From Gene to Protein Lecture Notes Biol 100 – K.Marr Topics for the next few lectures Transcription: From DNA to RNA Translation: From RNA to Protein Understanding Cystic Fibrosis Chapter 10 in Essential Biology by Campbell et al 2. Lab 7. Modeling DNA Structure, DNA Replication and Protein Synthesis Read the introduction carefully Part 1 (through page 9)—modeling DNA Structure and Replication Part 2—modeling transcription and translation

The Flow of Genetic Information: DNA to RNA to Protein  Phenotype Cytoplasm Transcription: DNA copied into mRNA molecule Translation: ribosomes translate mRNA into protein—a chain of amino acids Proteins control phenotype. How? Nucleus DNA Transcription mRNA Translation Protein

A few of the many roles played by proteins: The one gene–one protein hypothesis: The function of a gene is to dictate the production of a specific protein. Why are proteins so important? A few of the many roles played by proteins: Enzymes: catalysts for nearly all chemical reactions in cells; Determine what cells can make and digest Structural components: muscles (actin and myosin), connective tissue (collagen, elastin) Receptors on cell surface for growth factors, hormones, etc. Hormones: e.g. insulin, growth hormone, prolactin Transport: e.g. hemoglobin, spindle fibers Immune system: antibodies

CF phenotype Genes determine which proteins a cell can make Proteins control phenotype e.g. CFTR Gene codes for CFTR protein

Cytoplasm of cell lining duct or lungs CFTR Protein: The cystic fibrosis transmembrane regulator protein Carbohydrate CFTR Protein Pumps chloride ions (salt) into cells lining ducts or the lungs What are the consequences when CFTR doesn’t work? How does a gene control the production of a protein? Cytoplasm of cell lining duct or lungs Chloride ions CFTR Protein Water Inside of duct or Air sac in lungs Cell membrane Water

The order of Bases in a gene determines the order of amino acids in the protein it codes for Is the order of amino acids in a protein important?

Transcription: copying DNA into RNA View animation of transcription Questions to answer: 1. What do we start with and end with? 2. Where does transcription occur? When? 3. What is needed for transcription to occur? 4. What is the sequence of events?

An RNA Nucleotide Phosphate Base (Uracil, U) Sugar: ribose This oxygen is absent in deoxyribose

Transcription of a gene by RNA polymerase RNA nucleotides RNA polymerase Newly made RNA Direction of transcription Template strand of DNA

Transcription: copying DNA into RNA ( 1 of 2) (b) Transcription begins (a) Parent DNA (b) Transcription begins RNA polymerase Complementary base pairing Strand separation

Transcription: copying DNA into RNA ( 2 of 2) (c) Transcription continues (d) Products of transcription Non-coding strand Coding strand New RNA strand (actually several hundred base pairs long) Nucleotide joining Parent DNA totally conserved

Comparing DNA and RNA DNA RNA Number of Strands Sugars Bases

Transcription in Eukaryotic Cells: Differential RNA splicing can result in one gene producing more than one protein (a) Gene Intron 1 Intron 2 Intron 3 Intron 4 Intron 5 Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Transcription (b) Primary transcript RNA splicing: Differential splicing can result in different mRNA molecules and, therefore, different proteins (c) Spliced RNA RNA Processing (d) Mature RNA Translation Fig. 7.07 (d) protein

Processing of Eukaryotic RNA Intron Exon RNA Processing includes Adding a cap and tail Removing introns Splicing exons together Differential splicing produces different mRNA molecules Exon Intron Exon Gene (DNA) Transcription + the Addition of cap and tail Cap RNA transcript with cap and tail Introns removed Tail Exons spliced together mRNA Coding sequence Nucleus Cytoplasm

Translation: Ribosomes reading mRNA to produce a polypeptide View animation of translation Questions to answer What do we start with and end with? 2. Where does translation occur? 3. What is needed for translation to occur? What is the sequence of events? What are the roles of mRNA, ribosomes, start codon, tRNA, anticodons, stop codon?

Transfer RNA: tRNA tRNA Acts as a molecular interpreter Amino acid attachment site tRNA Acts as a molecular interpreter Carries amino acids Matches amino acids with codons in mRNA using anticodons Hydrogen bond RNA polynucleotide chain Anticodon Anticodon

A portion of an mRNA molecule attached to a tRNA Codon on mRNA mRNA Each Codon codes Specifies a specific tRNA—amino acid complex Amino acid

A ribosome translating mRNA into protein Small subunit Ribosomes Organelle that makes protein Reads mRNA 5’  3’ Made of rRNA and protein Consist of 2 subunits mRNA Large subunit Protein under construction

1. Initiation of Translation Codon mRNA Anticodon Ribosome tRNA Amino acid

2. Elongation Peptide bond forms

2. Elongation continues: Translocation of Ribosome moves tRNA ejected

3. Termination of Translation factor binds Ribosome moves tRNA ejected Peptide bond forms

3. Termination continued: Disassembly of Ribosome tRNA Polypeptide chain

Transcription & Translation of the CRTR Gene in Healthy People Part of a normal CFTR gene: 5’...ATCATCTTTGGTGTT...3’ non-coding strand 3’...TAGTAGAAACCACAA...5’ coding strand Transcribe this portion of the gene. The whole gene codes for 1480 amino acids in CFTR protein! What is the order of bases in the resulting mRNA molecule? Translate this portion of the gene. What is the order of amino acids in the resulting protein?

Table of Codons found on mRNA Each codon specifies a specific amino acid The same genetic code is used by nearly all organisms!!

Transcription & Translation of the CRTR Gene in Healthy People Part of a normal CFTR gene: 5’...ATCATCTTTGGTGTT...3’ non-coding strand 3’...TAGTAGAAACCACAA...5’ coding strand Transcription 5’...AUCAUCUUUGGUGUU...3’ Translation .....Ile-Ile-Phe-Gly-Val… (only 5 of the 1480 amino acids in protein!!)

Transcription & Translation of the CRTR Gene in People with CF Part of CFTR gene associated with Cystic Fibrosis: 5’...ATCATTGGTGTT...3’ non-coding strand 3’...TAGTAACCACAA...5’ coding strand Transcribe this portion of the gene. What is the order of bases in the resulting mRNA molecule? Translate this portion of the gene. What is the order of amino acids in the resulting protein? What is different about the gene and the protein in people with cystic fibrosis?

Transcription & Translation of the CRTR Gene in People with CF Part of CFTR gene associated with Cystic Fibrosis: 5’...ATCATTGGTGTT...3’ non-coding strand 3’...TAGTAACCACAA...5’ coding strand Transcription 5’...AUCAUUGGUGUU...3’ Translation .....Ile-Ile-Gly-Val…….. Phenylalanine (Phe) is missing

Explaining the symptoms of CF Why does CF only affect certain parts of the body? What do the characteristics of CF have in common? Mucus build-up in the lungs Lung infections (e.g. pneumonia) Male sterility (blocked vas deferens) Salty sweat Trouble digesting food (blocked pancreatic duct)

Explaining the symptoms of CF In CF, the faulty CFTR protein never makes it to cell membrane What builds up outside of cells? Why? Why salty sweat? Why does mucus collect in lungs? Why respiratory infections? Why problems with digestion? Why male sterility?

Understanding Cystic Fibrosis at the Cellular Level How does CFTR protein get from where it’s produced to its home in the cell membrane? Where is the CFTR protein produced? CFTR is a glycoprotein—where does it go for modification? How does it get there? How does the modified CFTR protein get to the plasma membrane? The defective CFTR protein is recognized at the ER as defective Where is the defective CFTR protein sent?

CF symptoms may be mild or severe CFTR Gene Several hundred different mutations are associated with CF

What’s a Mutation? Any change in the nucleotide sequence of DNA Types of Mutations Substitution, insertion or deletion Occur during DNA replication Mutations may Result from: Errors in DNA replication Mutagens physical or chemical agents that may cause errors during DNA replication chemicals in cigarette smoke Radiation (e.g. U.V. light, X-rays)

DF508 deletion: the most common cause of cystic fibrosis Why does isoleucine (Ile) at amino acid position 507 remain unchanged?

Mutations responsible for Sickle Cell Anemia Only one amino acid in 146 is incorrect in sickle-cell hemoglobin! Normal hemoglobin DNA Mutant hemoglobin DNA mRNA mRNA Normal hemoglobin Sickle-cell hemoglobin Glu Val

Types of Mutations: Base Substitutions, Insertions or deletions May result in changes in the amino acid sequence in a protein, or May be silent (have no effect) mRNA Protein Met Lys Phe Gly Ala (a) Base substitution Met Lys Phe Ser Ala

Types of Mutations: Base Insertions and deletions Can have disastrous effects Change the reading frame of the genetic message mRNA Protein Met Lys Phe Gly Ala (b) Nucleotide deletion Met Lys Leu Ala His

Although mutations are often harmful They are the source of the rich diversity of genes in the living world They contribute to the process of evolution by natural selection

SUMMARY OF KEY CONCEPTS DNA and RNA: Polymers of Nucleotides Nitrogenous base Phosphate group Sugar DNA Nucleotide Polynucleotide

Review: DNA RNA Protein RNA Polymerase 1. Transcription 1 Nucleus RNA transcript DNA Intron 2. RNA processing 2 Amino acid CAP Tail mRNA Intron Enzyme tRNA 3. Amino acid attachment Ribosomal subunits 4 4. Initiation of translation Stop codon Anticodon Codon 6. Termination 5 5. Elongation