From Gene to Protein Lecture Notes Biol 100 – K.Marr

1. From Gene to Protein Lecture NotesBiol 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

2. 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

3. 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

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

5. CFTR Protein: The cystic fibrosis transmembrane regulator protein 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? Carbohydrate Cytoplasm of cell lining duct or lungs Chloride ions CFTR Protein Water Inside of duct or Air sac in lungs Cell membrane Water

6. 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?

7. 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?

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

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

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

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

12. Comparing DNA and RNA

13. 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

14. 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 Processing of Eukaryotic RNA Tail Introns removed Exons spliced together mRNA Coding sequence Nucleus Cytoplasm

15. 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?

16. Transfer RNA: tRNA 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

17. 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

18. 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

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

20. 2. Elongation Peptide bond forms

21. 2. Elongation continues: Translocation of Ribosome Ribosome moves tRNA ejected

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

23. 3. Termination continued: Disassembly of Ribosome tRNA Polypeptide chain

24. 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?

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

26. 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!!)

27. 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?

28. 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

29. 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)

30. 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?

31. 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?

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

33. 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)

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

35. 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 Sickle-cell hemoglobin Normal hemoglobin Glu Val

36. Types of Mutations:Base Substitutions, Insertions or deletions • Base substitutions • 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

37. 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

38. 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

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

40. RNA Polymerase Review: DNA RNA Protein 1 1. Transcription Nucleus RNA transcript DNA 2. RNA processing 2 Intron 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. Elongation 5