From DNA to Protein: Gene Expression

1. From DNA to Protein: Gene Expression

2. 14 From DNA to Protein: Gene Expression 14.1 What Is the Evidence that Genes Code for Proteins? 14.2 How Does Information Flow from Genes to Proteins? 14.3 How Is the Information Content in DNA Transcribed to Produce RNA?

3. 14 From DNA to Protein: Gene Expression 14.4 How Is Eukaryotic DNA Transcribed and the RNA Processed? 14.5 How Is RNA Translated into Proteins? 14.6 What Happens to Polypeptides after Translation?

4. 14 From DNA to Protein: Gene Expression Methicillin-resistant Staphylococcus aureus (MRSA) is now a major cause of serious illness and death. It is treated with antibiotics such as tetracycline that target its gene expression, but many strains are now becoming resistant to tetracycline. Opening Question: Can new treatments focused on gene expression control MRSA?

5. 14.1 What Is the Evidence that Genes Code for Proteins? The molecular basis of phenotypes was discovered before it was known that DNA is the genetic material. Studies of many different organisms showed that major phenotypic differences were due to differences in specific proteins.

6. 14.1 What Is the Evidence that Genes Code for Proteins? Identification of gene products as proteins began with studies of alkaptonuria, a disease in children. It was more common in children of first cousins. A recessive mutant allele was inherited from both parents. The mutation produced homogentisic acid, which accumulated in blood, joints, and urine, and turned the urine dark brown.

7. 14.1 What Is the Evidence that Genes Code for Proteins? Homogentisic acid (HA) is a breakdown product of the amino acid tyrosine; it is normally converted to a harmless product by an enzyme.

8. 14.1 What Is the Evidence that Genes Code for Proteins? When the allele is mutated, the enzyme is inactive, and HA accumulates. Thus, the researchers correlated one gene to one enzyme. Confirmation required identification of the specific enzyme and gene mutation, which occurred much later. Biologists turned to model organisms to understand gene expression.

9. 14.1 What Is the Evidence that Genes Code for Proteins? Model organisms: • Easy to grow in the laboratory • Short generation times • Easy to manipulate genetically • Produce large numbers of progeny Examples: Pea plants, Drosophila, E. coli, and common bread mold—Neurospora crassa.

10. 14.1 What Is the Evidence that Genes Code for Proteins? Neurospora is haploid for most of its life cycle, so there are no dominant or recessive alleles. Beadle and Tatum used Neurospora to test the one-gene, one-enzyme hypothesis. Wild-type Neurospora strains have enzymes to catalyze all the reactions needed for growth.

11. 14.1 What Is the Evidence that Genes Code for Proteins? Mutations were induced with X-rays as the mutagens—something that damages DNA and causes mutations—heritable alterations in DNA sequences. The mutant strains needed additional nutrients, such as vitamins, to grow.

12. 14.1 What Is the Evidence that Genes Code for Proteins? Each mutant strain required only one additional nutrient. Results suggested that each mutation caused a defect in only one enzyme in a metabolic pathway, confirming the one-gene, one-enzyme hypothesis.

13. 14.1 What Is the Evidence that Genes Code for Proteins? Mutations are a powerful tool to determine cause and effect, and have been used to determine metabolic pathways. If a gene determines synthesis of one enzyme, mutating that gene will result in a nonfunctional enzyme, and the reaction doesn’t occur—stopping the pathway at that point.

14. Figure 14.1 One Gene, One Enzyme

15. 14.1 What Is the Evidence that Genes Code for Proteins? One-gene, one-enzyme has since been revised to the one-gene, one-polypeptide relationship. Many proteins have several polypeptides chains, or subunits. Example: Hemoglobin has four subunits, each specified by a separate gene. Not all genes code for polypeptides.

16. Working with Data 14.1: One Gene, One Enzyme To test the one-gene, one-enzyme hypothesis, X-rays were used to cause mutations in Neurospora. Fifteen mutant strains were produced that could not synthesize arginine, but some strains could grow if supplied with ornithine and citrulline. These compounds are intermediates in the metabolic pathway that synthesizes arginine.

17. Working with Data 14.1: One Gene, One Enzyme The 15 mutant strains were tested for growth in the presence of the other substances: Growth is expressed as dry weight of fungal material after five days.

18. Working with Data 14.1: One Gene, One Enzyme Question 1: Based on the biochemical pathway for arginine synthesis shown in Figure 14.1, which enzyme (A, B, or C) was mutated in each strain?

19. Figure 14.1 One Gene, One Enzyme

20. Working with Data 14.1: One Gene, One Enzyme Question 2: Why was there some growth in strains 34105 and 33442 even when there were no additions to the growth medium?

21. Working with Data 14.1: One Gene, One Enzyme Question 3: Nineteen other amino acids were tested as substitutes for arginine in the three strains. In all cases, there was no growth. Explain these results.

22. Working with Data 14.1: One Gene, One Enzyme Question 4: Sexual reproduction in Neurospora was used to create double mutants, which carried the mutations from both parental strains. A double mutant derived from strains 33442 and 36703 had the growth characteristics shown in the table. Explain these data in terms of the genes, mutations, and biochemical pathway.

23. 14.2 How Does Information Flow from Genes to Proteins? Gene expression occurs in two steps: • Transcription: DNA sequence is copied to a complementary RNA sequence • Translation: RNA sequence is template for an amino acid sequence

24. 14.2 How Does Information Flow from Genes to Proteins? This model was proposed by Crick and Watson, and called “The central dogma of molecular biology.”

25. 14.2 How Does Information Flow from Genes to Proteins? Three kinds of RNA are involved in gene expression: • Messenger RNA (mRNA) and transcription: One strand of DNA is copied to a complementary mRNA strand. In eukaryotes, the mRNA moves to the cytoplasm.

26. 14.2 How Does Information Flow from Genes to Proteins? 2. Ribosomal RNA (rRNA) and translation: Ribosomes are protein synthesis factories made up of proteins and rRNA. rRNA catalyzes peptide bond formation between amino acids, to form a polypeptide.

27. 14.2 How Does Information Flow from Genes to Proteins? 3. Transfer RNA (tRNA): Can bind a specific amino acid, and recognize specific sequences in mRNA. tRNA recognizes which amino acid should be added next to a growing polypeptide chain.

28. 14.2 How Does Information Flow from Genes to Proteins? The central dogma suggested that information flows from DNA to RNA to protein, which raised two questions: • How does genetic information get from the nucleus to the cytoplasm? • What is the relationship between a DNA sequence and an amino acid sequence?

29. Figure 14.2 From Gene to Protein

30. 14.2 How Does Information Flow from Genes to Proteins? Some viruses are exceptions: they have RNA instead of DNA. Most replicate by transcribing RNA to a complementary RNA strand, which then makes multiple copies of the viral genome.

31. 14.2 How Does Information Flow from Genes to Proteins? Retroviruses, such as HIV, make a DNA copy of their genome—reverse transcription. The host cell transcription machinery makes more RNA, resulting in new viral particles.

32. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? Transcription requires: • A DNA template for base pairings • The four ribonucleoside triphosphates (ATP,GTP,CTP,UTP) • An RNA polymerase • Salts and pH buffer, if done in a test tube

33. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? Transcription produces mRNA, tRNA, and rRNA. These RNAs are encoded by specific genes. Eukaryotes also make several small RNAs, including small nuclear RNA (snRNA), microRNA (miRNA), and small interfering RNA (siRNA).

34. Table 14.1

35. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? RNA polymerases catalyze synthesis of RNA: • Catalyze addition of nucleotides in a 5′-to-3′ direction • Processive—one enzyme-template binding results in polymerization of hundreds of RNA bases • They do not need primers

36. Figure 14.3 RNA Polymerase

37. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? Transcription occurs in three phases: 1. Initiation: RNA polymerase binds to a DNA sequence called a promoter. Promoters tell the enzyme where to start and which strand of DNA to transcribe. The promoter has an initiation site where transcription begins.

38. Figure 14.4 DNA Is Transcribed to Form RNA (A)

39. Figure 14.4 DNA Is Transcribed to Form RNA (A)

40. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? Sigma factors and transcription factors are proteins that bind to DNA sequences and to RNA polymerase. They help direct the polymerase onto the promoter, and help determine which genes are expressed at particular times.

41. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? 2. Elongation: RNA polymerase unwinds DNA about 10 base pairs at a time; reads template in 3′ to 5′ direction. The transcript is antiparallel to the DNA template strand. RNA polymerases do not proofread and correct mistakes.

42. Figure 14.4 DNA Is Transcribed to Form RNA (B)

43. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? RNA polymerase uses (ribo)nucleoside triphosphates (NTPs) as substrates. Two phosphate groups are removed from each substrate molecule; the energy released is used to drive the polymerization reaction.

44. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? 3. Termination: Specified by a specific DNA sequence. Mechanism in eukaryotes is not well understood. In bacteria, the transcript forms a loop and falls away from the DNA; or a helper protein binds to the transcript and causes it to detach from the DNA.

45. Figure 14.4 DNA Is Transcribed to Form RNA (C)

46. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? The genetic code specifies which amino acids will be used to build a protein. Codon: a sequence of three bases, something like a three-letter “word.” Each codon specifies a particular amino acid.

47. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? How was the code deciphered? How could 20 “code words” (amino acids) be written with only four “letters” (the four bases)? A triplet code seemed likely; it could result in 4 × 4 × 4 = 64 codons.

48. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? Nirenberg and Matthaei used simple artificial mRNAs of known composition to identify the polypeptide that resulted. This led to the identification of the first three codons.

49. Figure 14.5 Deciphering the Genetic Code

50. 14.3 How Is the Information Content in DNA Transcribed to Produce RNA? Later, scientists used artificial mRNAs only three nucleotides long (one codon). These would bind to a ribosome and a corresponding tRNA carrying an amino acid. Thus the codes for all the amino acids were determined.