation Complex

• The complete assembly of
• 1) transcription factors and
• 2) RNA Polymerase
• Bound to the promoter area of the DNA to be
  transcribed

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2nd Step Initiation

• 2nd step of transcription
• Actual unwinding of DNA to start RNA synthesis.
• Requires Initiation Factors

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Comment

• Getting Transcription started is complicated
• Gives many ways to control which genes are
  decoded and which proteins are synthesized

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3rd Step Elongation

• 3rd step in transcription
• RNA Polymerase untwists DNA 1 turn at a time
• Exposes 10 DNA bases for pairing with RNA
  nucleotides

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Elongation

• Adds nucleotides to 3 end of growing RNA strand
• Enzyme moves 5 ? 3 (of RNA strand)
• Rate is about 60 nucleotides per second

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Comment

• Each gene can be read by sequential RNA
  Polymerases giving several copies of RNA
• Result - several copies of the protein can be made

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4th Step Termination

• DNA sequence that tells RNA Polymerase to stop
• Ex AATAAA
• RNA Polymerase detaches from DNA after closing
  the helix

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Final Product

• Pre-mRNA
• This is a raw RNA that will need processing and
  modifications

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Modifications of RNA

• 1. 5 Cap
• 2. Poly-A Tail
• 3. Splicing

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5' Cap

• Modified Guanine nucleotide added to the 5' end
• Protects mRNA from digestive enzymes
• Recognition sign for ribosome attachment

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Poly-A Tail

• 150-200 Adenine nucleotides added to the 3' tail
• Protects mRNA from digestive enzymes.
• Aids in mRNA transport from nucleus.

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RNA Splicing

• Removal of non-protein coding regions of RNA
• Coding regions are then spliced back together

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Introns and Exons

• Introns
• Intervening sequences
• Removed from RNA.
• Exons
• Expressed sequences of RNA
• Translated into AAs

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Introns - Function

• Left-over DNA (?)
• Way to lengthen genetic message
• Old virus inserts (?)
• Way to create new proteins

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Translation Poster Requirements

• 1. What is translation? (definition)
• 2. What is needed?
• 3. Specifics Structure of tRNA
• 4. Where does it occur?
• 5. Ribosome specifics be sure to include the
  specifics of each subunit
• 6. Steps of translation details of each step
• 7. What bonds are formed?
• 8. Illustration

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2nd step of Protein Synthesis Translation

• Process by which a cell interprets a genetic
  message and builds a polypeptide
• Location mRNA moves from nucleus to cytoplasm
  and ribosomes

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Materials Required for translation

• tRNA
• Ribosomes
• mRNA

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Transfer RNA tRNA

• Made by transcription
• About 80 nucleotides long
• Carries AA for polypeptide synthesis

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Structure of tRNA

• Has double stranded regions and 3 loops.
• AA attachment site at the 3' end.
• 1 loop serves as the Anticodon.

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Anticodon

• Region of tRNA that base pairs to mRNA codon
• Usually is a compliment to the mRNA bases, so
  reads the same as the DNA codon
• Example
• DNA- GAC
• mRNA CUG
• tRNA anticodon - GAC

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Ribosomes

• Two subunits (large and small) made in the
  nucleolus
• Made of rRNA (60)and protein (40)
• rRNA is the most abundant type of RNA in a cell

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Large Subunit

• Has 3 sites for tRNA.
• P site Peptidyl-tRNA site - carries the growing
  polypeptide chain
• A site Aminoacyl-tRNA site -holds the tRNA
  carrying the next AA to be added
• E site Exit site

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Translation Steps

• 1. Initiation
• 2. Elongation
• 3. Termination

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Initiation

• Brings together
• mRNA
• A tRNA carrying the 1st AA
• 2 subunits of the ribosome

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Initiation Steps

• 1. Small subunit binds to the
  mRNA
• 2. Initiator tRNA (Met, AUG) binds to mRNA
• 3. Large subunit binds to mRNA
• Initiator tRNA is in the P-site

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Initiation

• Requires other proteins called "Initiation
  Factors
• GTP used as energy source

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Elongation Steps

• 1. Codon Recognition
• 2. Peptide Bond Formation
• 3. Translocation

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Codon Recognition

• tRNA anticodon matched to mRNA codon in the A site

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Peptide Bond Formation

• A peptide bond is formed between the new AA and
  the polypeptide chain in the P-site
• Bond formation is by rRNA acting as a ribozyme
• After bond formation
• The polypeptide is now transferred from the tRNA
  in the P-site to the tRNA in the A-site

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Translocation

• tRNA in P-site is released
• Ribosome advances 1 codon, 5 3
• tRNA in A-site is now in the P-site
• Process repeats with the next codon

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Termination

• Triggered by stop codons
• Release factor binds in the A-site instead of a
  tRNA
• H2O is added instead of AA, freeing the
  polypeptide
• Ribosome separates

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Polyribosomes

• Cluster of ribosomes all reading the same mRNA
• Another way to make multiple copies of a protein

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Prokaryotes Prok. vs. Euk. Protein Synthesis
Video
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Polypeptide vs. Protein

• Polypeptide usually needs to be modified before
  it becomes functional
• Ex
• Sugars, lipids, phosphate groups added
• Some AAs removed
• Protein may be cleaved
• Join polypeptides together (Quaternary Structure)

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Mutations

• Changes in the genetic make-up of a cell
• Chapter 15 covered large-scale chromosomal
  mutations
• (Hint - review these!)

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Mutation types - Cells

• Somatic cells or body cells not inherited
• Germ Cells or gametes - inherited

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Point or Spot Mutations

• Changes in one or a few nucleotides in the
  genetic code
• Effects - none to fatal

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Types of Point Mutations

• 1. Base-Pair Substitutions
• 2. Insertions
• 3. Deletions

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Base-Pair Substitution

• The replacement of 1 pair of nucleotides by
  another pair
• Ex Sickle cell anemia

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Types of Substitutions

• 1. Missense - altered codons, still code for AAs
  but not the right ones
• 2. Nonsense - changed codon becomes a stop codon

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Question?

• What will the "Wobble" Effect have on Missense?
• If the 3rd base is changed, the AA may still be
  the same and the mutation is silent

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Missense Effect

• Can be none to fatal depending on where the AA
  was in the protein
• Ex
• If in an active site - major effect
• If in another part of the enzyme - no effect

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Nonsense Effect

• Stops protein synthesis
• Leads to nonfunctional proteins unless the
  mutation was near the very end of the polypeptide

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Sense Mutations

• The changing of a stop codon to a reading codon
• Result - longer polypeptides which may not be
  functional
• Ex. heavy hemoglobin

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Insertions Deletions

• The addition or loss of a base in the DNA
• Cause frame shifts and extensive missense,
  nonsense or sense mutations

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Frame Shift

• The reading of the code is every three bases
• Ex the red cat ate the rat
• Ex thr edc ata tat her at
• The words only make sense if read in this
  grouping of three

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Question?

• Loss of 3 nucleotides is often not a problem
• Why?
• Because the loss of a 3 bases or one codon
  restores the reading frame

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Mutagens

• Materials that cause DNA changes
• 1. Radiation
• ex UV light, X-rays
• 2. Chemicals
• ex 5-bromouracil

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• Chernobyl video

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Summary

• Recognize the relationship between genes and
  enzymes (proteins) as demonstrated by the
  experiments of Beadle and Tatum.
• Identify the flow of genetic information from DNA
  to RNA to polypeptide (the Central Dogma).
• Read DNA or RNA messages using the genetic code.
• Recognize the steps and procedures in
  transcription.

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Summary

• Identify methods of RNA modification.
• Recognize the steps and procedures in
  translation.
• Recognize categories and consequences of
  base-pair mutations.
• Identify causes of mutations.
• Be able to recognize and discuss What is a gene?