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10 From DNA to Protein: Gene Expression Chapter 10 From DNA to Protein: Gene Expression Key Concepts 10.1 Genetics Shows That Genes Code for Proteins 10.2 DNA Expression Begins with Its Transcription to RNA 10.3 The Genetic Code in RNA Is Translated into the Amino Acid Sequences of Proteins Chapter 10 From DNA to Protein: Gene Expression 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes 10.5 Proteins Are Modified after Translation Chapter 10 Opening Question How do antibiotics target bacterial protein synthesis? Concept 10.1 Genetics Shows That Genes Code for Proteins Identification of proteins as gene products began in the early 20th century. Children with a rare disease called alkaptonuria had parents who were first cousins. Garrod realized they must be homozygous for a recessive mutant gene. He isolated homogentisic acid from the children, which accumulated in the blood, joints, and urine. Concept 10.1 Genetics Shows That Genes Code for Proteins Garrod speculated that homogentisic acid is normally broken down to a harmless product by an enzyme, but the dominant wild-type allele for the enzyme was mutated in alkaptonuria patients. These and other studies led him to correlate one gene to one enzyme. Concept 10.1 Genetics Shows That Genes Code for Proteins Homogentisic acid is part of a biochemical pathway of protein breakdown. Phenylketonuria is another genetic disease that involves this pathway. • The enzyme that converts phenylalanine to tyrosine is nonfunctional. • Untreated, it can lead to mental retardation, but is easily detected in newborns. Figure 10.1 Metabolic Diseases and Enzymes Concept 10.1 Genetics Shows That Genes Code for Proteins Study of alkapatonuria and phenylketonuria led to the “one gene–one enzyme” hypothesis. But studies of proteins made up of multiple polypeptides showed that this hypothesis was an oversimplification. Concept 10.1 Genetics Shows That Genes Code for Proteins Hemoglobin consists of 4 polypeptide chains. • Variations in human populations show hundreds of single amino acid alterations in the β-chains, such as the one that results in sickle-cell disease. The one gene–one enzyme relationship has since been revised to the one gene–one polypeptide relationship. Figure 10.2 Gene Mutations and Amino Acid Changes Concept 10.1 Genetics Shows That Genes Code for Proteins Not all genes code for polypeptides. • Some are transcribed to RNA but not translated to polypeptides; the RNAs have other functions. Molecular biology: the study of nucleic acids and proteins; often focuses on gene expression Concept 10.1 Genetics Shows That Genes Code for Proteins Gene are expressed in two steps: • Transcription: information from a DNA sequence is copied to a complementary RNA sequence • Translation: converts the RNA sequence to an amino acid sequence Concept 10.1 Genetics Shows That Genes Code for Proteins Three types of RNA: • Messenger RNA (mRNA)—a DNA sequence is copied to produce a complementary mRNA strand, which moves to a ribosome to be translated. § The nucleotide sequence of the mRNA determines the sequence of amino acids in the polypeptide chain. Concept 10.1 Genetics Shows That Genes Code for Proteins • Ribosomal RNA (rRNA), along with proteins, makes up ribosomes; one catalyzes peptide bond formation. • Transfer RNA (tRNA) binds to specific amino acids and to specific sequences on mRNA by base pairing. § tRNA recognizes which amino acid should be added next in a growing polypeptide and carries it to the ribosome. Figure 10.3 From Gene to Protein Concept 10.2 DNA Expression Begins with Its Transcription to RNA Transcription requires several components: • A DNA template for base pairings • The 4 nucleoside triphosphates (ATP, GTP, CTP, UTP) • An RNA polymerase enzyme Concept 10.2 DNA Expression Begins with Its Transcription to RNA RNA polymerases catalyze synthesis of RNA from the DNA template. RNA polymerases are processive—a single enzyme–template binding results in polymerization of hundreds of RNA bases. Unlike DNA polymerases, RNA polymerases do not need primers. Figure 10.4 RNA Polymerase Concept 10.2 DNA Expression Begins with Its Transcription to RNA Transcription occurs in three steps: • Initiation • Elongation • Termination Concept 10.2 DNA Expression Begins with Its Transcription to RNA Initiation requires a promoter—a control sequence of DNA that tells RNA polymerase where to start transcription and which strand to transcribe. Each promoter has a transcription initiation site. Other proteins (sigma factors and transcription factors) bind “upstream” from the initiation site and help RNA polymerase bind and determine which genes are expressed. Figure 10.5 DNA Is Transcribed to Form RNA (Part 1) Concept 10.2 DNA Expression Begins with Its Transcription to RNA Elongation: RNA polymerase unwinds DNA and reads template in 3′-to-5′ direction. RNA polymerase adds nucleotides to the 3′ end of the new strand; the RNA transcript is antiparallel to the DNA template strand. Uracil (not thymine) in the RNA molecule is paired with adenine in the DNA molecule. RNA polymerases can proofread but allow more mistakes. Concept 10.2 DNA Expression Begins with Its Transcription to RNA Termination is specified by a specific DNA sequence. Mechanisms of termination are complex and vary among different genes and organisms. In eukaryotes, multiple proteins are involved in recognizing the termination sequence and separating the newly formed RNA from the DNA template and RNA polymerase. Figure 10.5 DNA Is Transcribed to Form RNA (Part 2) Concept 10.2 DNA Expression Begins with Its Transcription to RNA Coding regions are sequences of DNA that are expressed as proteins. In prokaryotes, most of the DNA is made up of coding regions. In prokaryotes and viruses, several adjacent genes sometimes share one promoter. Table 10.1 Concept 10.2 DNA Expression Begins with Its Transcription to RNA In eukaryotes each gene has its own promoter and most have noncoding sequences called introns. The transcribed regions are exons. Introns and exons both appear in the primary mRNA transcript (pre-mRNA); introns are removed from the final mRNA. Figure 10.6 Transcription of a Eukaryotic Gene Concept 10.2 DNA Expression Begins with Its Transcription to RNA Nucleic acid hybridization can be used to locate introns. Target DNA is denatured by heat to separate the strands. It is then incubated with a probe—a nucleic acid strand from another source. If the probe has a complementary sequence, it forms a hybrid with the DNA by base pairing. Figure 10.7 Nucleic Acid Hybridization Concept 10.2 DNA Expression Begins with Its Transcription to RNA Researchers used this technique to examine the β-globin gene with previously isolated βglobin mRNA as the probe. Viewing the hybridized molecules by electron microscopy, they saw that the introns formed loops—stretches of DNA that did not have complementary base sequences on the mature mRNA. If pre-mRNA was used, the result was a linear matchup—complete hybridization. Figure 10.8 Demonstrating the Existence of Introns Concept 10.2 DNA Expression Begins with Its Transcription to RNA Introns interrupt, but do not scramble, the DNA sequence of a gene. Most eukaryotic genes contain introns. Concept 10.2 DNA Expression Begins with Its Transcription to RNA The pre-mRNA is modified: RNA splicing removes introns and splices exons together. Consensus sequences are short sequences between exons and introns; they are first bound by snRNPs—small nuclear ribonucleoprotein particles. Concept 10.2 DNA Expression Begins with Its Transcription to RNA Then other proteins accumulate to form a large RNA–protein complex called a spliceosome. The complex cuts the pre-mRNA, releases the introns, and joins the exons together to produce mature mRNA. Figure 10.9 The Spliceosome: An RNA Splicing Machine Concept 10.2 DNA Expression Begins with Its Transcription to RNA In the disease beta thalassemia, a mutation at an intron consensus sequence in the βglobin gene prevents the pre-mRNA from being spliced correctly. Nonfunctional β-globin mRNA is produced, resulting in severe anemia. This is an example of how mutations are used to elucidate cause-and-effect relationships. Concept 10.2 DNA Expression Begins with Its Transcription to RNA The pre-mRNA is also modified at both ends: A 5′ cap (G cap) is added to the 5′ end. It facilitates binding to a ribosome and prevents digestion by ribonucleases. A poly A tail is added to the 3′ end. It assists in export from the nucleus and contributes to the stability of the mRNA. IN-TEXT ART, p. 202 Concept 10.3 The Genetic Code in RNA Is Translated into the Amino Acid Sequences of Proteins Translation of the nucleotide sequence of an mRNA into an amino acid sequence occurs at ribosomes. In prokaryotes, transcription and translation are coupled: ribosomes often bind to an mRNA as it is being transcribed. In eukaryotes, the nuclear envelope separates mRNA production and translation. Concept 10.3 The Genetic Code in RNA Is Translated into the Amino Acid Sequences of Proteins The genetic information is a series of sequential, nonoverlapping, three-letter “words” (3 bases) called codons. Each codon specifies an amino acid. Codons were first identified by using short artificial polynucleotides instead of complex mRNAs. Figure 10.10 Deciphering the Genetic Code (Part 1) Figure 10.10 Deciphering the Genetic Code (Part 2) Concept 10.3 The Genetic Code in RNA Is Translated into the Amino Acid Sequences of Proteins There are more codons than amino acids. AUG codes for methionine and is also the start codon. UAA, UAG, and UGA are stop codons. For most amino acids, there is more than one codon; the genetic code is redundant. But it is not ambiguous—each codon specifies only one amino acid. Figure 10.11 The Genetic Code Concept 10.3 The Genetic Code in RNA Is Translated into the Amino Acid Sequences of Proteins The genetic code is nearly universal: the codons that specify amino acids are the same in all organisms. A few exceptions occur in mitochondria, chloroplasts, and some protists. The genetic code is ancient; it unifies life and indicates that all life came from a common ancestor. It is also the basis for genetic engineering. Concept 10.3 The Genetic Code in RNA Is Translated into the Amino Acid Sequences of Proteins Point mutations also confirm the genetic code. Point mutations in a coding region are compared with the amino acid sequences in the resulting polypeptides. • Silent mutations can occur because of redundancy § Example: CCG and CCU are both translated as proline (Pro). Figure 10.12 Mutations (Part 1) Figure 10.12 Mutations (Part 2) Concept 10.3 The Genetic Code in RNA Is Translated into the Amino Acid Sequences of Proteins • Missense mutations result in a change in the amino acid sequence. § Example: GAU is translated as aspartic acid (Asp), whereas a mutation that results in GUU is translated as valine (Val). Figure 10.12 Mutations (Part 3) Concept 10.3 The Genetic Code in RNA Is Translated into the Amino Acid Sequences of Proteins • Nonsense mutations result in a premature stop codon, resulting in a shortened protein that is usually not functional. Figure 10.12 Mutations (Part 4) Concept 10.3 The Genetic Code in RNA Is Translated into the Amino Acid Sequences of Proteins • Frame-shift mutations are insertions or deletions of one or more bases. § This causes new sets of triplets to be read and an altered sequence of amino acids in the resulting polypeptide. Figure 10.12 Mutations (Part 5) Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes tRNAs link information in mRNA codons with specific amino acids. Two events must occur: • tRNAs must read mRNA codons correctly • tRNAs must deliver the correct amino acids that correspond to each codon For each amino acid, there is at least one specific type of tRNA. Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes The structure of tRNAs facilitates their three functions: • Bind to a specific amino acid and become “charged” • Bind to mRNA at the anticodon—a triplet that is complementary to the mRNA codon for the particular amino acid • Interact with ribosomes noncovalently Figure 10.13 Transfer RNA Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes Wobble: specificity for the base at the 3′ end of the codon is not always observed. • Example: Codons for alanine—GCA, GCC, and GCU—are recognized by the same tRNA. Wobble allows cells to produce fewer tRNA species but does not allow the genetic code to be ambiguous. Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes tRNAs are charged by specific aminoacyltRNA synthetases. Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes An experiment demonstrated the importance of the specificity between a tRNA and its amino acid: • Cysteine already bound to tRNA was chemically changed to alanine. • During protein synthesis, the tRNA anticodon was recognized, not the amino acid: § The cysteine-specific tRNA delivered its cargo (alanine) to every mRNA codon for cysteine. Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes Translation occurs at ribosomes. All prokaryotic and eukaryotic ribosomes consist of one large and one small subunit. The subunits exist separately when not translating. Ribosomes are not specific; they can make any type of protein. Figure 10.14 Ribosome Structure Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes The large subunit has 3 tRNA binding sites: • A (amino acid) site binds with the anticodon of a charged tRNA • P (polypeptide) site is where tRNA adds its amino acid to the polypeptide chain • E (exit) site is where tRNA sits before being released from the ribosome Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes Ribosomes have a fidelity function: when proper binding occurs, hydrogen bonds form between the base pairs. Small subunit validates the match—if hydrogen bonds have not formed between all three base pairs, the tRNA is rejected. Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes Translation also occurs in three steps: • Initiation • Elongation • Termination Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes Initiation: Initiation complex—the small subunit binds to its recognition sequence on mRNA. Methionine-charged tRNA then binds to the AUG start codon. The large subunit then joins the complex with the charged tRNA in the P site. The complex is put together by a group of proteins called initiation factors. Figure 10.15 The Initiation of Translation (Part 1) Figure 10.15 The Initiation of Translation (Part 2) Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes Elongation: Another charged tRNA enters the A site and the large subunit catalyzes two reactions: • Breaks bond between tRNA in P site and its amino acid • Forms peptide bond between that amino acid and the amino acid on tRNA in the A site Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes The large subunit is said to have peptidyl transferase activity. The component with this activity is an rRNA, so the catalyst is an example of a ribozyme (from ribonucleic acid and enzyme). Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes When the first tRNA has released its methionine, the ribosome moves so that the first tRNA is at the E site. The first tRNA is released and can become charged again. Elongation occurs as the steps are repeated. Figure 10.16 The Elongation of Translation (Part 1) Figure 10.16 The Elongation of Translation (Part 2) Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes Termination: Translation ends when a stop codon enters the A site. It binds a protein release factor, which allows hydrolysis of the bond between the polypeptide chain and the tRNA in the P site. The newly completed polypeptide then separates from the ribosome. Figure 10.17 The Termination of Translation (Part 1) Figure 10.17 The Termination of Translation (Part 2) Figure 10.17 The Termination of Translation (Part 3) Table 10.2 Concept 10.4 Translation of the Genetic Code Is Mediated by tRNA and Ribosomes Several ribosomes can simultaneously translate a single mRNA molecule, producing multiple polypeptides at the same time. A strand of mRNA with associated ribosomes is called a polyribosome, or polysome. Figure 10.18 A Polysome Concept 10.5 Proteins Are Modified after Translation Proteins can undergo modifications both during and after translation. As a polypeptide emerges from the ribosome it may fold into its 3-D shape and perform its role in the cytosol. Or, polypeptides may contain a signal sequence that “targets” them to the nucleus, mitochondria, plastids, or peroxisomes. Concept 10.5 Proteins Are Modified after Translation The signal sequence binds to a receptor protein on the organelle surface. A channel forms in the membrane and the protein moves into the organelle. Figure 10.19 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell (Part 1) Concept 10.5 Proteins Are Modified after Translation A nuclear localization signal (NLS) targets the protein to the nucleus. Function of NLS was established by experiments in which proteins with or without it were put into cells and then located by labeling the proteins with fluorescent dyes. Only proteins with the NLS were found in the nucleus. Figure 10.20 Testing the Signal (Part 1) Figure 10.20 Testing the Signal (Part 2) Concept 10.5 Proteins Are Modified after Translation If a polypeptide has a signal directing it to the rough endoplasmic reticulum (RER), translation will pause, and the ribosome will bind to a receptor at the RER membrane. Then translation resumes, and as elongation continues, the polypeptide crosses into the RER lumen. It may be retained in the lumen, sent to other regions in the endomembrane system, or be secreted from the cell. Figure 10.19 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell (Part 2) Concept 10.5 Proteins Are Modified after Translation Most polypeptides are modified after translation: Proteolysis—cutting off signal sequences; or cutting a long chain (a polyprotein) into final products by proteases Glycosylation—addition of carbohydrates to form glycoproteins Concept 10.5 Proteins Are Modified after Translation Phosphorylation—addition of phosphate groups by protein kinases The charged phosphate groups change the conformation of the protein, often exposing the active site or other binding site. Figure 10.21 Posttranslational Modifications of Proteins Figure 10.22 An Antibiotic at the Ribosome Answer to Opening Question Strains of MRSA with resistance to tetracyclines are emerging. Genes that confer resistance are carried on plasmids, which can move between bacteria during conjugation. Some resistance genes encode proteins that transfer tetracyclines out of the cell; others encode proteins that prevent antibiotics from binding to the ribosomes. Answer to Opening Question To overcome resistance, new antibiotics are being developed and tried. The evolutionary race between drug resistance and development of antibiotics continues.