Polypeptide Chain Elongation

1. Polypeptide Chain Elongation http://www.molecularmovies.com/showcase/ • 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.

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

9. 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.

10. No tRNA exists for stop codons!

12. Dissociation upon finish of protein synthesis

13. Fig1

14. 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.

15. 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 :)

16. A Triplet Code*

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

18. A suppressor mutation restores the original reading frame.*

19. Insertion of 3 base pairs does not change the reading frame.*

20. Evidence of a Triplet Code:In Vitro Translation Studies • Trinucleotides were sufficient to stimulate specific binding of aminoacyl-tRNAs to ribosomes. • Chemically synthesized mRNAs containing repeated dinucleotide sequences directed the synthesis of copolymers with alternating amino acid sequences. • mRNAs with repeating trinucleotide sequences directed the synthesis of a mixture of three homopolymers.

21. Deciphering the Genetic Code

22. You must know single letter codes and some triplets! What does Degree of Degeneracy Reflect?

23. The Genetic Code • Initiation and termination Codons • Initiation codon: AUG • Termination codons: UAA, UAG, UGA • Degeneracy: partial and complete • Ordered • Nearly Universal (exceptions: mitochondria and some protozoa)

24. Key Points • Each of the 20 amino acids in proteins is specified by one or more nucleotide triplets in mRNA. (20 amino acids refers to what is attached to the tRNAs!) • Of the 64 possible triplets, given the four bases in mRNA, 61 specify amino acids and 3 signal chain termination. (have no tRNAs!)

25. Key Points • The code is nonoverlapping, with each nucleotide part of a single codon, degenerate, with most amino acids specified by two to four codons, and ordered, with similar amino acids specified by related codons. • The genetic code is nearly universal; with minor exceptions, the 64 triplets have the same meaning in all organisms. (this is funny)

26. Do all cells/animals make the same Repertoire of tRNAs?

27. The Wobble Hypothesis:Base-Pairing Involving the Third Base of the Codon is Less Stringent.

28. Base-Pairing with Inosine at the Wobble Position

29. Suppressor Mutations • Some mutations in tRNA genes alter the anticodons and therefore the codons recognized by the mutant tRNAs. • These mutations were initially detected as suppressor mutations that suppressed the effects of other mutations. • Example: tRNA mutations that suppress amber mutations (UAG chain-termination mutations) in the coding sequence of genes.

30. Making a (UAG) Mutation

31. Translation of an amber (UAG) Mutation in the Absence of a Suppressor tRNA

32. Translation of an amber Mutation in the Presence of a Suppressor tRNA Note it is amber su3…why?????????

33. Translation of an amber Mutation in the Presence of a Suppressor tRNA If there was a single tRNATyr gene, then could one have a amber supressor of it?

34. Historical Comparisons • Comparison of the amino acid sequence of bacteriophage MS2 coat protein and the nucleotide sequence of the gene encoding the protein (Walter Fiers, 1972). Was this first???? • Sickle-cell anemia: comparison of the sequence of the normal and sickle-cell alleles at the amino acid level and at the nucleotide level.

35. Are the proteins produced a pure reflection of the mRNA sequence???? tRNA environment, protein modifications post-translationally

36. Evolution?

40. Alpha and Beta chain mutants…some of them

41. Phylogenetic relationships

44. How could we use GFP fluorescence to figure out-codon optimize GFP?

46. CsCl centrifugation of DNA over time developed by Meselson and Stahl

48. In class question (extra credit) for Quiz #4 Question 1: (0.5pts) Why does one add EtBr to CsCl gradients for the isolation of plasmid DNA? Question 2: (0.5pts-All or None credit) Is an 8kb supercoiled plasmid more dense than a 3kb supercoiled plasmid. Yes/No (circle one) Will an 8kb supercoiled plasmid have more EtBr bound to it? Yes/No (circle one)

49. We will talk about this again in a later lecture: But CsCl gradients are not the same thing as Sucrose Gradients or Agarose Gel Electrophoresis.