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From DNA to Protein Chapter 14 Hsueh-Fen Juan Oct 23, 2012 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 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 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 Ribonucleotides and Nucleotides Ribonucleotides and Nucleotides adenine A HC NH 2 N CC N N C N CH guanine G O N CC NH HC NC C N NH 2 DNA deoxyribonucleic acid RNA ribonucleic acid nucleotide base sugar– phosphate backbone cytosine C NH 2 HC C N HC thymine T N N CC N HC N C CH N guanine G O N CC NH HC NC C N NH 2 NH 2 HC C N HC N C O base pair CH 3 C C NH HC N C O Nucleotide bases of DNA NH 2 cytosine C C O O adenine A uracil U O HC C NH HC N C O 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 RNA Fig. 14-3, p. 217 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 microRNAs • Endogenous approximately 23 nt RNAs Bartel DP Cell 2009. • Control gene expression at the post-transcriptional level by degrading or repressing target messenger RNAs (mRNAs) Chang YM & Juan HF et al PNAS 2008. Bartel DP Cell 2004. • Involved in diverse physiological and pathological processes. Suzuki HI et al Nature 2009. • Oncomirs: miRNAs which is closely related to tumor Esquela-Kerscher A & Slack FJ. Nat Rev Cancer 2006. 9 Dicer Drosha Pre-miRNA unwind mature miRNA Pri-miRNA RISC 3’UTR 5’UTR miRNA gene AAAAA Translational Repression 5’UTR 3’UTR mRNA Degradation AAAAA 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 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 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 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 Base-Pairing in DNA Synthesis and Transcription 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) Transcription gene region newly forming RNA transcript 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 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 Animation: Gene transcription details Transcription Many RNA polymerases can transcribe a gene at the same time 14.3 RNA and the Genetic Code Base triplets in an mRNA are words in a proteinbuilding message Two other classes of RNA (rRNA and tRNA) translate those words into a polypeptide chain 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 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 polyA tail at the 3’ end (cap用來幫助mRNA附著核醣 體,tail用來防水解) Post-Transcriptional Modifications Animation: Pre-mRNA transcript processing 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 Genetic Information From DNA to mRNA to amino acid sequence 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) Codons of the Genetic Code 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) Ribosomes tRNA 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 Translation Translation occurs in the cytoplasm of cells Translation occurs in three stages • Initiation • Elongation • Termination 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 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 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 Translation in Eukaryotes Initiation A A mature mRNA mRNA leaves the nucleus and enters cytoplasm, which has many free amino acids, tRNAs, and initiator small ribosomal subunits. tRNA ribosomal An initiator tRNA binds subunit 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 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 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 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 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 Animation: Translation 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 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 Common Mutations part of DNA mRNA transcribed from DNA resulting amino THREONINE PROLINE acid sequence A Part of the DNA, mRNA, and amino acid sequence of the beta chain of a normal hemoglobin molecule. GLUTAMATE GLUTAMATE LYSINE base substitution in DNA altered mRNA altered amino THREONINE acid sequence PROLINE VALINE GLUTAMATE deletion in DNA altered mRNA altered amino THREONINE acid sequence PROLINE GLYCINE ARGININE LYSINE B A base-pair substitution in DNA replaces a thymine with an adenine. 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). C Deletion of the same thymine causes a frameshift. 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. Stepped Art Fig. 14-13, p. 224 Animation: Base-pair substitution Animation: Frameshift mutation What Causes Mutations? Transposable elements (轉置子) • Segments of DNA that can insert themselves anywhere in a chromosomes • 幾乎所有物種都有它的存在,人類有45%DNA與其 相關 • Barbara McClintick 的各種奇怪玉米 Spontaneous mutations • Uncorrected errors in DNA replication • 這很正常,DNA複製速度超快,偶爾出錯也不意外 Harmful environmental agents • Ionizing radiation, UV radiation, chemicals • Thymine dimer 胸腺嘧啶二聚體 McClintock’s Transposable Elements Mutations Caused by Radiation Ionizing radiation damages chromosomes, nonionizing (UV) radiation forms thymine dimers (胸腺嘧啶二聚體) Inherited Mutations Mutations in somatic cells of sexually reproducing species are not inherited Mutations in a germ cell or gamete may be inherited, with evolutionary consequences Transcription Assembly of RNA on unwound Summary: Protein Synthesis in Eukaryotic Cells regions of DNA molecule mRNA processing mRNA rRNA proteins mature mRNA ribosomal transcripts subunits Translation At an intact ribosome, synthesis of a polypeptide chain at the binding sites for mRNA and tRNAs Convergence of RNAs tRNA mature tRNA cytoplasmic pools of amino acids, ribosomal subunits, and tRNAs Protein Fig. 14-16, p. 226 Animation: Protein synthesis summary