From DNA to Protein

1. From DNA to Protein Chapter 14 Hsueh-Fen Juan Oct 23, 2012

2. 14.1 DNA, RNA, and Gene Expression • What is genetic information and how does a cell use it? • Ricin:蓖麻毒素，相當可怕又易取得，毒性來自於它可使核醣體失去功能

3. The Nature of Genetic Information • Each strand of DNA consists of a chain of four kinds of nucleotides: A, T, G and C • The sequence of the four bases in the strand is the genetic information

4. Converting a Gene to an RNA • Transcription • Enzymes use the nucleotide sequence of a gene to synthesize a complementary strand of RNA • DNA is transcribed to RNA • Most RNA is single stranded • RNA uses uracil in place of thymine • RNA uses ribose in place of deoxyribose

5. Ribonucleotides and Nucleotides

6. Ribonucleotides and Nucleotides

7. adenine A adenine A DNA RNA deoxyribonucleic acid ribonucleic acid NH 2 NH 2 N N C C C C N N nucleotide base HC HC N CH CH C C N N N sugar– phosphate backbone guanine G guanine G O O N C C N C C NH NH HC HC N N C C C C N N NH 2 NH 2 cytosine C cytosine C NH 2 NH 2 C C HC HC N N C O O HC C HC N N thymine T base pair uracil U O O CH 3 C C C NH HC NH O C O C HC HC N N DNA has one function: It permanently stores a cell’s genetic information, which is passed to offspring. RNAs have various functions. Some serve as disposable copies of DNA’s genetic message; others are catalytic. Nucleotide bases of DNA Nucleotide bases of RNA Fig. 14-3, p. 217

8. RNA in Protein Synthesis • Messenger RNA (mRNA) • Contains information transcribed from DNA • Ribosomal RNA (rRNA) • Main component of ribosomes, where polypeptide chains are built • Transfer RNA (tRNA) • Delivers amino acids to ribosomes

9. microRNAs • Endogenous approximately 23 nt RNAs • Control gene expression at the post-transcriptional level by degrading or repressing target messenger RNAs (mRNAs) • Involved in diverse physiological and pathological processes. • Oncomirs: miRNAs which is closely related to tumor Bartel DP Cell2009. Chang YM & Juan HF et al PNAS2008. Bartel DP Cell2004. Suzuki HI et al Nature2009. 9 Esquela-Kerscher A & Slack FJ. Nat Rev Cancer2006.

10. Dicer Pre-miRNA unwind Drosha mature miRNA Pri-miRNA RISC miRNA gene 3’UTR 5’UTR 3’UTR 5’UTR AAAAA AAAAA Translational Repression mRNA Degradation

11. Converting mRNA to Protein • Translation • The information carried by mRNA is decoded into a sequence of amino acids, resulting in a polypeptide chain that folds into a protein • mRNA is translated to protein • rRNA and tRNA translate the sequence of base triplets in mRNA into a sequence of amino acids

12. Gene Expression • A cell’s DNA sequence (genes) contains all the information needed to make the molecules of life • Gene expression • A multistep process including transcription and translation, by which genetic information encoded by a gene is converted into a structural or functional part of a cell or body

13. 14.2 Transcription: DNA to RNA • RNA polymerase assembles RNA by linking RNA nucleotides into a chain, in the order dictated by the base sequence of a gene • A new RNA strand is complementary in sequence to the DNA strand from which it was transcribed

14. DNA Replication and Transcription • DNA replication and transcription both synthesize new molecules by base-pairing • In transcription, a strand of mRNA is assembled on a DNA template using RNA nucleotides • Uracil (U) nucleotides pair with A nucleotides • RNA polymerase adds nucleotides to the transcript • 同DNA複製，能量來自水解高能磷酸鍵

15. Base-Pairing in DNA Synthesis and Transcription

16. The Process of Transcription • RNA polymerase and regulatory proteins attach to a promoter (a specific binding site in DNA close to the start of a gene) 這句很重要，說明了轉錄的起始準備所有所需物質 • RNA polymerase moves over the gene in a 5‘ to 3’ direction, unwinds the DNA helix, reads the base sequence, and joins free RNA nucleotides into a complementary strand of mRNA (RNA聚合酶超強多功能，不需解旋酶自己就可解)

17. Transcription

18. newly forming RNA transcript gene region RNA polymerase, the enzyme that catalyzes transcription DNA template winding up DNA template unwinding A RNA polymerase binds to a promoter in the DNA, along with regulatory proteins. The binding positions the polymerase near a gene in the DNA. B The polymerase begins to move along the DNA and unwind it. As it does, it links RNA nucleotides into a strand of RNA in the order specified by the base sequence of the DNA. In most cases, the nucleotide sequence of the gene occurs on only one of the two strands of DNA. Only the complementary strand will be translated into RNA. The DNA double helix winds up again after the polymerase passes. The structure of the “opened” DNA molecule at the transcription site is called a transcription bubble, after its appearance. Fig. 14-5a, p. 218

19. transcription site 5’ 3’ growing RNA transcript C What happened in the gene region? RNA polymerase catalyzed the covalent bonding of many nucleotides to one another to form an RNA strand. The base sequence of the new RNA strand is complementary to the base sequence of its DNA template—a copy of the gene. (接的方式同DNA複製) Fig. 14-5b, p. 219

20. Animation: Gene transcription details

21. Transcription • Many RNA polymerases can transcribe a gene at the same time

22. 14.3 RNA and the Genetic Code • Base triplets in an mRNA are words in a protein-building message • Two other classes of RNA (rRNA and tRNA) translate those words into a polypeptide chain

23. Post-Transcriptional Modifications • In eukaryotes, RNA is modified before it leaves the nucleus as a mature mRNA (真核在核內修飾) • Introns • Nucleotide sequences that are removed from a new RNA • Exons • Sequences that stay in the RNA

24. Alternative Splicing • Alternative splicing (選擇性/可變 剪接) • Allows one gene to encode different proteins • Some exons are removed from RNA and others are spliced together in various combinations • After splicing, transcripts are finished with a modified guanine “cap” at the 5‘ end and a poly-A tail at the 3’ end (cap用來幫助mRNA附著核醣體，tail用來防水解)

25. Post-Transcriptional Modifications

26. Animation: Pre-mRNA transcript processing

27. mRNA – The Messenger • mRNA carries protein-building information to ribosomes and tRNA for translation • Codon • A sequence of three mRNA nucleotides that codes for a specific amino acid • The order of codons in mRNA determines the order of amino acids in a polypeptide chain

28. Genetic Information • From DNA to mRNA to amino acid sequence

29. Genetic Code • Genetic code • Consists of 64 mRNA codons (triplets) • Some amino acids can be coded by more than one codon • Some codons signal the start or end of a gene • AUG (methionine) is a start codon (同時也決定met) • UAA, UAG, and UGA are stop codons (不決定胺基酸)

30. Codons of the Genetic Code

31. rRNA and tRNA – The Translators • tRNAs deliver amino acids to ribosomes • tRNA has an anticodon complementary to an mRNA codon, and a binding site for the amino acid specified by that codon • Ribosomes, which link amino acids into polypeptide chains, consist of two subunits of rRNA and proteins (負責催化合成長肽鍊的是核醣體的rRNA，而非蛋白質)

32. Ribosomes

33. tRNA

34. 14.4 Translation: RNA to Protein • Translation converts genetic information carried by an mRNA into a new polypeptide chain • The order of the codons in the mRNA determines the order of the amino acids in the polypeptide chain

35. Translation • Translation occurs in the cytoplasm of cells • Translation occurs in three stages • Initiation • Elongation • Termination

36. Initiation • An initiation complex is formed • A small ribosomal subunit binds to mRNA (小次單元附著mRNA) • The anticodon of initiator tRNA base-pairs with the start codon (AUG) of mRNA • A large ribosomal subunit joins the small ribosomal subunit

37. Elongation • The ribosome assembles a polypeptide chain as it moves along the mRNA • Initiator tRNA carries methionine, the first amino acid of the chain • The ribosome joins each amino acid to the polypeptide chain with a peptide bond

38. Termination • 一條mRNA上可以有多個核醣體同時進行轉譯，此多核醣體+mRNA之複合物稱為polysome • 轉譯能量來自mRNA之GTP-cap的高能磷酸鍵 • ATP用來將胺基酸附著在free tRNA上 • When the ribosome encounters a stop codon, polypeptide synthesis ends • Release factors bind to the ribosome • Enzymes detach the mRNA and polypeptide chain from the ribosome

39. Translation in Eukaryotes

40. Initiation A A mature mRNA leaves the nucleus and enters cytoplasm, which has many free amino acids, tRNAs, and ribosomal subunits. mRNA initiator tRNA small ribosomal subunit An initiator tRNA binds to a small ribosomal subunit and the mRNA. large ribosomal subunit B A large ribosomal subunit joins, and the cluster is now called an initiation complex. Stepped Art Fig. 14-12 (a-b), p. 222

41. Elongation C An initiator tRNA carries the amino acid methionine, so the first amino acid of the new polypeptide chain will be methionine. A second tRNA binds the second codon of the mRNA (here, that codon is GUG, so the tRNA that binds carries the amino acid valine). A peptide bond forms between the first two amino acids (here, methionine and valine). Fig. 14-12c, p. 223

42. D The first tRNA is released and the ribosome moves to the next codon in the mRNA. A third tRNA binds to the third codon of the mRNA (here, that codon is UUA, so the tRNA carries the amino acid leucine). A peptide bond forms between the second and third amino acids (here, valine and leucine). Fig. 14-12d, p. 223

43. E The second tRNA is released and the ribosome moves to the next codon. A fourth tRNA binds the fourth mRNA codon (here, that codon is GGG, so the tRNA carries the amino acid glycine). A peptide bond forms between the third and fourth amino acids (here, leucine and glycine). Fig. 14-12e, p. 223

44. Termination F Steps d and e are repeated over and over until the ribosome encounters a stop codon in the mRNA. The mRNA transcript and the new polypeptide chain are released from the ribosome. The two ribosomal subunits separate from each other. Translation is now complete. Either the chain will join the pool of proteins in the cytoplasm or it will enter rough ER of the endomembrane system (Section 4.9). Fig. 14-12f, p. 223

45. Animation: Translation

46. 14.5 Mutated Genes and Their Protein Products • If the nucleotide sequence of a gene changes, it may result in an altered gene product, with harmful effects • 因為密碼子有退化性，不同的密碼子也可能指向相同胺基酸，這稱為細胞的margin of safety • Mutations • Small-scale changes in the nucleotide sequence of a cell’s DNA that alter the genetic code

47. Common Mutations • Base-pair-substitution (點突變) • May result in a premature stop codon or a different amino acid in a protein product • Example:sickle-cell anemia • Deletion or insertion • Can cause the reading frame of mRNA codons to shift, changing the genetic message • Example:Huntington’s disease

48. Common Mutations

49. A Part of the DNA, mRNA, and amino acid sequence of the beta chain of a normal hemoglobin molecule. part of DNA mRNA transcribed from DNA B A base-pair substitution in DNA replaces a thymine with an adenine. resulting amino acid sequence THREONINE PROLINE GLUTAMATE GLUTAMATE LYSINE base substitution in DNA When the altered mRNA is translated, valine replaces glutamate as the sixth amino acid of the new polypeptide chain. Hemoglobin with this chain is HbS—sickle hemoglobin (Section 3.6). altered mRNA altered amino acid sequence THREONINE PROLINE VALINE GLUTAMATE LYSINE C Deletion of the same thymine causes a frameshift. deletion in DNA The reading frame for the rest of the mRNA shifts, and a different protein product forms. This mutation results in a defective hemoglobin molecule. The outcome is thalassemia, a type of anemia. altered mRNA altered amino acid sequence THREONINE PROLINE GLYCINE ARGININE Stepped Art Fig. 14-13, p. 224

50. Animation: Base-pair substitution