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Transformation-Griffith’s Expt

Transformation-Griffith’s Expt. 1928. DNA Mediates Transformation. Convert IIR to IIIS By DNA?. Avery MacLeod and McCarty Experiment. Circa 1943. Transforming Principle. DNAse activity. + means that activity is present. All RNA gets degraded during enzyme preparation.

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Transformation-Griffith’s Expt

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  1. Transformation-Griffith’s Expt 1928

  2. DNA Mediates Transformation Convert IIR to IIIS By DNA?

  3. Avery MacLeod and McCarty Experiment Circa 1943

  4. Transforming Principle

  5. DNAse activity + means that activity is present All RNA gets degraded during enzyme preparation

  6. Chapter 12Translation and the Genetic Code

  7. Protein Structure Proteins are complex macromolecules composed of 20 (?) different amino acids.

  8. Amino Acids • Proteins are made of polypeptides. • A polypeptide is a long chain of amino acids. • Amino acids have a free amino group, a free carboxyl group, and a side group (R).

  9. Peptide Bonds • Amino acids are joined by peptide bonds. • The carboxyl group of one amino acid is covalently attached to the amino group of the next amino acid.

  10. Protein Synthesis: Translation The genetic information in mRNA molecules is translated into the amino acid sequences of polypeptides according to the specifications of the genetic code.

  11. The Macromolecules of Translation • Polypeptides and rRNA molecules Euk: 28S, 18S, 5.8S, 5S • Amino-acid Activating Enzymes • tRNA Molecules • Soluble proteins involved in polypeptide chain initiation, elongation, and termination

  12. Why does one need the ribosome to translate mRNA?

  13. Ribosomes What does “S” mean? Why do sizes get bigger?

  14. The Nucleolus • In eukaryotes, the nucleolus is the site of rRNA synthesis and ribosome assembly

  15. Synthesis and Processing of the 30S rRNA Precursor in E. coli ProKaryote numbers

  16. Synthesis and Processing of the 45S rRNA Precursor in Mammals

  17. rRNA Genes • rRNA Genes in E. coli • Seven rRNA genes distributed among three sites on the chromosome • rRNA Genes in Eukaryotes • rRNA genes are present in hundreds to thousands of copies • The 5.8S-18S-28S rRNA genes are present in tandem arrays in the nucleolar organizer regions of the chromosomes. • The 5S rRNA genes are distributed over several chromosomes.

  18. Transfer RNAs (tRNAs) • tRNAs are adapters between amino acids and the codons in mRNA molecules. • The anticodon of the tRNA base pairs with the codon of mRNA. • The amino acid is covalently attached to the 3’ end of the tRNA. • tRNAs often contain modified nucleosides.

  19. What is Inosine?

  20. Inosine

  21. tRNA Structure

  22. Specificity of tRNAs • tRNA molecules must have the correct anticodon sequence. • tRNA molecules must be recognized by the correct aminoacyl-tRNA synthetase. • tRNA molecules must bind to the appropriate sites on the ribosomes.

  23. Codon Specificity Resides in the tRNA, Not the Attached Amino Acid.

  24. tRNA Binding Sites on the Ribosome (Ribosme moves like an enzyme)

  25. Stages of Translation • Polypeptide Chain Initiation • Chain Elongation • Chain Termination

  26. Translation Initiation in E. coli • 30S subunit of the ribosome • Initiator tRNA (tRNAMet) • mRNA • Initiation Factors IF-1, IF-2, and IF-3 • One molecule of GTP • 50S subunit of the ribosome

  27. The Shine-Dalgarno Sequence

  28. Translation Initiation in Eukaryotes • The amino group of the methionine on the initiator tRNA is not formylated. • The initiation complex forms at the 5’ terminus of the mRNA, not at the Shine-Dalgarno/AUG translation start site. • The initiation complex scans the mRNA for an AUG initiation codon. Translation usually begins at the first AUG. • Kozak’s Rules describe the optimal sequence for efficient translation initiation in eukaryotes.

  29. Polypeptide Chain Elongation • An aminoacyl-tRNA binds to the A site of the ribosome. • The growing polypeptide chain is transferred from the tRNA in the P site to the tRNA in the A site by the formation of a new peptide bond. • The ribosome translocates along the mRNA to position the next codon in the A site. At the same time, • The nascent polypeptide-tRNA is translocated from the A site to the P site. • The uncharged tRNA is translocated from the P site to the E site.

  30. Elongation of Fibroin Polypeptides (A mRNA can have multiple Ribosomes

  31. Polypeptide Chain Termination • Polypeptide chain termination occurs when a chain-termination codon (stop codon) enters the A site of the ribosome. • The stop codons are UAA, UAG, and UGA. • When a stop codon is encountered, a release factor binds to the A site. • A water molecule is added to the carboxyl terminus of the nascent polypeptide, causing termination.

  32. No tRNA exists for stop codons!

  33. Dissociation upon finish of protein synthesis

  34. The Genetic Code The genetic code is a nonoverlapping code, with each amino acid plus polypeptide initiation and termination specified by RNA codons composed of three nucleotides.

  35. Properties of the Genetic Code • The genetic code is composed of nucleotide triplets. • The genetic code is nonoverlapping. (?) • The genetic code is comma-free. (?) • The genetic code is degenerate. (yes) • The genetic code is ordered. (5’ to 3’) • The genetic code contains start and stop codons. (yes) • The genetic code is nearly universal. YES :)

  36. A Triplet Code*

  37. A Single-Base Pair Insertion Alters the Reading Frame*

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