104225 104311 Biotechnology involves the use of living

生物技術學-許濤老師授課內容 104/2/25 -104/3/11 Biotechnology involves the use of living organisms in industrial processes-particularly in agriculture, food processing and medicine.

生物技術 Biotechnology 運用生命科學方法如基因重組等為基礎，進行研 發、製造或提升產品品質，以改善人類生活的科 學技術 Biotechnology involves the use of living organisms in industrial processes-particularly in agriculture, food processing, and medicine Traditional Biotechnology Modern Biotechnology (Molecular Biotechnology-after 1970)

Selected developments in the history of molecular biotechnology • • • 1917 Karl Ereky coins the term biotechnology 1943 Penicillin is produced on an industrial scale 1953 Watson and Crick determines the structure of DNA 1961 The journal: Biotechnology & Bioengineering published 1961 -1966 Entire genetic code is deciphered 1970 First restriction endonuclease is isolated 1973 Boyer & Cohen establish recombinant DNA technology 1988 PCR method is published 1990 Human genome project is officially initiated 1996 First recombinant protein, erythropoietin, exceeds $ 1 billion in annual sales • 2001 Human genome is sequenced, 2002 human gene microarrays become commercially available

FIGURE 1. 1 Traditional Biotechnology Products Bread, cheese, wine, and beer have been made worldwide for many centuries using microorganisms, such as yeast

Fundamentals of Molecular Biotechnology • • Recombinant DNA technology Chemical synthesis & amplification (PCR) Genomics & gene expression (proteins) Proteomics: mass spectral analysis Bioinformatics: computer technology Nanotechnology Unicellular & multicellular organisms as research models

Application of Molecular Biotechnology • • • Molecular diagnostics (mutation, disease) Protein therapeuticals (monoclonal Ab) Production of transgenic organisms Bioremediation Synthesis of commercial compounds by recombinant microorganisms (antibiotics, enzymes)

Chapter 1 Companion site for Biotechnology Author: Clark

Mendel’s Laws of Inheritance • A gene can exist in different forms called alleles • One allele can be dominant over the other, recessive, allele • The first filial generation (F 1) contains offspring of the original parents • If each parent carries two copies of a gene, the parents are diploid for that gene

Mendel’s Gene Transmission • Heterozygotes have one copy of each allele • Parents in 1 st mating are homozygotes, having 2 copies of one allele • Sex cells, or gametes, are haploid, containing only 1 copy of each gene • Heterozygotes produce gametes having either allele • Homozygotes produce gametes having only one allele

The Chromosome Theory of Inheritance • Chromosomes are discrete physical entities that carry the genes • Thomas Hunt Morgan used the fruit fly, Drosophila melanogaster, to study genetics • Autosomes occur in pairs in a given individual • Sex chromosomes are identified as X and Y – Female has two X chromosomes – Male has one X and one Y chromosome

UNN 1. 1 Relationship of Genotype and Phenotype (A) Each parent has two alleles, either two yellow or two green. Any offspring will be heterozygous, each having a yellow and a green allele. Since the yellow allele is dominant, the peas look yellow. (B) When the heterozygous F 1 offspring self-fertilize, the green phenotype re-emerges in one-fourth of the F 2 generation. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 13

Outline of Griffith’s Transformation Experiments 2 -14

Procedure for the Hershey-Chase Transformation Experiments 2 -15

FIGURE 1. 3 Nucleic Acid Structure (A) DNA has two strands antiparallel to each other. The structure of the subcomponents is shown to the sides. (B) RNA is usually single-stranded and has two chemical differences from DNA. First, an extra hydroxyl group (-OH) is found at the 2’ position of ribose, and second, thymine is replaced by uracil. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 16

Purines and Pyrimidines • Adenine and guanine are related structurally to the parent molecule purine • Cytosine, thymine and uracil resemble pyrimidine 2 -17

A Trinucleotide The example trinucleotide has polarity – Top of molecule has a free 5’-phosphate group = 5’ end – Bottom has a free 3’hydroxyl group = 3’ end 2 -18

Analytical Tools Physical-chemical analysis has often used: 1. Ultracentrifugation Used to estimate size of material 2. Electrophoresis Indicated high charge-to-mass ratio 3. Ultraviolet Absorption Spectrophotometry Absorbance of UV light matched that of DNA 4. Elementary Chemical Analysis Nitrogen-to-phosphorus ratio of 1. 67, not found in protein

FIGURE 1. 4 Packaging of DNA in Bacteria and Eukaryotes (A) Bacterial DNA is supercoiled and attached to a scaffold to condense its size to fit inside the cell. (B) Eukaryotic DNA is wrapped around histones to form a nucleosome. Nucleosomes are further condensed into a 30 -nm fiber attached to proteins at MAR sites. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 20

13. 1 Histones • Eukaryotic cells contain 5 kinds of histones – – – H 1 H 2 A H 2 B H 3 H 4 • Each histone type isn’t homogenous – Gene reiteration – Posttranslational modification Source: Panyim and Chalkley, Arch. Biochem. & Biophys. 130, 1969, f. 6 A, p. 343.

Histones in the Nucleosome • Chemical cross-linking in solution: – H 3 to H 4 – H 2 A to H 2 B • H 3 and H 4 exist as a tetramer (H 3 -H 4)2 • Chromatin is composed of roughly equal masses of DNA and histones – Corresponds to 1 histone octamer per 200 bp of DNA – Octamer composed of: • 2 each H 2 A, H 2 B, H 3, H 4 • 1 each H 1

1. 3 The Three Domains of Life Current research theories support the division of living organisms into three domains 1. Bacteria 2. Eukaryota 3. Archaea living in the most inhospitable regions of the earth • Thermophiles tolerate extremely high temperatures • Halophiles tolerate very high salt concentrations • Methanogens produce methane as a by-product of metabolism 1 -23

FIGURE 1. 5 Hydrothermal Vent Tubeworms These hydrothermal vent tubeworms from the Pacific Ocean get energy from symbiotic bacteria that live inside them. Courtesy of National Oceanic & Atmospheric Administration/National Undersea Research Program (NURP). Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 24

FIGURE 1. 6 Subcellular Structure of Escherichia coli (A) Scanning electron micrograph of E. coli. The rod-shaped bacteria are approximately 0. 6 microns by 1– 2 microns. Courtesy of Rocky Mountain Laboratories, NIAID, NIH. (B) Gram-negative bacteria have three structural layers surrounding the cytoplasm. The outer membrane and cytoplasmic membrane are lipid bilayers, and the cell wall is made of peptidoglycan. Unlike eukaryotes, no membrane surrounds the chromosome, leaving the DNA readily accessible to the cytoplasm. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 25

FIGURE 1. 7 Bacteria are Easy to Grow (A) Bacteria growing in liquid culture. (B) Bacteria growing on agar. This photo shows a mixture of bacterial colonies from the blue/white method for screening plasmid insertions—see Chapter 3 and Fig. 3. 15 for a full explanation. (C) Fast-growing bacteria can double in numbers in short periods. Here, the number of bacteria double after approximately 45 minutes and reach a density of 5 × 109 cells/m. L in about 5 hours. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 26

FIGURE 1. 8 The E. coli chromosome is divided into 100 map units, arbitrarily starting at the thr. ABC operon. Various genes and their locations are shown. The replication origin (ori. C) and termination zone (ter. B and ter. C) are indicated. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 27

FIGURE 1. 9 Plasmids Encode the Genes for Colicin Col. E 1 plasmids are extrachromosomal DNA elements that are maintained by bacteria for producing a toxin (cea gene). They also carry genes for toxin release and immunity. These plasmids have been modified to carry genes useful in genetic engineering. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 28

FIGURE 1. 10 Somatic versus Germline Cells During development, cells either become somatic cells, which form the body, or germline cells, which form either eggs or sperm. The germline cells are the only cells whose genes are passed on to future generations. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 29

FIGURE 1. 10 Somatic versus Germline Cells During development, cells either become somatic cells, which form the body, or germline cells, which form either eggs or sperm. The germline cells are the only cells whose genes are passed on to future generations. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 30

FIGURE 1. 11 Somatic Mutations The early embryo has the same genetic information in every cell. During division of a somatic cell, a mutation may occur that affects the organ or tissue it gives rise to. Because the mutation was isolated in a single precursor cell, other parts of the body and the germline cells will not contain the mutation. Consequently, the mutation will not be passed on to any offspring. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 31

FIGURE 1. 12 Structure of Yeast Cell This yeast cell, undergoing division, is starting to partition components into the bud. Eventually, the bud will grow in size and be released from the mother (lower oval), leaving a scar on the surface of the cell wall. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 32

FIGURE 1. 13 The 2 -Micron Plasmid of Yeast Two different forms of the 2 -micron plasmid are shown. The enzyme Flp recombinase recognizes the FRT sites and recombines them, thus flipping one half of the plasmid relative to the other half. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 33

FIGURE 1. 14 Alternating Haploid and Diploid Phases of Yeast Haploid cells come in two different forms, a and a. These express mating pheromones, a factor and alpha (a) factor, which attract the two forms to each other. When the pheromones bind to receptors on the opposite cell type, the two haploid cells become competent to fuse into a diploid cell. Diploid cells sporulate under growth limiting conditions. Otherwise, the diploid cells form genetic clones by budding. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 34

FIGURE 1. 15 Caenorhabditis elegans Courtesy of Jill Bettinger, Virginia Commonwealth University, Richmond, VA. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 35

FIGURE 1. 16 Life Cycle of Caenorhabditis elegans When the C. elegans sperm fuses with an egg, a small worm develops (L 1). The larva goes through multiple stages until it reaches the sexually mature adult phase. C. elegans has six different chromosomes: five autosomes and one X chromosome. The worms are diploid, with two sets of chromosomes. When the embryo has two X chromosomes, it becomes a hermaphrodite. If the embryo has only one X, it becomes a male, but males make up only 0. 05% of a normal population. The genome is 97 Mb and was completely sequenced in 1998. Approximately 27% of the genome is coding sequence with about 19, 000 genes, more than 900 of which are RNA coding genes. The average gene contains five introns and is about 3000 base pairs long. Intronic DNA accounts for 26% of the total genome. The remaining 47% of the genome is intergenic and noncoding. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 36

FIGURE 1. 17 Life Cycle of Drosophila melanogaster Drosophila fruit flies start as a tiny egg that develops into a worm (maggot). After a series of larval stages, the worm forms a pupa where the adult form develops. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 37

FIGURE 1. 22 Arabidopsis thaliana The plant most used as a model for molecular biology research is A. thaliana, a member of the mustard family (Brassicaceae). Courtesy of Dr. Jeremy Burgess, Science Photo Library. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 38

FIGURE 1. 18 Polytene Chromosome Fluorescent staining of polytene chromosome from Drosophila. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 39

FIGURE 1. 19 The Zebrafish, Danio rerio This fish is used as a model vertebrate to study genetics, cell biology, and developmental biology. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 40

FIGURE 1. 20 Human He. La Cells Grown in Vitro He. La cells were taken from the tumor of Henrietta Lacks, a woman suffering from cervical cancer, in the 1950 s and have been cultured continuously ever since. (A) Viewed under phase contrast. (B) Viewed under differential interference contrast. Courtesy of Michael W. Davidson, Optical Microscopy Group, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 41

FIGURE 1. 21 Insect Cells in Culture (A) Hv. T 1 cells from tobacco budworm testes are strongly attached to the surface of the dish. (B) TN 368 cells from cabbage looper ovary are only loosely attached. Courtesy of Dwight E. Lynn, Insect Biocontrol Lab, USDA, Beltsville, MD. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 42

FIGURE 1. 23 Virus Life Cycle The life cycle of a virus starts when the viral DNA or RNA enters the host cell. Once inside, the virus uses the host cell to manufacture more copies of the virus genome and to make the protein coats for assembly of virus particles. Once multiple copies of the virus have been assembled, the host cell bursts open, allowing the progeny to escape and find other hosts to invade Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 43

FIGURE 2. 6 Components of the lac Operon The lac operon consists of three structural genes, lac. ZYA, which are all transcribed from a single promoter, designated lac. P. The promoter is regulated by binding of the repressor at the operator, lac. O, and of Crp protein at the Crp site. Note that in reality, the operator partly overlaps both the promoter and the lac. Z structural gene. The single lac m. RNA is translated to produce the Lac. Z, Lac. Y, and Lac. A proteins. The lac. I gene that encodes the Lac. I repressor has its own promoter and is transcribed in the opposite direction from the lac. ZYA operon. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 44

FIGURE 1. 24 Examples of Different Viruses come in a variety of shapes and sizes that determine whether the entire virus or only its genome enters the host cells. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 45

FIGURE 1. 25 Retroviral Life Cycle Retroviral genomes are made of positive RNA. Once the RNA enters the host, a DNA copy of the genome is made using reverse transcriptase. The original RNA strand is then degraded and replaced with DNA. Then the entire double-stranded DNA version of the retrovirus genome can integrate into the host genome. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 46

FIGURE 1. 26 Conjugation in E. coli Bacteria with a transferable plasmid can make a sex pilus that attaches to a recipient cell. When the two cells touch, a conjugation bridge forms, and a copy of the plasmid transfers from one cell to another. If the plasmid is integrated into the donor cell genome, segments of genomic DNA may also pass across. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 47

FIGURE 1. 27 Transposons Move by Replicative or Conservative Transposition (A) Replicative transposition leaves the original transposon in its original place, and a copy is inserted at another site within the host genome. (B) During conservative transposition, the original transposon excises from its original site and integrates at a different location. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 48

Chapter 2 Companion site for Biotechnology Author: Clark

FIGURE 2. 2 The Structure of a Typical Genes are regions of DNA that are transcribed to give RNA. In most cases, the RNA is translated into protein, but some RNA is not. The gene has a promoter region plus transcriptional start and stop points that flank the actual message. After transcription, the RNA has a 5’ untranslated region (5’ UTR) and 3’ untranslated region (3’ UTR), which are not translated; only the ORF is translated into protein. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 50

Structural Relationship Between Gene, m. RNA and Protein Transcription of DNA (top) does not begin or end at same places as translation – Transcription begins at first G – Translation begins 9 -bp downstream – This m. RNA has a 9 -bp leader or 5’-untranslated region / 5’-UTR 3 -51

DNA Size and Genetic Capacity How many genes are in a piece of DNA? – Start with basic assumptions • Gene encodes protein • Protein is abut 40, 000 D – How many amino acids does this represent? • • Average mass of an amino acid is about 110 D Average protein – 40, 000 / 110 = 364 amino acids Each amino acid = 3 DNA base pairs 364 amino acids requires 1092 base pairs 2 -52

DNAs of Various Sizes and Shapes • Phage DNA is typically circular • Some DNA will be linear • Supercoiled DNA coils or wraps around itself like a twisted rubber band 2 -53

DNA Content and the C-Value Paradox • C-value is the DNA content per haploid cell • Might expect that more complex organisms need more genes than simple organisms • For the mouse or human compared to yeast this is correct • Yet the frog has 7 times more per cell than humans 2 -54

C-Value Paradox • The observation that more complex organisms will not always need more genes than simple organisms is called the C-value paradox • Most likely explanation for the paradox is that DNA that does not code for genes is present when the less complex organism has more DNA 2 -55

FIGURE 2. 3 RNA Polymerase Synthesizes RNA at the Transcription Bubble RNA polymerase is a complex enzyme with two grooves. The first groove holds a single strand of DNA, and the second groove holds the growing RNA polymerase travels down the DNA, adding ribonucleotides that complement each of the bases on the DNA template strand. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 56

FIGURE 2. 4 Monocistronic versus Polycistronic Eukaryotes transcribe genes in single units, where each m. RNA encodes for only one protein. Prokaryotes transcribe genes in operons as one single m. RNA, and then translate the proteins as separate units. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 57

Making the RNA • The strand used by RNA polymerase is called the template (noncoding or antisense) and is complementary to the resulting m. RNA. The opposite strand of DNA is called the coding strand (nontemplate or sense strand) and its sequence is identical to the RNA except for the replacement of T with U in RNA. In vitro transcription of a c. DNA fragment placed behind a promotor using a viral polymerase can produce antisense or sense m. RNA without the requirement of transcription factors. The sequence of antisense m. RNA is complementary to that of sense m. RNA produced in living organisms.

FIGURE 2. 5 Eukaryotic Transcription Many different general transcription factors help RNA polymerase II find the TATA and initiator box region of a eukaryotic promoter. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 59

Four Distinct Preinitiation Complexes • TFIID with help from TFIIA binds to the TATA box forming the DA complex • TFIIB binds next generating the DAB complex • TFIIF helps RNA polymerase bind to a region from -34 to +17, now it is DABPol. F complex • Last the TFIIE then TFIIH bind to form the complete preinitiation complex = DABPol. FEH • In vitro the participation of TFIIA seems to be optional 11 -60

Class II Promoters • Promoters recognized by RNA polymerase II (class II promoters) are similar to prokaryotic promoters • Considered to have two parts: – Core promoter having 4 elements – Upstream promoter element 10 -61

Core Promoter Elements – TATA Box • TATA box – Found on the nontemplate strand – Very similar to the prokaryotic -10 box – There are frequently TATA-less promoters • Housekeeping genes that are constitutively active in nearly all cells as they control common biochemical pathways • Developmentally regulated genes 10 -62

The Class II Preinitiation Complex • Class II preinitiation complex contains: – Polymerase II – 6 general transcription factors: • • • TFIIA TFIIB TFIID TFIIE TFIIH • The transcription factors (TF) and polymerase bind the preinitiation complex in a specific order 11 -63

• Detection of heat shock protein (Hsc 70 and Hsp 70) m. RNA expression in zebrafish embryos by whole mount in situ hybridization-an example illustrating the use of antisense riboprobes obtained from in vitro transcription of cloned c. DNAs using viral RNA polymerases

36 hpf 60 hpf 84 hpf H 108 hpf hsc 70 hsp 70 Analysis of heat shock cognate 70 (Hsc 70) and heat shock protein 70 (Hsp 70) m. RNA expressions in developing zebrafish using whole mount in situ hybridization. H stands for heat shock treatment at 37℃ for 30 min. (Hsu et al. 2010)

36 hpf 60 hpf 84 hpf H Lateral view ↓ Ventral view Analysis of Hsc 70/Hsp 70 protein production in developing zebrafish using whole mount immunohistochemistry. (Hsu et al. 2010) 108 hpf

Discovery of the Operon During the 1940 s and 1950 s, Jacob and Monod studied the metabolism of lactose by E. coli • Three enzyme activities / three genes were induced together by galactosides • Constitutive mutants need no induction, genes are active all the time • Merodiploids are partial diploid bacteria 7 -67

FIGURE 2. 7 Control of Lactose Operon The lactose operon is turned on only when glucose is absent but lactose is present. When glucose is available, the global activator protein, Crp, does not activate binding of RNA polymerase. When there is no glucose, Crp binds to the promoter and stimulates RNA polymerase to bind. The lack of lactose keeps Lac. I protein bound to the operator site and prevents RNA polymerase from transcribing the operon. Only when lactose is present is Lac. I released from the DNA. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 68

Catabolite Repression of the lac Operon • When glucose is present, lac operon is in a relatively inactive state • Selection in favor of glucose attributed to role of a breakdown product, catabolite • Process known as catabolite repression uses a breakdown product to repression the operon 7 -69

Positive Control of lac Operon • Positive control of lac operon by a substance sensing lack of glucose that responds by activating lac promoter – The concentration of nucleotide, cyclic-AMP, rises as the concentration of glucose drops 7 -70

Proposed CAP-c. AMP Activation of lac Transcription • The CAP-c. AMP dimer binds to its target site on the DNA • The a. CTD (a-carboxy terminal domain) of polymerase interacts with a specific site on CAP • Binding is strengthened between promoter and polymerase 7 -71

Inducer of the lac Operon • Inducer (one molecule) of lac operon binds the repressor • The inducer is allolactose, an alternative form of lactose 7 -72

FIGURE 2. 8 Structures of Lactose, allo-Lactose, and IPTG is a nonmetabolizable analog of the lactose operon inducer, allo-lactose. β-galactosidase cannot break the sulfur linkage, and therefore, does not cleave IPTG in two. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 73

FIGURE 2. 9 Model of Two-Component Regulatory System The two-component regulatory system includes a membrane component (sensor kinase) and a cytoplasmic component (regulator). Outside the cell, the sensor domain of the kinase detects an environmental change, which leads to phosphorylation of the transmitter domain. The response regulator protein receives the phosphate group, and as a consequence, changes configuration so as to bind the DNA. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 74

FIGURE 2. 10 Transcription Factors Have Two Independent Domains (A) One domain of the GAL 4 transcription factor normally binds to the GAL 4 DNA recognition sequence and the other binds the transcription apparatus. (B) If the Lex. A sequence is substituted for the GAL 4 site, the transcription factor does not recognize or bind the DNA. (C) An artificial protein made by combining a Lex. A binding domain with a GAL 4 activator domain will not recognize the GAL 4 site, but (D) will bind to the Lex. A recognition sequence and activate transcription. Thus, the GAL 4 activator domain acts independently of any particular recognition sequence. It works as long as it is held in close contact with the DNA. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 75

FIGURE 2. 11 Somatic Mutations The early embryo has the same genetic information in every cell. During division of a somatic cell, a mutation may occur that affects the organ or tissue it gives rise to. Because the mutation was isolated in a single precursor cell, other parts of the body and the germline cells will not contain the mutation. Consequently, the mutation will not be passed on to any offspring. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 76

Enhancers • Enhancers are nonpromoter DNA elements that bind protein factors and stimulate transcription – Can act at a distance – Originally found in eukaryotes – Recently found in prokaryotes 9 -77

FIGURE 2. 12 Eukaryotic Regulation of Transcription (A) AP-1 is a eukaryotic transcription factor that consists of Fos and Jun. These two proteins interact through their leucine zippers. (B) To activate transcription, AP-1 must itself first be activated by phosphorylation by the kinase, JNK. Only then does Jun stimulate RNA polymerase II to transcribe the appropriate genes. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 78

FIGURE 2. 13 DNA Methylation Induces Gene Silencing Gene expression in eukaryotes can be turned off by chromatin condensation. First, the area to be silenced is methylated. The methyl groups attract methyl cytosine binding protein, which in turn attracts histone deacetylases. Once HDAC removes the acetyl groups from the histone tails, the histones aggregate tightly. The closeness of histones excludes any DNA binding proteins and hence turns off gene expression in the area. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 79

FIGURE 2. 14 Processing Eukaryotic m. RNA Eukaryotic RNA is processed before exiting the nucleus for translation into protein. A guanine with a methyl group is added to the 5’ end of the message, a poly(A) tail is added to the 3’ end, and the introns are spliced out. These modifications stabilize the message and make it much shorter than the original RNA transcribed from the DNA. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 80

FIGURE 2. 15 The Genetic Code The 64 codons found in m. RNA are shown with their corresponding amino acids. As usual, bases are read from 5’ to 3’ so that the first base is at the 5’ end of the codon. Three codons (UAA, UAG, UGA) have no cognate amino acid but signal stop. AUG (encoding methionine) and, much less often, GUG (encoding valine) act as start codons. To locate a codon, find the first base in the vertical column on the left, the second base in the horizontal row at the top, and the third base in the vertical column on the right. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 81

FIGURE 2. 16 Structure of t. RNA Allows Wobble in the Third Position Transfer RNA recognizes the codons along m. RNA and presents the correct amino acid for each codon. The first position of the anticodon on t. RNA matches the third position of the codon. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 82

FIGURE 2. 17 Translation in Prokaryotes (A) Initiation of translation begins with the association of the small ribosome subunit with the Shine-Dalgarno sequence (S-D sequence) on the m. RNA. Next, the initiator t. RNA that reads AUG is charged with f. Met. The charged initiator t. RNA associates with the small ribosome subunit and finds the start codon. Assembly is helped by initiation factors (IF 1, IF 2, and IF 3)—not shown. (B) During elongation peptide bonds are formed between the amino acids at the A-site and the P-site. The movement of the ribosome along the m. RNA and addition of a new t. RNA to the A-site are controlled by elongation factors (also not shown). (C) Termination requires release factors. The various components dissociate. The completed protein folds into its proper three-dimensional shape. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 83

FIGURE 2. 18 Translation in Eukaryotes (A) Assembly of the small subunit plus initiator Met-t. RNA involves the binding of factors e. IF 3 and e. IF 2. (B) The cap binding protein of e. IF 4 attaches to the m. RNA before it joins the small subunit. (C) The m. RNA binds to the small subunit via cap binding protein and the 40 S initiation complex is assembled. (D) Assembly of the large subunit requires factor e. IF 5. After assembly, e. IF 2 and e. IF 3 depart. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 84

FIGURE 2. 19 Human Mitochondrial DNA The mitochondrial DNA of humans contains the genes for ribosomal RNA (16 S and 12 S), some transfer RNAs (single-letter amino acid codes mark these on the genome), and some proteins of the electron transport chain. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 85