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Quiz. Nucleotide? Added to which end? Lagging strand?. DNA pol III synthesizes leading strand continuously. 3 . 5 . Parental DNA. DNA pol III starts DNA synthesis at 3  end of primer, continues in 5   3  direction. 5 . 3 . 5 .

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  1. Quiz • Nucleotide? • Added to which end? • Lagging strand?

  2. DNA pol III synthesizes leading strand continuously 3 5 Parental DNA DNA pol III starts DNA synthesis at 3 end of primer, continues in 5 3 direction 5 3 5 Lagging strand synthesized in short Okazaki fragments, later joined by DNA ligase Primase synthesizes a short RNA primer 3 5

  3. Chapter 17 From Gene to Protein

  4. Big Questions • How does your body produce insulin? • How does your offspring know how to make insulin? • Central “Dogma” DNA > RNA > “Protein” (Info > Message > Product)

  5. Chapter 17 Study Guide Questions The Connection between Genes and Proteins 1. Explain how RNA differs from DNA. 2. Briefly explain how information flows from gene to protein. Is the central dogma ever violated? 3. Distinguish between transcription and translation. 4. Compare where transcription and translation occur in bacteria and in eukaryotes. 5. Define “codon” and explain the relationship between the linear sequence of codons on mRNA and the linear sequence of amino acids in a polypeptide. 6. Explain what it means to say that the genetic code is redundant and unambiguous. 7. Explain the significance of the reading frame during translation.

  6. Chapter 17 Study Guide Questions The Synthesis and Processing of RNA 8. Explain how RNA polymerase recognizes where transcription should begin. Describe the role of the promoter, the terminator, and the transcription unit. 9. Explain the general process of transcription, including the three major steps of initiation, elongation, and termination. 10. Explain how RNA is modified after transcription in eukaryotic cells. 11. Define and explain the role of ribozymes. What three properties allow some RNA molecules to function as ribozymes? 12. Describe the functional and evolutionary significance of introns. 13. Explain why, due to alternative RNA splicing, the number of different protein products an organism can produce is much greater than its number of genes.

  7. Chapter 17 Study Guide Questions The Synthesis of Protein 14. Describe the structure and function of tRNA. 15. Explain how tRNA is joined to the appropriate amino acid. 16. Describe the structure and functions of ribosomes. 17. Describe the process of translation (including initiation, elongation, and termination) and explain which enzymes, protein factors, and energy sources are needed for each stage. 18. Describe the significance of polyribosomes. 19. Explain what determines the primary structure of a protein and describe how a polypeptide must be modified before it becomes fully functional. 20. Define “point mutations”. Distinguish between base-pair substitutions and base-pair insertions. Give an example of each and note the significance of such changes.

  8. Today’s Tip: Ricin will kill you Georgi Ivanov Markov Inhibits ribosome translation of mRNA

  9. 1. Explain how RNA differs from DNA. DNA • Double-stranded • ATCG • Deoxyribose RNA • Single-stranded • AUCG • Ribose

  10. 2. Briefly explain how information flows from gene to protein. Is the central dogma ever violated? Genes Specific sequences of nucleotides Proteins Instructions carried in genes Gene expression • DNA directs protein synthesis Includes two stages: • transcription and translation Cellular chain of command: DNA RNA protein

  11. 3. Distinguish between transcription and translation. Transcription • synthesis of RNA under the direction of DNA Messenger RNA (mRNA) Product of transcription Translation • synthesis of a polypeptide • occurs under the direction of mRNA • _________are the sites of translation

  12. 4. Compare where transcription and translation occur in bacteria and in eukaryotes. Prokaryotes Transcription – cytoplasm Translation – cytoplasm Eukaryotes Transcription – Nucleus Translation – cytoplasm

  13. LE 17-3-1 DNA TRANSCRIPTION Prokaryotic cell

  14. LE 17-3-2 DNA TRANSCRIPTION mRNA Ribosome Prokaryotic cell Polypeptide Prokaryotic cell

  15. LE 17-3-3 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Prokaryotic cell Nuclear envelope DNA TRANSCRIPTION Eukaryotic cell

  16. LE 17-3-4 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Prokaryotic cell Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Eukaryotic cell

  17. LE 17-3-5 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Prokaryotic cell Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Polypeptide Eukaryotic cell

  18. 5. Define “codon” and explain the relationship between the linear sequence of codons on mRNA and the linear sequence of amino acids in a polypeptide. Codons • mRNA base triplets • specifies specific amino acid • amino acid placed at the corresponding position along a polypeptide Template strand • provides a template for ordering the sequence of nucleotides in an RNA transcript

  19. 5. Define “codon” and explain the relationship between the linear sequence of codons on mRNA and the linear sequence of amino acids in a polypeptide. Triplet code • nonoverlapping • three-nucleotide words • code for amino acids Example: • AGT on DNA strand • results in the placement of the amino acid serine • at the corresponding position of the polypeptide to be produced

  20. 6. Explain what it means to say that the genetic code is redundant and unambiguous. How many amino acids? 20 How many codons? 64 What does that imply?

  21. 6. Explain what it means to say that the genetic code is redundant and unambiguous. Redundant but not ambiguous (?) 1. One amino acid can have more than one codon e.g., UCU, UCC, UCA, etc., all code for Serine 2. No codon specifies more than one amino acid

  22. 7. Explain the significance of the reading frame during translation. The fat cat ate the rat Reading Frame • Codons must be read in the correct reading frame • What happens if you are off by one letter? • Frame Shift Error

  23. Practice What amino acid does CAC code for? • AGA? • CGU? • Three code for stop • AUG codes for begin

  24. Evolution of the Genetic Code The genetic code is nearly universal What does that mean? • shared by the simplest bacteria to the most complex animals What does that imply? Luciferase

  25. 9. Explain the general process of transcription, including the three major steps of initiation, elongation, and termination. The three stages of transcription: 1) Initiation 2) Elongation 3) Termination

  26. Molecular Components of Transcription RNA Polymerase pries the DNA strands apart and hooks together the RNA nucleotides • RNA synthesis follows the same base-pairing rules as DNA • except __?__ substitutes for thymine

  27. 8. Explain how RNA polymerase recognizes where transcription should begin. Describe the role of the promoter, the terminator, and the transcription unit. Initiation Promoter • DNA sequence where RNA polymerase attaches Transcription Unit • stretch of DNA that is transcribed Terminator • DNA sequence found only on prokaryotes Animation: Transcription

  28. LE 17-7 Promoter Transcription unit 5¢ 3¢ 5 3¢ DNA Start point RNA polymerase Initiation 5¢ 3¢ 5 3¢ Template strand of DNA RNA tran- script Unwound DNA Elongation Rewound DNA 5¢ 3¢ 3¢ 5 3¢ 5¢ RNA transcript

  29. LE 17-7 Promoter Transcription unit 5 3 3¢ 5¢ DNA Start point RNA polymerase Initiation 5¢ 3¢ 5¢ 3¢ Template strand of DNA RNA tran- script Unwound DNA Elongation Rewound DNA 5¢ 3¢ 3¢ 3¢ 5¢ 5¢ RNA transcript Termination 5¢ 3¢ 3¢ 5¢ 5¢ 3¢ Completed RNA transcript

  30. Elongation of the RNA Strand RNA polymerase • untwists the double helix • 10 to 20 bases at a time • 40 nucleotides per second (eukaryotes) • can be transcribed simultaneously by several RNA polymerases Why?

  31. Termination of Transcription Mechanisms of termination are different in prokaryotes and eukaryotes Prokaryotes • polymerase stops transcription at the end of the terminator sequence Eukaryotes • polymerase continues transcription after the pre-mRNA is cleaved • Polymerase eventually falls off the DNA

  32. 10. Explain how RNA is modified after transcription in eukaryotic cells. • Does the pre-mRNA now leave the nucleus? • How is the pre-mRNA modified?

  33. Alteration of mRNA Ends Each end of a pre-mRNA molecule is modified in a particular way: • The 5 end receives a modified nucleotide cap • The 3 end gets a poly-A tail These modifications share several functions: • They seem to facilitate the export of mRNA • They protect mRNA from hydrolytic enzymes • They help ribosomesattach to the 5’ end

  34. Split Genes and RNA Splicing Intervening sequences (Introns) • long noncoding stretches - removed Exons • shorter coding sections – will be expressed RNA splicing • removesintrons and joins exons • creating an mRNA molecule with a continuous coding sequence

  35. RNA duplicating RNA, a step closer to the origin of lifeBy YunXie |http://arstechnica.com/science/news/2011/04/investigations-into-the-ancient-rna-world.ars NASA JPL According to the “RNA world” model of life's origin, RNA performed all of the operations that are essential to life. RNA alone passed on genetic information and catalyzed the reactions of basic metabolism; DNA and proteins were not in the picture. The RNA world hypothesis is an appealingly simple model for simple early life forms, since it allows the complex array of biochemical interactions among proteins, DNA, and RNA to evolve gradually. Our current natural world no longer uses RNA enzymes that act on their own to perform most biological functions. To better understand ancient RNA enzymes, modern scientists have to rely on proxies, like engineered RNA "ribozymes" that have catalytic functions without the need for proteins. However, scientists have had trouble creating a proxy for the first self-replicating molecule, or even an RNA ribozyme that can copy an RNA that's long enough to have further biological functions. Aniela Wochner and her coauthors have overcome that difficulty. In a recent issue of Science, they report the creation of an RNA ribozyme that synthesizes complex RNAs, including RNAs that act as ribozymes and perform a biological function. Previously, the leading RNA polymerase ribozyme, called R18, could only transcribe RNAs up to 14 bases long (as a frame of reference, R18 itself is about 196 bases long). It was also highly template-dependent, meaning it could only copy certain sequences of RNA. To establish early life on Earth, a ribozyme would need to be able to make a variety of RNA sequences of adequate length, including something long enough to synthesize itself. Wochner and her colleagues sought to engineer a superior RNA ribozyme by modifying R18. First, they wanted to improve the interactions among the template RNA, the ribozyme, and the primer sequence that starts the copying. To make RNA, the ribozyme has to recognize the primer and the template, which base-pair with one another. Then, the ribozyme catalyzes the addition of new bases onto the primer, making an RNA sequence that is complementary to the template.  Scientists have proposed that the one end of the R18 ribozyme interacts with the primer and template. So, Wochner and her colleagues appended a random sequence into the 5’-end of R18 and selected for improved RNA polymerase activity. They found one ribozyme (named C19) that did better than R18 on a specific, short template, but it didn't work well on longer templates. They further modified C19 by making truncated variants of its sequence and screened for improved activity on longer templates. They found one variant (the ribozyme tC19) that can extend primers by up to 95 bases with favorable templates. The final obstacle was the fact that it only worked well on favorable template sequences—the researchers wanted a ribozyme that will be able to copy a diverse range of RNA templates, not just a few favorable ones. To find one, they made 50 million randomly mutated R18 sequences, did numerous rounds of selections, and found a combination of mutations that improved the recognition of diverse templates. They applied those mutations to the tC19 ribozyme, creating the RNA polymerase ribozyme tC19Z (198-bases long). Ribozyme tC19Z synthesizes longer RNA sequences and can work with a greater range of primer-template combinations than any of the previous ribozymes. Wochner and her colleagues were able to use tC19Z to synthesize a minimal version of the hammerhead ribozyme (an RNA that binds to and cleaves an RNA substrate). The synthesized hammerhead ribozyme had catalytic activity, as it was able to cleave an RNA substrate at the expected location in the sequence. Wochner and her coauthors have significantly expanded our abilities to engineer RNA polymerase ribozymes; however, further improvements are still necessary. For example, tC19Z probably cannot synthesize something of its own size in a reasonable amount of time. Nevertheless, it's impressive that the researchers were able to select for such drastic improvements on R18, as the sequence hasn't seen a significant upgrade since its creation almost a decade ago. Their work lead us closer to understanding ribozymes that could have existed in early Earth. Science, 2011. DOI: 10.1126/science.1200752 (About DOIs)

  36. Transfer RNA (tRNA) translates an mRNA message into protein Molecules of tRNA are not identical: Each carries a specific amino acid on one end Each has an anticodon on the other end Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look

  37. 14. Describe the structure and function of tRNA. tRNA molecule • single RNA strand • ~ 80 nucleotides long • cloverleaf shape • twists and folds into a three-dimensional molecule How? • hydrogen bonds A C C

  38. “problems” A. How do you make sure that the correct amino acid to connects to the correct tRNA? B. How do you make sure that the correct tRNA matches up with the correct codon?

  39. 15. Explain how tRNA is joined to the appropriate amino acid. Accurate translation requires two steps: First step: • correct match between a tRNA and an amino acid • done by the enzyme aminoacyl-tRNAsynthetase (20 types) Second step: - correct match between the tRNAanticodon and an mRNA codon

  40. 16. Describe the structure and functions of ribosomes. Ribosomes facilitate specific coupling of tRNAanticodons with mRNA codons in protein synthesis Two ribosomal subunits(large and small) made of proteins and ribosomal RNA (rRNA)

  41. You have brucellosis. What do you do? • Tetracycline: • works by binding specifically to the 30S ribosome of the bacteria • preventing attachment of the aminoacyltRNA to the RNA-ribosome complex

  42. A ribosome has three binding sites for tRNA: • The P site • holds the growing polypeptide chain • The A site • holds the next amino acid to be added • The E site • is the exit site, where discharged tRNAs leave the ribosome

  43. The three stages of translation: Initiation Elongation Termination 17. Describe the process of translation (including initiation, elongation, and termination) and explain which enzymes, protein factors, and energy sources are needed for each stage.

  44. Ribosome Association and Initiation of Translation Initiation stage • brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits

  45. Ribosome Association and Initiation of Translation • First, a small ribosomal subunit binds with mRNA and a special initiator tRNA

  46. Ribosome Association and Initiation of Translation Then the small subunit moves along the mRNA until it reaches the start codon (AUG)

  47. Ribosome Association and Initiation of Translation Proteins called initiation factors bring in the large subunit so the initiator tRNA occupies the P site

  48. Elongation of the Polypeptide Chain Elongation • Amino acids added one by one • Each addition involves proteins called elongation factors • Occurs in three steps: • codon recognition • peptide bond formation • translocation

  49. LE 17-18 Amino end of polypeptide E 3¢ mRNA P site A site Ribosome ready for next aminoacyl tRNA 5¢ GTP 2 2 GDP E E P A P A GDP GTP three steps: 1- codon recognition 2 -peptide bond formation 3- translocation E P A

  50. Release factor Free polypeptide 5¢ 3¢ 3¢ 5¢ 5¢ Stop codon (UAG, UAA, or UGA) When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. LE 17-19 Termination of Translation 3¢ The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. The two ribosomal subunits and the other components of the assembly dissociate. Ribosome acts as one large Ribozyme

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