12. From DNA to Protein: Genotype to Phenotype

From DNA to Protein: Genotype to Phenotype12 From DNA to Protein: Genotype to Phenotype12.1 What Is the Evidence that Genes Code for Proteins?12.2 How Does Information Flow from Genes to Proteins?12.3 How Is the Information Content in DNA Transcribed to Produce RNA?12.4 How Is RNA Translated into Proteins?12.5 What Happens to Polypeptides after Translation?12.6 What Are Mutations?12.1 What Is the Evidence that Genes Code for Proteins?The molecular basis of phenotypes was known before it was known that DNA is the genetic material.Studies of many different organisms showed that major phenotypic differences were due to specific proteins.12.1 What Is the Evidence that Genes Code for Proteins?Model organisms: easy to grow or observe; show the phenomenon to be studiedAssume that results from one organism can be applied to othersExamples: pea plants, Drosophila, E. coli, common bread mold Neurospora crassa12.1 What Is the Evidence that Genes Code for Proteins?Neurospora is haploid for most of its life cycle.Wild-type strains have enzymes to catalyze all reactions needed to make cell constituents—prototrophs.Beadle and Tatum used X-rays as mutagens. Mutants were auxotrophs—needed additional nutrients to grow.12.1 What Is the Evidence that Genes Code for Proteins?For each auxotrophic strain, they found a single compound that would support growth of that strain.Suggested the one-gene, one-enzyme hypothesisFigure 12.1 One Gene, One Enzyme (Part 1)Figure 12.1 One Gene, One Enzyme (Part 2)12.1 What Is the Evidence that Genes Code for Proteins?Beadle and Tatum found several different arg mutant strains—had to be supplied with arginine.arg mutants could have mutations in the same gene; or in different genes that governed steps of a biosynthetic pathway.12.1 What Is the Evidence that Genes Code for Proteins?arg mutants were grown in the presence of compounds suspected to be intermediates in the biosynthetic pathway for arginine.This confirmed that each mutant was missing a single enzyme in the pathway.12.1 What Is the Evidence that Genes Code for Proteins?The gene-enzyme relationship has been revised to the one-gene, one-polypeptide relationship.Example: In hemoglobin, each polypeptide chain is specified by a separate gene.Other genes code for RNA that is not translated to polypeptides; some genes are involved in controlling other genes.12.2 How Does Information Flow from Genes to Proteins?Expression of a gene to form a polypeptide:Transcription—copies information from gene to a sequence of RNA.Translation—converts RNA sequence to amino acid sequence.12.2 How Does Information Flow from Genes to Proteins?RNA, ribonucleic acid differs from DNA:Usually one strandThe sugar is riboseContains uracil (U) instead of thymine (T)12.2 How Does Information Flow from Genes to Proteins?RNA can pair with a single strand of DNA, except that adenine pairs with uracil instead of thymine.Single-strand RNA can fold into complex shapes by internal base pairing.Figure 12.2 The Central DogmaThe central dogma of molecular biology: information flows in one direction when genes are expressed (Francis Crick).12.2 How Does Information Flow from Genes to Proteins?The central dogma raised two questions:How does genetic information get from the nucleus to the cytoplasm?What is the relationship between a DNA sequence and an amino acid sequence?12.2 How Does Information Flow from Genes to Proteins?Messenger hypothesis—messenger RNA (mRNA) forms as a complementary copy of DNA and carries information to the cytoplasm.This process is transcription.Figure 12.3 From Gene to Protein12.2 How Does Information Flow from Genes to Proteins?Adapter hypothesis—an adapter molecule that can bind amino acids, and recognize a nucleotide sequence—transfer RNA (tRNA).tRNA molecules carrying amino acids line up on mRNA in proper sequence for the polypeptide chain—translation.12.2 How Does Information Flow from Genes to Proteins?Exception to the central dogma:Viruses: acellular particles that reproduce inside cells; many have RNA instead of DNA. 12.2 How Does Information Flow from Genes to Proteins?Synthesis of DNA from RNA is reverse transcription.Viruses that do this are retroviruses.12.3 How Is the Information Content in DNA Transcribed to Produce RNA?Within each gene, only one strand of DNA is transcribed—the template strand.Transcription produces mRNA; the same process is used to produce tRNA and rRNA.12.3 How Is the Information Content in DNA Transcribed to Produce RNA?RNA polymerases catalyze synthesis of RNA.RNA polymerases are processive—a single enzyme-template binding results in polymerization of hundreds of RNA bases.Figure 12.4 RNA Polymerase12.3 How Is the Information Content in DNA Transcribed to Produce RNA?Transcription occurs in three phases:InitiationElongationTermination12.3 How Is the Information Content in DNA Transcribed to Produce RNA?Initiation requires a promoter—a special sequence of DNA.RNA polymerase binds to the promoter.Promoter tells RNA polymerase where to start, which direction to go in, and which strand of DNA to transcribe.Part of each promoter is the initiation site.Figure 12.5 DNA Is Transcribed to Form RNA (A)12.3 How Is the Information Content in DNA Transcribed to Produce RNA?Elongation: RNA polymerase unwinds DNA about 10 base pairs at a time; reads template in 3′ to 5′ direction.The RNA transcript is antiparallel to the DNA template strand.RNA polymerases do not proofread and correct mistakes.Figure 12.5 DNA Is Transcribed to Form RNA (B)12.3 How Is the Information Content in DNA Transcribed to Produce RNA?Termination: specified by a specific DNA base sequence.Mechanisms of termination are complex and varied.Eukaryotes—first product is a pre-mRNA that is longer than the final mRNA and must undergo processing.Figure 12.5 DNA Is Transcribed to Form RNA (C)12.3 How Is the Information Content in DNA Transcribed to Produce RNA?The genetic code: specifies which amino acids will be used to build a proteinCodon: a sequence of three bases. Each codon specifies a particular amino acid.Start codon: AUG—initiation signal for translationStop codons: stops translation and polypeptide is releasedFigure 12.6 The Genetic Code12.3 How Is the Information Content in DNA Transcribed to Produce RNA?For most amino acids, there is more than one codon; the genetic code is redundant.But not ambiguous—each codon specifies only one amino acid.12.3 How Is the Information Content in DNA Transcribed to Produce RNA?The genetic code is nearly universal: the codons that specify amino acids are the same in all organisms.Exceptions: within mitochondria and chloroplasts, and in one group of protists.12.3 How Is the Information Content in DNA Transcribed to Produce RNA?This common genetic code is a common language for evolution.The code is ancient and has remained intact throughout evolution.The common code also facilitates genetic engineering.12.3 How Is the Information Content in DNA Transcribed to Produce RNA?How was the code deciphered?20 “code words” (amino acids) are written with only four “letters.”Triplet code seemed likely: could account for 4 × 4 × 4 = 64 codons.12.3 How Is the Information Content in DNA Transcribed to Produce RNA?Nirenberg and Matthaei used artificial polynucleotides instead of mRNA as a messenger.Then they identified the polypeptide that resulted.Figure 12.7 Deciphering the Genetic Code12.4 How Is RNA Translated into Proteins?tRNA, the adaptor molecule: for each amino acid, there is a specific type or “species” of tRNA.Functions of tRNA:Carries an amino acidAssociates with mRNA moleculesInteracts with ribosomesFigure 12.8 Transfer RNA12.4 How Is RNA Translated into Proteins?The conformation (three-dimensional shape) of tRNA results from base pairing (H bonds) within the molecule.3′ end is the amino acid attachment site—binds covalently. Always CCA.Anticodon: site of base pairing with mRNA. Unique for each species of tRNA.12.4 How Is RNA Translated into Proteins?Example:DNA codon for arginine: 3′-GCC-5′Complementary mRNA: 3′-CGG-5′Anticodon on the tRNA: 3′-GCC-5′ This tRNA is charged with arginine.12.4 How Is RNA Translated into Proteins?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.Allows cells to produce fewer tRNA species; but not in all cases—the genetic code remains unambiguous.12.4 How Is RNA Translated into Proteins?Charging a tRNA with the correct amino acid—amino-acyl-tRNA synthetases.Each enzyme is specific for one amino acid and its corresponding tRNA.The enzymes have three-part active sites: they bind a specific amino acid, a specific tRNA, and ATP.Figure 12.9 Charging a tRNA Molecule (Part 1)Figure 12.9 Charging a tRNA Molecule (Part 2)12.4 How Is RNA Translated into Proteins?The activating enzymes are highly specific. Amino acid is attached to the 3′ end of tRNA by an energy-rich bond—this will provide energy for synthesis of the peptide bond to join amino acids.12.4 How Is RNA Translated into Proteins?Experiment by Benzer and others:Chemically changed cysteine already bound to tRNA to alanine. Resulting polypeptide had alanine in every place that cysteine should be.Protein synthesis machinery recognizes the anticodon, not the amino acid.12.4 How Is RNA Translated into Proteins?Ribosome: the workbench—holds mRNA and tRNA in the correct positions to allow assembly of polypeptide chain.Ribosomes are not specific, they can make any type of protein.12.4 How Is RNA Translated into Proteins?Ribosomes have two subunits, large and small.In eukaryotes, the large subunit has three molecules of ribosomal RNA (rRNA) and 45 different proteins in a precise pattern.The small subunit has one rRNA and 33 proteins.12.4 How Is RNA Translated into Proteins?Subunits are held together by ionic and hydrophobic forces (not covalent bonds).When not active in translation, the subunits exist separately.Figure 12.10 Ribosome Structure12.4 How Is RNA Translated into Proteins?Large subunit has three tRNA binding sites:A site binds with anticodon of charged tRNA.P site is where tRNA adds its amino acid to the growing chain.E site is where tRNA sits before being released.12.4 How Is RNA Translated into Proteins?Hydrogen bonds form between the anticodon of tRNA and the codon of mRNA.Small subunit rRNA validates the match—if hydrogen bonds have not formed between all three base pairs, it must be an incorrect match, and the tRNA is rejected.12.4 How Is RNA Translated into Proteins?Translation also occurs in three steps:InitiationElongationTermination12.4 How Is RNA Translated into Proteins?Initiation:An initiation complex forms—charged tRNA and small ribosomal subunit, both bound to mRNA.rRNA binds to recognition site on mRNA—the Shine-Dalgarno sequence, “upstream” from the start codon.Figure 12.11 The Initiation of Translation (Part 1)Figure 12.11 The Initiation of Translation (Part 2)12.4 How Is RNA Translated into Proteins?Start codon is AUG; first amino acid is always methionine, which may be removed after translation.The large subunit joins the complex, the charged tRNA is now in the P site of the large subunit.Initiation factors are responsible for assembly of the initiation complex.12.4 How Is RNA Translated into Proteins?Elongation: the second charged tRNA enters the A site.Large subunit catalyzes two reactions:Breaks bond between tRNA in P site and its amino acid.Peptide bond forms between that amino acid and the amino acid on tRNA in the A site.Figure 12.12 The Elongation of Translation (Part 1)Figure 12.12 The Elongation of Translation (Part 2)12.4 How Is RNA Translated into Proteins?The large subunit has peptidyl transferase activity.RNA acts as the catalyst; normally proteins are catalysts. Supports the idea that catalytic RNA evolved before DNA.12.4 How Is RNA Translated into Proteins?When the first tRNA has released its methionine, it moves to the E site and dissociates from the ribosome—can then become charged again.Elongation occurs as the steps are repeated, assisted by proteins called elongation factors.12.4 How Is RNA Translated into Proteins?Termination: translation ends when a stop codon enters the A site.Stop codon binds a protein release factor—allows hydrolysis of bond between polypeptide chain and tRNA on the P site.Polypeptide chain—C terminus is the last amino acid added.Figure 12.13 The Termination of Translation (Part 1)Figure 12.13 The Termination of Translation (Part 2)Figure 12.13 The Termination of Translation (Part 3)Table 12.112.4 How Is RNA Translated into Proteins?Several ribosomes can work together to translate the same mRNA, producing multiple copies of the polypeptide.A strand of mRNA with associated ribosomes is called a polyribosome or polysome.Figure 12.14 A Polysome (Part 1)Figure 12.14 A Polysome (Part 2)12.5 What Happens to Polypeptides after Translation?Posttranslational aspects of protein synthesis:Polypeptide may be moved from synthesis site to an organelle, or out of the cell.Polypeptides are often modified with more chemical groups.12.5 What Happens to Polypeptides after Translation?Polypeptide folds as it emerges from the ribosome.The amino acid sequence determines the pattern of folding.Amino acid sequence also contains a signal sequence—an “address label.”12.5 What Happens to Polypeptides after Translation?Amino acid sequence gives a set of instructions:“Finish translation and send to an organelle.” OR“Stop translation, go to the ER, finish synthesis there.”Figure 12.15 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell12.5 What Happens to Polypeptides after Translation?Conformation of signal sequences allow them to bind specific receptor proteins—docking proteins—on outer membranes of organelles. Receptor forms a channel that the protein passes through. May be unfolded at this time by chaperonins.12.5 What Happens to Polypeptides after Translation?If the protein is sent to the ER:Signal sequence binds to a signal receptor particle, before translation is done.Ribosome attaches to a receptor on the ER, the growing polypeptide chain passes through the channel.An enzyme removes the signal sequence.Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER (Part 1)Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER (Part 2)12.5 What Happens to Polypeptides after Translation?Sugars may be added in the Golgi apparatus—the resulting glycoproteins end up in the plasma membrane, lysosomes, or vacuoles.12.5 What Happens to Polypeptides after Translation?Protein modifications:Proteolysis: cutting the polypeptide chain, by proteases.Glycosylation: addition of sugars to form glycoproteins.Phosphorylation: addition of phosphate groups by kinases. Charged phosphate groups change the conformation.Figure 12.17 Posttranslational Modifications of Proteins12.6 What Are Mutations?Somatic mutations occur in somatic (body) cells. Mutation is passed to daughter cells, but not to sexually produced offspring.Germ line mutations occur in cells that produce gametes. Can be passed to next generation.12.6 What Are Mutations?Conditional mutants: express phenotype only under restrictive conditions. Example: the allele may code for an enzyme that is unstable at certain temperatures.12.6 What Are Mutations?All mutations are alterations of the nucleotide sequence.Point mutations: change in a single base pair—loss, gain, or substitution of a base.Chromosomal mutations: change in segments of DNA—loss, duplication, or rearrangement.12.6 What Are Mutations?Point mutations can result from replication and proofreading errors, or from environmental mutagens.Silent mutations have no effect on the protein because of the redundancy of the genetic code.Silent mutations result in genetic diversity not expressed as phenotype differences.12.6 What Are Mutations?12.6 What Are Mutations?Missense mutations: base substitution results in amino acid substitution.12.6 What Are Mutations?Sickle allele for human β-globin is a missense mutation.Sickle allele differs from normal by only one base—the polypeptide differs by only one amino acid.Individuals that are homozygous have sickle-cell disease.Figure 12.18 Sickled and Normal Red Blood Cells12.6 What Are Mutations?Nonsense mutations: base substitution results in a stop codon.12.6 What Are Mutations?Frame-shift mutations: single bases inserted or deleted—usually leads to nonfunctional proteins.12.6 What Are Mutations?Chromosomal mutations:Deletions—severe consequences unless it affects unnecessary genes or is masked by normal alleles.Duplications—if homologous chromosomes break in different places and recombine with the wrong partners.Figure 12.19 Chromosomal Mutations (A, B)12.6 What Are Mutations?Chromosomal mutations:Inversions—breaking and rejoining, but segment is “flipped.”Translocations—segment of DNA breaks off and is inserted into another chromosome. Can cause duplications and deletions. Meiosis can be prevented if chromosome pairing is impossible.Figure 12.19 Chromosomal Mutations (C, D)12.6 What Are Mutations?Spontaneous mutations—occur with no outside influence. Several mechanisms:Bases can form tautomers—different forms; rare tautomer can pair with the wrong base.Chemical reactions may change bases (e.g., loss of amino group).12.6 What Are Mutations?Replication errors—some escape detection and repair.Nondisjunction in meiosis.12.6 What Are Mutations?Induced mutation—due to an outside agent, a mutagen.Chemicals can alter bases (e.g., nitrous acid can cause deamination).Some chemicals add other groups to bases (e.g., benzpyrene adds a group to guanine and prevents base pairing). DNA polymerase will then add any base there.12.6 What Are Mutations?Ionizing radiation such as X-rays create free radicals—highly reactive—can change bases, break sugar phosphate bonds.UV radiation is absorbed by thymine, causing it to form covalent bonds with adjacent nucleotides—disrupts DNA replication. Figure 12.20 Spontaneous and Induced Mutations (Part 1) Figure 12.20 Spontaneous and Induced Mutations (Part 2)12.6 What Are Mutations?Mutation provides the raw material for evolution in the form of genetic diversity.Mutations can harm the organism, or be neutral.Occasionally, a mutation can improve an organism’s adaptation to its environment, or become favorable as conditions change.12.6 What Are Mutations?Complex organisms tend to have more genes than simple organisms.If whole genes are duplicated, the new genes would be surplus genetic information.Extra copies could lead to the production of new proteins. New genes can also arise from transposable elements (see Chapters 13 and 14).