Chapter 17: From Gene to Protein (Protein Synthesis)

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).

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

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 Symptoms reflect person’s inability to make proteins/enzymes

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

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

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

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

Central Dogma Transcription Translation Polypeptide chain DNA Transcription RNA Translation Polypeptide chain (will become protein)

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

Why is there an RNA intermediate? Evolutionary adaptation: Check-point in process Provides protection for DNA code More copies can be made simultaneously

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

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 1960’s)

Codon A 3-nucleotide “word” in the Genetic Code 64 possible codons known

Codon Amino acid

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

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 doesn’t matter This allows for mistakes during DNA replication

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)

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

Protein Synthesis Intro Intro movie

Protein Synthesis Intro Step 1: Transcription: DNA  mRNA Step 2: Translation: mRNA  tRNA  Am. Acid  Polypep. chain Polypeptide chain then becomes protein

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

Transcription Steps 1. RNA Polymerase Binding 2. Initiation 3. Elongation 4. Termination

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)

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 Doesn’t require an RNA primer

RNA Polymerase Binding Needs: Promoter Regions (on the DNA) Special sequences of DNA nucleotides that “tell” cell where transcription begins Transcription Factors Proteins

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

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

Transcription Factors Proteins that bind to DNA before RNA Polymerase Recognizes TATA box, attaches, and “flags” the spot for RNA Polymerase RNA poly won’t attach unless these are present

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

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

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

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

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

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

4th Step: Termination DNA sequence that tells RNA Polymerase to stop Ex: AATAAA RNA Polymerase detaches from DNA after closing the helix

Final Product Pre-mRNA This is a “raw” RNA that will need processing and modifications

Modifications of RNA 1. 5’ Cap 2. Poly-A Tail 3. Splicing

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

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

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

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

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

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

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

Materials Required for translation tRNA Ribosomes mRNA

Transfer RNA = tRNA Made by transcription About 80 nucleotides long Carries AA for polypeptide synthesis

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

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

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

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

Translation Steps 1. Initiation 2. Elongation 3. Termination

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

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

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

Elongation Steps: 1. Codon Recognition 2. Peptide Bond Formation 3. Translocation

Codon Recognition tRNA anticodon matched to mRNA codon in the A site

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

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

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

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

Prokaryotes: Prok. vs. Euk. Protein Synthesis Video

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)

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

Mutation types - Cells Somatic cells or body cells – not inherited Germ Cells or gametes - inherited

Point or Spot Mutations Changes in one or a few nucleotides in the genetic code Effects - none to fatal

Types of Point Mutations 1. Base-Pair Substitutions 2. Insertions 3. Deletions

Base-Pair Substitution The replacement of 1 pair of nucleotides by another pair Ex: Sickle cell anemia

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

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”

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

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

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

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

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

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

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

Chernobyl video

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.

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?”