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15 FROM DNA TO PROTEIN Chapter Outline 15.1 THE CONNECTION BETWEEN DNA, RNA, AND PROTEIN Proteins are specified by genes The pathway form gene to polypeptide involves transcription and translation The genetic code is written in three-letter words using a four letter alphabet 15.2 TRANSCRIPTION: DNA-DIRECTED RNA SYNTHESIS RNA polymerases work like DNA polymerases, but require no primer Specific sequences of nucleotides in the DNA indicate where transcription of a gene begins and ends 15.3 PRODUCTION OF mRNAs IN EUKARYOTES Eukaryotic protein-coding genes are transcribed into precursor-mRNAs that are modified in the nucleus Introns are removed during pre-mRNA processing to produce the translatable mRNA Introns contribute to protein variability 15.4 TRANSLATION: mRNA-DIRECTED POLYPEPTIDE SYNTHESIS tRNAs are small, highly specialized RNAs that bring amino acids to the ribosome Ribosomes are rRNA-protein complexes that work as automated protein assembly machines Translation initiation brings the ribosomal subunits, an mRNA, and the first aminoacyl-tRNA together Polypeptide chains grow during the elongation stage of translation Termination releases a completed polypeptide from the ribosome Multiple ribosomes simultaneously translate a single mRNA Newly synthesized polypeptides are processed and folded into finished form Finished proteins contain sorting signals that direct them to cellular locations Base-pair mutations can affect protein structure and function Objectives After reading the chapter, you should be able to: 1. Know how the structure and behavior of DNA determines the structure and behavior of the three forms of RNA during transcription. 2. Understand the role the three forms of RNA play in determining the primary structure of polypeptide chains during translation. 3. Understand the types of mutations and their role in genetic variation. 4. Explain why the central dogma is too simple a model 5. Understand the importance of the degeneracy of the genetic code 6. Understand the sequence and location of the events in transcription and translation Woelker 2009 From DNA to Protein 128 Key Terms one gene-one enzyme hypothesis degenerate code exon shuffling wobble hypothesis one gene-one polypeptide hypothesis universal code transfer RNAs (tRNAs) RNA polymerases anticodon signal peptide (or signal sequence) transcription promoter signal recognition particle (SRP) translation transcription unit aminoacylation (or charging) aminoacyl-tRNA mutations template strand TATA box messenger RNA (mRNA) precursor-mRNA (premRNA) aminoacyl-tRNA synthetase base-pair substitution mutations ribosomes missense mutation genetic code 5' cap codon ribosomal RNA (rRNA) nonsense mutation poly(A) tail sense codons introns ribosome binding site frameshift mutation start codon mRNA splicing peptidyl-tRNA initiator codon spliceosome peptidyl transferase stop codons ribozyme nonsense codons small ribonucleoprotein particles termination codons alternative splicing polysome (polyribosome) silent mutation release factor (or termination factor) Lecture Outline 15.1 The Connection between DNA, RNA, and Protein A. Genes specify proteins as demonstrated by alkaptonuria, which is a genetic mutation in a gene for a key enzyme. B. The use of Neurospora, a fungi that mutates easily, confirmed that mutations in a gene result in changes in the protein encoded (Figure 15.2). C. The one gene–one polypeptide hypothesis represents the analysis of this study. D. The pathway from gene to polypeptide uses transcription and translation (Figure 15.3). 1. Transcription is the process by which DNA is used to create an RNA copy. 2. Translation is the use of the RNA strand to create a polypeptide. E. The process of going from “DNA RNA Protein” is called the central dogma (Figure 15.3). F. The genetic code is written in three-letter words using a four-letter alphabet. 1. The alphabet for DNA is made up of adenine, thymine, guanine, and cytosine (A, T, G, and C). 2. The alphabet for RNA is made up of adenine, uracil, guanine, and cytosine (A, U, G, and C). 3. The genetic code had to be at least three letters coding for one amino acid, and it was proven to be a triplet code (Figure 15.4). 4. The exact genetic code has 64 possible codons (Figure 15.5). a. Some codons code for start sites. b. Some code for stop codons. c. Only two amino acids, tryptophan and methionine, have a single codon. d. Most are redundant and are coded by multiple codon combinations. e. The universal code, with few exceptions, is the same in all living organisms. 15.2 Transcription: DNA-Directed RNA Synthesis (Figure 15.6) A. RNA transcription is similar to DNA replication. B. Only one of the two DNA strands acts a template for RNA synthesis. 1. RNA polymerases catalyze the assembly of RNA without a primer. 2. Specific sequences indicate where gene transcription should begin. C. The structural organization of the gene and the outline of how it is transcribed is depicted in Figure 15.7. 1. A promoter initiates transcription (Figure 15.7, step 1). 2. An RNA polymerase molecule binds to the DNA at the beginning of the gene to be transcribed. 3. The DNA begins to unwind at the front of the RNA polymerase. Woelker 2009 From DNA to Protein 129 4. 5. During transcription, RNA nucleotides are base paired one after another with the template DNA bases. The RNA copy is released when the entire gene has been transcribed. The unwound region of the DNA rewinds into a double helix. D. Three main steps of transcription: 1. Initiation: RNA polymerase binds to the promoter, unwinds the DNA, and initiates transcription at the start point. 2. Elongation: RNA polymerase moves along the DNA, unwinding it and adding new RNA nucleotides to the transcript in the 5' to 3' direction. Behind the enzyme, the DNA strands reform into a double helix. 3. Termination: The complete RNA molecule is released from the template DNA, RNA polymerase leaves the DNA, and the double helix reforms. E. The promoter of protein-coding genes and transcription initiates and specifies where transcription begins. 1. In eukaryotes, RNA pol II transcribes protein-coding genes. 2. RNA pol I and III transcribe genes for non-protein coding RNAs. 3. Promoters are upstream of the genes and more complex in eukaryotes. 4. A key element in eukaryotes is the TATA box, which is recognized by transcription factors. F. A protein binding at a termination sequence releases RNA and RNA polymerase from the template and is called a terminator. 15.3 Production of mRNAs in Eukaryotes A. mRNAs have noncoding regions that do not code for protein elements. B. Eukaryotic protein-coding genes make a precursor-mRNA that is modified in the nucleus (Figure 15.8). C. Modifications of pre-mRNA and mRNA ends include a 5' cap and an mRNA tail. D. RNA digesting enzymes degrade the poly (A) tail, which extends the functional life of the mRNA. E. Eukaryotic cells have introns that interrupt the protein coding sequence and exons that are the protein coding regions. F. Introns are removed during pre-mRNA processing to produce translatable mRNA (Figure 15.9). G. Small nuclear ribonucleoprotein plus the pre-mRNA form a spliceosome to “loop out” the intron (Figure 15.9). H. Introns increase protein variability by alternative splicing and exon shuffling. 1. Alternative splicing can join exons in different combinations to produce different mRNAs from a single gene (Figure 15.10). 2. Exon shuffling mixes functional regions to allow the evolution of new proteins. 15.4 Translation: mRNA-Directed Polypeptide Synthesis A. The ribosome binds to the mRNA strand, and tRNA brings amino acids to complex into a polypeptide (Figure 15.11). B. Transfer RNAs (tRNA) are small, highly specialized RNAs that bring amino acids to the ribosome. 1. tRNA have a highly distinctive structure (Figure 15.12). 2. It has an anticodon region that binds to the codon and brings an amino acid on the other end. C. Addition of amino acids to their corresponding tRNAs is called aminoacylation or “charging” (Figure 15.13). 1. There are twenty different enzymes that catalyze this charging, and they are collectively called aminoacyl-tRNA synthetases. 2. This process requires energy from an ATP molecule. D. Ribosomes have one large and one small subunit (Figure 15.14). E. Ribosomes are rRNA-protein complexes that work as automated protein assembly machines (Figure 15.14). F. Translation initiation brings the ribosomal subunits, an mRNA, and the first aminoacyl-tRNA together (Figure 15.15). 1. Each step in translation initiation is aided by proteins termed initiation factors. 2. Met-tRNA with GTP bound to it and the small ribosomal subunit form a complex. 3. The complex binds to the 5' cap of the mRNA and scans along it until it reaches the AUG start codon. 4. The large ribosomal subunit binds, and GTP is hydrolyzed, completing initiation. G. Steps to the elongation phase of translation (Figure 15.16) 1. An aminoacyl-tRNA binds the A site. 2. Peptidyl transferase cleaves the amino acid from the P site tRNA and bonds it to the amino acid on the A site tRNA. Woelker 2009 From DNA to Protein 130 3. H. I. J. K. L. M. Woelker 2009 The ribosome translocates along the mRNA to the next codon, thereby bringing the tRNA with the growing polypeptide to the P site and moving the empty tRNA to the E site. 4. When translocation is complete, the empty tRNA in the E site is released, and the cycle is ready to go again. Termination releases a completed polypeptide from the ribosome (Figure 15.17). 1. The ribosome reaches a termination codon (UAA, UAG, or UGA). 2. A release factor binds to the termination codon in the A site and causes the ribosome to disassemble. Multiple ribosomes can simultaneously translate a single mRNA (Figure 15.19). Newly synthesized polypeptides are processed and folded into a finished form. 1. The final shape is not always random and may require the assistance of a chaperone protein to reach the correct 3D shape. 2. Some proteins are processed to an inactive form and then wait to be activated. Finished proteins contain sorting signals that direct them to cellular locations (Figure 15.20). Base-pair mutations can affect protein structure and function (Figure 15.21). A missense mutation in a gene for one of the two polypeptides of hemoglobin is the cause of sickle-cell disease (Figure 15.22). From DNA to Protein 131