8.0 Gene expression and mutation

Related Sadava’s chapters:

1) From DNA to protein

2) Gene mutation

8.1 From DNA to protein: gene expression

• What Is the Evidence that Genes Code for Proteins? • How Does Information Flow from Genes to Proteins? 8.1 From DNA to protein: gene expression

• Identification of a gene product as a protein began with a mutation. • Garrod saw a disease phenotype— alkaptonuria—occurring in children who shared more alleles as first cousins. • A substance in their blood (HA, homogentisic acid) accumulated—was not catalyzed—the gene for the enzyme was mutated. • Garrod correlated one gene to one enzyme. 8.1 From DNA to protein: gene expression

• Beadle and Tatum used Neurospora to test hypothesis that specific gene expression → specific enzyme activity. • Neurospora is haploid for most of its life cycle—all alleles are expressed as phenotypes. • Wild-type strains like Neurospora are prototrophs—have enzymes to catalyze all reactions to make cell constituents. • Beadle and Tatum used X rays as mutagens to cause mutations—inherited genotypic changes. • Mutants were auxotrophs—needed additional nutrients to grow. • For each auxotrophic mutant strain, the addition of just one compound supported growth. • Results suggested that each mutation caused a defect in only one enzyme in a metabolic pathway. 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

• The gene-enzyme relationship has since 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 are not translated to polypeptides; some genes are involved in controlling other genes. 8.1 From DNA to protein: gene expression

Gene expression to form a specific polypeptide occurs in two steps: • Transcription—copies information from a DNA sequence (a gene) to a complementary RNA sequence • Translation—converts RNA sequence to amino acid sequence of a polypeptide “The central dogma of molecular biology.” 8.1 From DNA to protein: gene expression

RNA (ribonucleic acid) differs from DNA: • Usually one polynucleotide strand • The sugar is ribose • Contains uracil (U) instead of thymine (T) 8.1 From DNA to protein: gene expression

Bases in 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. 8.1 From DNA to protein: gene expression

Three kinds of RNA in protein synthesis: • Messenger RNA (mRNA)—carries copy of a DNA sequence to site of protein synthesis at the ribosome • Transfer RNA (tRNA)—carries amino acids for polypeptide assembly • Ribosomal RNA (rRNA)—catalyzes peptide bonds and provides structure 8.1 From DNA to protein: gene expression

The central dogma suggested that information flows from DNA to RNA to protein, which 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? 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

Exception to the central dogma: • Viruses: Non-cellular particles that reproduce inside cells; many have RNA instead of DNA. • Viruses can replicate by transcribing from RNA to RNA, and then making multiple copies by transcription.

8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

• RNA polymerases catalyze synthesis of RNA. • 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 and lack a proofreading function. 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

• The genetic code: Specifies which amino acids will be used to build a protein • Codon: A sequence of three bases—each codon specifies a particular amino acid. • Start codon: AUG—initiation signal for translation. • Stop codons: UAA, UAG, UGA—stop translation and polypeptide is released. 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

• For most amino acids, there is more than one codon; the genetic code is redundant. • Wobble base pair • The genetic code is not ambiguous—each codon specifies only one amino acid. • 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, there are differences. • The frequency of synonymous codons varies between species. • Three reading frames coexist on the DNA/RNA coding sequence.

8.1 From DNA to protein: gene expression

• Prokaryotes and eukaryotes differ in gene structure—in the organization of nucleotide sequences. • In eukaryotes a nucleus separates transcription and translation. • Eukaryotic genes may have noncoding sequences—introns. • The coding sequences are exons. • Introns and exons appear in the primary mRNA transcript—pre- mRNA; introns are removed from the final mRNA. Figure 14.7 Transcription of a Eukaryotic Gene (1) 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

• Introns interrupt, but do not scramble, the DNA sequence that encodes a polypeptide. • Sometimes, the separated exons code for different domains (functional regions) of the protein.

8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

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Roberts & Smith (2002) 8.1 From DNA to protein: gene expression

• In the disease β-thalassemia, a mutation may occur at an intron consensus sequence in the β-globin gene—the pre-mRNA can not be spliced correctly. • Non-functional β-globin mRNA is produced. 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

• Mature mRNA leaves the nucleus through nuclear pores. • TAP protein binds to the 5′ end; this binds to other proteins that are recognized by receptors at the nuclear pore. • These proteins lead the mRNA through the pore—unused pre-mRNAs stay in the nucleus. 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

• tRNA, the adapter molecule, links information in mRNA codons with specific amino acids.

• For each amino acid, there is a specific type or “species” of tRNA.

Three functions of tRNA: • It binds to an amino acid, and is then “charged” • It associates with mRNA molecules • It interacts with ribosomes 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

• 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. 8.1 From DNA to protein: gene expression

• Activating enzymes—aminoacyl-tRNA synthetases—charge tRNA with the correct amino acids. • Each enzyme is highly specific for one amino acid and its corresponding tRNA; the process of tRNA charging is called the second genetic code. • The enzymes have three-part active sites: They bind a specific amino acid, a specific tRNA, and ATP. 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

Experiment by Benzer and others: • Cysteine already bound to tRNA was chemically changed to alanine. • Which would be recognized—the amino acid or the tRNA in protein synthesis? • Answer: Protein synthesis machinery recognizes the anticodon, not the amino acid. 8.1 From DNA to protein: gene expression

• Ribosome: the workbench—holds mRNA and charged tRNAs in the correct positions to allow assembly of polypeptide chain. • Ribosomes are not specific, they can make any type of protein. • Ribosomes have two subunits, large and small. • In eukaryotes, the large subunit has three molecules of ribosomal RNA (rRNA) and 49 different proteins in a precise pattern. • The small subunit has one rRNA and 33 proteins. 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression Figure 14.17 The Termination of Translation (Part 1) Figure 14.17 The Termination of Translation (Part 2) Figure 14.17 The Termination of Translation (Part 3) 8.1 From DNA to protein: gene expression

• The large subunit has peptidyl transferase activity—if rRNA is destroyed, the activity stops • Therefore rRNA is the catalyst in peptidyl transferase activity. • This supports the idea that catalytic RNA evolved before DNA. 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

• 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 14.18 A Polysome (A) Figure 14.18 A Polysome (B) 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression

• Posttranslational aspects of protein synthesis: • Polypeptide emerges from the ribosome and folds into its 3-D shape. • Its conformation allows it to interact with other molecules—it may contain a signal sequence indicating where in the cell it belongs. 8.1 From DNA to protein: gene expression 8.1 From DNA to protein: gene expression Figure 14.21 A Signal Sequence Moves a Polypeptide into the ER (1) 8.1 From DNA to protein: gene expression 14.6 What Happens to Polypeptides after Translation?

If finished protein enters ER lumen, it receives signals of two types: • Sequences of amino acids allow protein to stay in ER • Sugars are added—glycoproteins end up at the plasma membrane, lysosome, (vacuole in plants), or are secreted 14.6 What Happens to Polypeptides after Translation?

Protein modifications: • Proteolysis: Cutting of a long polypeptide chain into final products, by proteases • Glycosylation: Addition of sugars to form glycoproteins • Phosphorylation: Addition of phosphate groups catalyzed by protein kinases— charged phosphate groups change the conformation 15.1 What Are Mutations?

Genetic mutations are changes in the nucleotide sequences of DNA that are passed on to the next generation. Mutations may or may not have a phenotypic effect. 15.1 What Are Mutations?

Mutations occur in two types: • Somatic mutations occur in somatic (body) cells—passed on by mitosis but not to sexually produced offspring • Germ line mutations—occur in germ line cells, the cells that give rise to gametes. A gamete passes a mutation on at fertilization 15.1 What Are Mutations?

Mutations have different phenotypic effects: • Silent mutations do not affect protein function • Loss of function mutations affect protein function and may lead to structural proteins or enzymes that no longer work—almost always recessive 15.1 What Are Mutations?

Mutations have different phenotypic effects: • Gain of function mutations lead to a protein with altered function • Conditional mutations cause phenotypes under restrictive conditions but are not detectable under permissive conditions 15.1 What Are Mutations?

At the molecular level, mutations or alterations in the nucleotide sequence are in two categories: • A point mutation—results from the gain, loss, or substitution of a single nucleotide • Chromosomal mutations are more extensive—may change the position or cause a DNA segment to be duplicated or lost 8.1 From DNA to protein: gene expression 8.2 Gene mutation and molecular medicine

Mutations occur in two types: • Somatic mutations occur in somatic (body) cells—passed on by mitosis but not to sexually produced offspring • Germ line mutations—occur in germ line cells, the cells that give rise to gametes. A gamete passes a mutation on at fertilization 8.2 Gene mutation and molecular medicine 8.2 Gene mutation and molecular medicine Figure 15.4 Chromosomal Mutations (A,B) 8.2 Gene mutation and molecular medicine Figure 15.5 Spontaneous and Induced Mutations (B,C) 8.2 Gene mutation and molecular medicine 8.2 Gene mutation and molecular medicine 8.2 Gene mutation and molecular medicine

• New methods of human genetic analysis include reverse genetics. • A clinical phenotype is related to a DNA variation—then the protein is identified. • Previously, as in sickle-cell anemia:

• Clinical phenotype→ protein phenotype→ gene 8.2 Gene mutation and molecular medicine

Mutations may have benefits: • Provide the raw material for evolution in the form of genetic diversity • Diversity may benefit the organism immediately—if mutation is in somatic cells • Or may cause an advantageous change in offspring 8.2 Gene mutation and molecular medicine

• Most human diseases are multifactorial—caused by interactions of many genes and proteins and the environment. • Susceptibility to disease is determined by these complex interactions. • 60 percent of people are affected by diseases that are genetically influenced. 8.2 Gene mutation and molecular medicine

• DNA testing is direct analysis of DNA for mutation; the most accurate way of detecting an abnormal allele. • Preimplantation screening of a zygote can be used for parents of a child with a disease like cystic fibrosis. • Fetal cells and newborns can be tested for sickle-cell disease and others. 8.2 Gene mutation and molecular medicine

Two main approaches to treating genetic diseases: • Modifying the disease phenotype • Replacing the defective gene 8.2 Gene mutation and molecular medicine

Modifying the disease phenotype can be done in three ways: • Restricting the substrate—as in PKU, reducing phenylalanine in the diet • Metabolic inhibitors, such as drugs that can target specific proteins • Supplying the missing protein—blood factor VIII in hemophilia 8.2 Gene mutation and molecular medicine

• In gene therapy, the aim is to supply the missing allele(s) by inserting a new gene that will be expressed in the host. • The challenges: Must find appropriate vector, ensure precise insertion into host DNA, ensure appropriate expression, and select cells to target. • The nonfunctional alleles cannot be replaced in every cell of the body. • Ex vivo techniques—cells are removed from the body, new genes inserted in the laboratory, cells returned to the body so that correct gene products are made.