Chapter 17 From Gene to Protein

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Chapter 17From Gene to Protein
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Question?

• How does DNA control a cell?
• By controlling Protein Synthesis.
• 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.

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Example

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

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George Beadle and Edward Tatum

• Worked with Neurospora and proved the link
  between genes and enzymes.

Neurospora Pink bread mold
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Experiment

• Grew Neurospora on agar.
• Varied the nutrients.
• Looked for mutants that failed to grow on minimum
  agar.

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Results

• Three classes of mutants for Arginine Synthesis.
• Each mutant had a different block in the Arginine
  Synthesis pathway.

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Conclusion

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

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Current Hypothesis

• One Gene - One Polypeptide Hypothesis.
• We now know proteins may have 4th degree
  structure.

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Central Dogma

• DNA
• Transcription
• RNA
• Translation
• Polypeptide

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Explanation

• DNA - the Genetic code or genotype.
• RNA - the message or instructions.
• Polypeptide - the product for the phenotype.

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Genetic Code

• Sequence of DNA bases that describe which Amino
  Acid to place in what order in a polypeptide.
• The genetic code gives the primary protein
  structure.

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Code Basis

• If you use
• 1 base 1 amino acid
• 4 bases 4 amino acids
• 41 4 combinations, which are not enough for 20
  AAs.

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If you use

• 2 bases 1 amino acid
• 42 16 amino acids
• Still not enough combinations.

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If you use

• 3 bases 1AA
• 43 64 combinations
• More than enough for 20 amino acids.

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Genetic Code

• Is based on triplets of bases.
• Has redundancy some AA's have more than 1 code.
• 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 Dictionary

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

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For Testing

• Be able to read a DNA or RNA message and give
  the AA sequence.
• RNA Genetic Code Table will be provided.

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Code Redundancy

• Third base in a codon shows "wobble.
• First two bases are the most important in reading
  the code and giving the correct AA. The third
  base often doesnt matter.

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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 only make sense if read in this
  grouping of three.

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Code Evolution

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

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Transcription

• Process of making RNA from a DNA template.

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Movie - preview
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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

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RNA Polymerase Binding

• Requires that the enzyme find the proper place
  on the DNA to attach and start transcription.

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RNA Polymerase Binding Needs

• Promoter Regions on the DNA.
• Transcription Factors.

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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.

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TATA Box

• Short segment of T,A,T,A
• Located 25 nucleotides upstream for 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.

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Transcription Initiation Complex

• The complete assembly of transcription factors
  and RNA Polymerase bound to the promoter area of
  the DNA to be transcribed.

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Initiation

• 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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Elongation

• RNA Polymerase untwists DNA 1 turn at a time.
• Exposes 10 DNA bases for pairing with RNA
  nucleotides.

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Elongation

• Enzyme moves 5 3.
• 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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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.

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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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Comment

• The head and tail areas often contain leaders
  and trailers, areas of RNA that are not read.
• Similar to leaders or trailers on cassette tapes.

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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

• Intervening sequences.
• Removed from RNA.

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Exons

• Expressed sequences of RNA.
• Translated into AAs.

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Spliceosome

• Cut out Introns and join Exons together.
• Made of snRNA and snRNP.

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snRNA

• Small Nuclear RNA.
• 150 nucleotides long.
• Structural part of spliceosomes.

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snRNPs

• ("snurps")
• Small Nuclear Ribonucleoprotiens
• Made of snRNA and proteins.
• Join with other proteins to form a spliceosome.

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Result
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Ribozymes

• RNA molecules that act as enzymes.
• Are sometimes Intron RNA and cause splicing
  without a spliceosome.

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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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Final RNA Transcript
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Translation

• Process by which a cell interprets a genetic
  message and builds a polypeptide.

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Materials Required

• 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.

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Example

• DNA - GAC
• mRNA - CUG
• tRNA anticodon - GAC

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Comment

• "Wobble" effect allows for 45 types of tRNA
  instead of 61.
• Reason - in the third position, U can pair with A
  or G.
• Inosine (I), a modified base in the third
  position can pair with U, C, or A.

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Importance

• Allows for fewer types of tRNA.
• Allows some mistakes to code for the same AA
  which gives exactly the same polypeptide.

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Aminoacyl-tRNA Synthetases

• Family of Enzymes.
• Add AAs to tRNAs.
• Active site fits 1AA and 1 type of tRNA.
• Uses a secondary genetic code to load the
  correct AA to each tRNA.

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Ribosomes

• Two subunits 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
Proteins
rRNA
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Both sununits
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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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Movie - preview
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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

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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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Comment

• Elongation takes 60 milliseconds for each AA
  added.

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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
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Comment

• Polypeptide usually needs to be modified before
  it becomes functional.

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Examples

• Sugars, lipids, phosphate groups added.
• Some AAs removed.
• Protein may be cleaved.
• Join polypeptides together (Quaternary
  Structure).

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Signal Hypothesis

• Clue on the growing polypeptide that causes
  ribosome to attach to ER.
• All ribosomes are free ribosomes unless clued
  by the polypeptide to attach to the ER.

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Result

• Protein is made directly into the ER .
• Protein targeted to desired location (e.g.
  secreted protein).
• Clue (the first 20 AAs are removed by
  processing).

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Mutations

• Changes in the genetic makeup 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.

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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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Mutagenesis

• Process of causing mutations or changes in the
  DNA.

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Spontaneous Mutations

• Random errors during DNA replication.

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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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Comment

• Any material that can chemically bond to DNA,
  or is chemically similar to the nitrogen bases,
  will often be a very strong mutagen.

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The Ames Test

• Measures the mutagenic strength of various
  chemicals.
• Looks for back-mutations in bacteria.

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Ames Results

• The more back mutations, the more colonies
  appear, the stronger the mutagenic effect of the
  material.
• Usually compared to and - controls.

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Summary

• Know Beadle and Tatum.
• Know the central dogma.
• Be able to read the genetic code.
• Be able to describe the events of transcription
  and translation.

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Summary

• Be able to discuss RNA and protein processing.
• Be able to describe and discuss DNA mutations.