BIOL 132 Gene Expression Chapter 17 PDF

Chapter 17 Expression of Genes Lecture Presentations by Nicole Tunbridge and © 2021 Pearson Education Ltd. Kathl...

Chapter 17 Expression of Genes Lecture Presentations by Nicole Tunbridge and © 2021 Pearson Education Ltd. Kathleen Fitzpatrick Figure 17.1a A population of albino donkeys grazes on vegetation on the hillsides of Ashinara, an Italian island. Several centuries ago, a recessive mutation that disables pigment synthesis arose in the DNA of one donkey and was passed down through the generations. Inbreeding has resulted in a large number of homozygous albino donkeys living on the island today © 2021 Pearson Education Ltd. Figure 17.1b © 2021 Pearson Education Ltd. CONCEPT 17.1: Genes specify proteins via transcription and translation The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins Proteins are the links between genotype and phenotype Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation How was the relationship between proteins and DNA discovered? © 2021 Pearson Education Ltd. Evidence from the Study of Metabolic Defects In 1902, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions He thought symptoms of an inherited disease reflect an inability to synthesize a certain enzyme Cells synthesize and degrade molecules in a series of steps, a metabolic pathway © 2021 Pearson Education Ltd. Nutritional Mutants in Neurospora: Scientific Inquiry George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal media Some cells grew when minimal media was supplemented with the amino acid arginine Their colleagues Adrian Srb and Norman Horowitz identified three classes of arginine-deficient mutants Each lacked a different enzyme necessary for synthesizing arginine © 2021 Pearson Education Ltd. Figure 17.2 Beadle and Tatum’s experimental approach © 2021 Pearson Education Ltd. Figure 17.3 Data from A. M. Srb and N. H. Horowitz, The ornithine cycle in Neurospora and its genetic control, Journal of Biological Chemistry 154:129–139 (1944). © 2021 Pearson Education Ltd.  The results of the experiments provided support for the one gene–one enzyme hypothesis The hypothesis states that the function of a gene is to dictate production of a specific enzyme © 2021 Pearson Education Ltd. The Products of Gene Expression: A Developing Story Not all proteins are enzymes, so researchers later revised the hypothesis: one gene–one protein Many proteins are composed of several polypeptides, each of which has its own gene Therefore, Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis It is common to refer to gene products as proteins rather than more precisely as polypeptides © 2021 Pearson Education Ltd. Basic Principles of Transcription and Translation RNA is the bridge between genes and protein synthesis Transcription is the synthesis of RNA using information in DNA Transcription produces messenger RNA (mRNA) Translation is the synthesis of a polypeptide, using information in the mRNA Ribosomes are the sites of translation © 2021 Pearson Education Ltd.  In prokaryotes, translation of mRNA can begin before transcription has finished In a eukaryotic cell, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield the finished mRNA © 2021 Pearson Education Ltd.  A primary transcript is the initial RNA transcript from any gene before processing The central dogma is the concept that cells are governed by a cellular chain of command: © 2021 Pearson Education Ltd. The Genetic Code How are the instructions for assembling amino acids into proteins encoded into DNA? There are 20 amino acids, but there are only four nucleotide bases in DNA How many nucleotides correspond to an amino acid? © 2021 Pearson Education Ltd. Codons: Triplets of Nucleotides The flow of information from gene to protein is based on a triplet code The words of a gene are transcribed into complementary nonoverlapping 3-nucleotide words of mRNA These words are then translated into a chain of amino acids, forming a polypeptide © 2021 Pearson Education Ltd.  One of the two DNA strands, the template strand, provides a template for ordering the sequence of complementary nucleotides in an RNA transcript The template strand is always the same strand for a given gene However, further along the chromosome, the opposite strand may be the template strand for a different gene © 2021 Pearson Education Ltd.  Specific DNA sequences associated with the gene direct which strand is used as the template The mRNA molecule produced is complementary to the template strand The nontemplate strand is called the coding strand because the nucleotides of this strand are identical to the codons, except that T is present in the DNA in place of U in the RNA coding strand template strand © 2021 Pearson Education Ltd.  During translation, the mRNA base triplets, called codons, are read in the 5′ → 3′ direction Each codon specifies the amino acid (one of 20) to be placed at the corresponding position along a polypeptide © 2021 Pearson Education Ltd. Cracking the Code All 64 codons were deciphered by the mid-1960s Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation The genetic code is redundant (more than one codon may specify a particular amino acid) but not ambiguous (no codon specifies more than one amino acid) Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced © 2021 Pearson Education Ltd. Evolution of the Genetic Code The genetic code is nearly universal, shared by the simplest bacteria and the most complex animals Genes can be transcribed and translated after being transplanted from one species to another A language shared by all living things must have been operating very early in the history of life © 2021 Pearson Education Ltd. Figure 17.7 Evidence for evolution: expression of genes from different species © 2021 Pearson Education Ltd. CONCEPT 17.2: Transcription is the DNA- directed synthesis of RNA: a closer look Transcription is the first stage of gene expression Molecular Components of Transcription RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and joins together the RNA nucleotides The mRNA is complementary to the DNA template strand RNA polymerase does not need any primer RNA synthesis follows the same base-pairing rules as DNA, except that uracil substitutes for thymine © 2021 Pearson Education Ltd.  The DNA sequence where RNA polymerase attaches is called the promoter In bacteria, the sequence signaling the end of transcription is called the terminator The stretch of DNA that is transcribed is called a transcription unit © 2021 Pearson Education Ltd. Synthesis of an RNA Transcript The three stages of transcription: – Initiation – Elongation – Termination © 2021 Pearson Education Ltd. Figure 17.8 The stages of transcription © 2021 Pearson Education Ltd. BioFlix® Animation: Transcription © 2021 Pearson Education Ltd. BioFlix® Animation: Overview of Transcription © 2021 Pearson Education Ltd. RNA Polymerase Binding and Initiation of Transcription Promoters signal the transcription start point and usually extend several dozen nucleotide pairs upstream of the start point Transcription factors help guide the binding of RNA polymerase and the initiation of transcription The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes © 2021 Pearson Education Ltd. Figure 17.9 The initiation of transcription at a eukaryotic promoter © 2021 Pearson Education Ltd. Elongation of the RNA Strand As RNA polymerase moves along the DNA, it untwists the double helix, 10–20 nucleotides at a time Nucleotides are added to the 3′ end of the growing RNA molecule Transcription progresses at a rate of 40 nucleotides per second in eukaryotes A gene can be transcribed simultaneously by several RNA polymerases © 2021 Pearson Education Ltd. Figure 17.10 © 2021 Pearson Education Ltd. Termination of Transcription The mechanisms of termination are different in bacteria and eukaryotes In bacteria, the polymerase stops transcription at the end of the terminator and the mRNA can be translated without further modification In eukaryotes, RNA polymerase II transcribes the polyadenylation signal sequence; the RNA transcript is released 10–35 nucleotides past this polyadenylation sequence © 2021 Pearson Education Ltd. © 2021 Pearson Education Ltd. CONCEPT 17.3: Eukaryotic cells modify RNA after transcription Enzymes in the eukaryotic nucleus modify pre- mRNA (RNA processing) before the genetic messages are dispatched to the cytoplasm During RNA processing, both ends of the primary transcript are changed Also, in most cases, certain interior sections of the molecule are cut out and the remaining parts spliced together © 2021 Pearson Education Ltd. Alteration of mRNA Ends Each end of a pre-mRNA molecule is modified in a particular way – The 5′ end receives a modified nucleotide 5′ cap – The 3′ end gets a poly-A tail These modifications share several functions – They seem to facilitate the export of mRNA to the cytoplasm – They protect mRNA from hydrolytic enzymes – They help ribosomes attach to the 5′ end © 2021 Pearson Education Ltd. Figure 17.11 UTR: untranslated region © 2021 Pearson Education Ltd. Split Genes and RNA Splicing Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions The noncoding segments in a gene are called intervening sequences, or introns The other regions are called exons because they are eventually expressed, usually translated into amino acid sequences Introns are removed through RNA splicing © 2021 Pearson Education Ltd. Figure 17.12 RNA splicing Exon Exon © 2021 Pearson Education Ltd.  The removal of introns is accomplished by spliceosomes Spliceosomes consist of a variety of proteins and several small RNAs that recognize the splice sites The RNAs of the spliceosome also catalyze the splicing reaction © 2021 Pearson Education Ltd. Ribozymes Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA Three properties of RNA enable it to function as an enzyme – It can form a three-dimensional structure because of its ability to base-pair with itself – Some bases in RNA contain functional groups that may participate in catalysis – RNA may hydrogen-bond with other nucleic acid molecules © 2021 Pearson Education Ltd. The Functional and Evolutionary Importance of Introns Some introns contain sequences that regulate gene expression and many affect gene products Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during splicing This is called alternative RNA splicing Consequently, the number of different proteins an organism can produce is much greater than its number of genes © 2021 Pearson Education Ltd.  Proteins often have a modular architecture consisting of discrete regions called domains In many cases, different exons code for the different domains in a protein Exon shuffling may result in the evolution of new proteins by mixing and matching exons between different genes © 2021 Pearson Education Ltd. Figure 17.14 © 2021 Pearson Education Ltd. CONCEPT 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look Genetic information flows from mRNA to protein through the process of translation Molecular Components of Translation A cell translates an mRNA message into protein with the help of transfer RNA (tRNA) tRNAs transfer amino acids to the growing polypeptide in a ribosome Translation is a complex process in terms of its biochemistry and mechanics © 2021 Pearson Education Ltd. Figure 17.15 © 2021 Pearson Education Ltd. The Structure and Function of Transfer RNA Each tRNA molecule enables translation of a given mRNA codon into a certain amino acid – Each carries a specific amino acid on one end – Each has an anticodon on the other end; the anticodon base-pairs with a complementary codon on mRNA © 2021 Pearson Education Ltd.  A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf © 2021 Pearson Education Ltd.  Because of hydrogen bonds, tRNA actually twists and folds into a three-dimensional molecule tRNA is roughly L-shaped with the 5′ and 3′ ends both located near one end of the structure The protruding 3′ end acts as an attachment site for an amino acid © 2021 Pearson Education Ltd. Figure 17.16 © 2021 Pearson Education Ltd. Video: Stick and Ribbon Rendering of a tRNA © 2021 Pearson Education Ltd.  Accurate translation requires two instances of molecular recognition – First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase – Second: a correct match between the tRNA anticodon and an mRNA codon Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon © 2021 Pearson Education Ltd. Figure 17.17 © 2021 Pearson Education Ltd. The Structure and Function of Ribosomes Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis Eukaryotic ribosomes are somewhat larger than bacterial ribosomes and differ in their molecular composition Some antibiotic drugs specifically inactivate bacterial ribosomes without harming eukaryotic ribosomes The two ribosomal subunits (large and small) are made of proteins and ribosomal RNAs (rRNAs) © 2021 Pearson Education Ltd.  A ribosome has three binding sites for tRNA – The P site holds the tRNA that carries the growing polypeptide chain – The A site holds the tRNA that carries the next amino acid to be added to the chain – The E site is the exit site, where discharged tRNAs leave the ribosome © 2021 Pearson Education Ltd. Figure 17.18 © 2021 Pearson Education Ltd. Building a Polypeptide The three stages of translation: – Initiation – Elongation – Termination All three stages require protein “factors” that aid in the translation process Energy is required for some steps, too © 2021 Pearson Education Ltd. Ribosome Association and Initiation of Translation The initiation of translation starts when the small ribosomal subunit binds with mRNA and a special initiator tRNA The initiator tRNA carries the amino acid methionine Then the small subunit moves along the mRNA until it reaches the start codon (AUG) Proteins called initiation factors bring in the large subunit that completes the translation initiation complex © 2021 Pearson Education Ltd. Figure 17.19 © 2021 Pearson Education Ltd. Elongation of the Polypeptide Chain During elongation, amino acids are added one by one to the C-terminus of the growing chain Each addition involves proteins called elongation factors Elongation occurs in three steps: codon recognition, peptide bond formation, and translocation Energy expenditure occurs in the first and third steps © 2021 Pearson Education Ltd.  Translation proceeds along the mRNA in a 5′ → 3′ direction The ribosome and mRNA move relative to each other, codon by codon The elongation cycles takes less than a tenth of a second in bacteria Empty tRNAs released from the E site return to the cytoplasm, where they will be reloaded with the appropriate amino acid © 2021 Pearson Education Ltd. Figure 17.20 © 2021 Pearson Education Ltd. Termination of Translation Elongation continues until a stop codon in the mRNA reaches the A site The A site accepts a protein called a release factor The release factor causes the addition of a water molecule instead of an amino acid This reaction releases the polypeptide, and the translation assembly comes apart © 2021 Pearson Education Ltd. Figure 17.21 © 2021 Pearson Education Ltd. Completing and Targeting the Functional Protein Often translation is not sufficient to make a functional protein Polypeptide chains are modified after translation or targeted to specific sites in the cell © 2021 Pearson Education Ltd. Protein Folding and Post-Translational Modifications During synthesis, a polypeptide chain begins to coil and fold spontaneously into a specific shape: a three-dimensional molecule with secondary and tertiary structure A gene determines the primary structure, and the primary structure in turn determines shape Post-translational modifications may be required before the protein can begin doing its particular job in the cell © 2021 Pearson Education Ltd. Targeting Polypeptides to Specific Locations Two populations of ribosomes are evident in cells: free ribosomes (in the cytosol) and bound ribosomes (attached to the ER) Free ribosomes mostly synthesize proteins that function in the cytosol Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell Ribosomes are identical and can switch from free to bound © 2021 Pearson Education Ltd.  Polypeptide synthesis always begins in the cytosol Synthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER Polypeptides destined for the ER or for secretion are marked by a signal peptide The signal peptide is a sequence of about 20 amino acids at or near the leading end of the polypeptide © 2021 Pearson Education Ltd.  A signal-recognition particle (SRP) binds to the signal peptide The SRP escorts the ribosome to a receptor protein built into the ER membrane The signal peptide is removed by an enzyme Other kinds of signal peptides target polypeptides to other organelles © 2021 Pearson Education Ltd. Figure 17.22 © 2021 Pearson Education Ltd. Making Multiple Polypeptides in Bacteria and Eukaryotes Multiple ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or polysome) Polyribosomes enable a cell to make many copies of a polypeptide very quickly © 2021 Pearson Education Ltd. Figure 17.23 © 2021 Pearson Education Ltd.  A bacterial cell ensures a streamlined process by coupling transcription and translation In this case the newly made protein can quickly diffuse to its site of function © 2021 Pearson Education Ltd. Figure 17.24 © 2021 Pearson Education Ltd. Animation: Overview of Protein Synthesis in Bacteria © 2021 Pearson Education Ltd.  In eukaryotes, the nuclear envelope separates the processes of transcription and translation RNA undergoes processing before leaving the nucleus © 2021 Pearson Education Ltd. Figure 17.25 © 2021 Pearson Education Ltd. Animation: Overview of Protein Synthesis in Eukaryotes © 2021 Pearson Education Ltd. CONCEPT 17.5: Mutations of one or a few nucleotides can affect protein structure and function Mutations are changes in the genetic information of a cell Point mutations are changes in just one nucleotide pair of a gene The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein © 2021 Pearson Education Ltd.  If a mutation has an adverse effect on the phenotype of the organism, the condition is referred to as a genetic disorder or hereditary disease © 2021 Pearson Education Ltd. Figure 17.26 © 2021 Pearson Education Ltd. Types of Small-Scale Mutations Point mutations within a gene can be divided into two general categories: – Single nucleotide-pair substitutions – Nucleotide-pair insertions or deletions © 2021 Pearson Education Ltd. Substitutions A nucleotide-pair substitution replaces one nucleotide and its partner with another pair of nucleotides Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code Missense mutations still code for an amino acid, but not the correct amino acid Nonsense mutations change an amino acid codon into a stop codon; most lead to a nonfunctional protein © 2021 Pearson Education Ltd. Figure 17.27a © 2021 Pearson Education Ltd. Insertions and Deletions Insertions and deletions are additions or losses of nucleotide pairs in a gene These mutations have a disastrous effect on the resulting protein more often than substitutions do Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation Insertions or deletions outside the coding part of a gene could affect how the gene is expressed © 2021 Pearson Education Ltd. Figure 17.27b © 2021 Pearson Education Ltd. Animation: Mutation Types © 2021 Pearson Education Ltd. New Mutations and Mutagens Spontaneous mutations can occur during errors in DNA replication or recombination Mutagens are physical or chemical agents that can cause mutations Chemical mutagens fall into a variety of categories Most carcinogens (cancer-causing chemicals) are mutagens, and most mutagens are carcinogenic © 2021 Pearson Education Ltd. Using CRISPR to Edit Genes and Correct Disease-Causing Mutations Biologists who study disease-causing mutations have sought techniques for gene editing–altering genes in a specific way The powerful technique called CRISPR-Cas9 is transforming the field of genetic engineering In bacteria, the protein Cas9 acts together with a guide RNA to help defend bacteria from viral infection © 2021 Pearson Education Ltd.  The Cas9 protein will cut any sequence to which it is targeted Scientists can introduce a Cas9–guide RNA complex into a cell they wish to alter The guide RNA is engineered to target a gene Cas9 cuts both strands of the targeted gene © 2021 Pearson Education Ltd.  The broken ends trigger a DNA repair system The repair enzymes remove or add some random nucleotides while joining the broken ends This is a way for researchers to “knock out” (disable) a given gene, to study what the gene does in an organism © 2021 Pearson Education Ltd.  To treat genetic disease, researchers have modified this technique They can introduce a template with a normal (functional) copy of the gene to be corrected In this way, the CRISPR-Cas9 system edits the defective gene and corrects it © 2021 Pearson Education Ltd. Figure 17.28 https://www.youtube.com/watch?v=6tw_JVz_IEc © 2021 Pearson Education Ltd.  Some genetic conditions like human sickle-cell disease have been somewhat successfully treated using mice There are still concerns about using the technique in humans There is the possibility of unintended effects on genes that have not been targeted Biologists have agreed to use extreme caution as the field moves forward © 2021 Pearson Education Ltd. What Is a Gene? Revisiting the Question The idea of the gene has evolved through the history of genetics We have considered a gene as – a discrete unit of inheritance – a region of specific nucleotide sequence in a chromosome – a DNA sequence that codes for a specific polypeptide chain ❖A gene can be defined as a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule © 2021 Pearson Education Ltd.