chapter 17 from gene to protein - biolympiads.com · •one gene - one enzyme hypothesis. current...
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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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For tests:
• Name(s) of experimenters
• Outline of the experiment
• Result of the experiment and its importance
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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 can’t 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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1941 - 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 (because of quaternary (4th degree) structure).
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Central DogmaDNA
RNA
Polypeptide
Transcription
Translation
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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
• Ex – AT, TA, CA, GC
• 42 = 16 amino acids
• Still not enough combinations.
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If you use:
• 3 bases = 1AA
• Ex – CAT, AGC, TTT
• 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 (codon).
• Proof - make artificial RNA and see what AAs are used in protein synthesis (early 1960’s).
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Codon
• A 3-nucleotide “word” in the Genetic Code.
• 64 possible codons known.
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DNA vs RNA
DNA RNA
Sugar – deoxyribose ribose
Bases – ATGC AUGC
Backbones – 2 1
Size – very large small
Use – genetic code varied
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Codon Dictionary
• Start- AUG (Met)
• Stop- UAAUAGUGA
• 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 doesn’t matter.
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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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Reading Frame and Frame Shift
• The “reading” of the code is every three bases (Reading Frame)– Ex: the red cat ate the rat
• Frame shift – improper groupings of the bases– Ex: thr edc ata tet her at
• The “words” only make sense if “read” in this grouping of three.
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Transcription
• Process of making RNA from a DNA template.
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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.
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Binding
• Requires that the enzyme find the “proper” place on the DNA to attach and start transcription.
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Binding
• Is a complicated process
• Uses Promoter Regions on the DNA (upstream from the information for the protein)
• Requires proteins called Transcription Factors.
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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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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.
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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 (noncoding).
• Removed from RNA.
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Exons
• Expressed sequences of RNA (coding).
• Translated into AAs.
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Spliceosome
• Cuts out Introns and join Exons together.
• Made of snRNA and snRNPs.
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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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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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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 – How is this different?
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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.
• May be at chromosome (review chapter 15) or DNA level
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DNA or Point 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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Comment
• Silent mutations may still have an effect by slowing down the “speed” of making the protein.
• Reason – harder to find some tRNAs than others.
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Missense Effect
• Can be none to fatal depending on where the AA was in the protein.
• Ex: if in an enzyme 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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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 and the protein may still be able to function.
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Mutagenesis
• Process of causing mutations or changes in the DNA.
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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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Spontaneous Mutations
• Random errors during DNA replication.
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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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What is a gene?
• A gene is a region of DNA that can be expressed to produce a final functional product.
• The product can be a protein or a RNA molecule.
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Protein vs RNA
• Protein – usually structure or enzyme for phenotype
• RNA – often a regulatory molecule which will be discussed in future chapters
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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.
• Be able to discuss RNA and protein processing.
• Be able to describe and discuss mutations.
• Be able to discuss “what is a gene?”.