Molecular Biology And Genetics Lecture Notes PDF

MOLECULAR BIOLOGY AND GENETICS Department of Biochemistry  What is molecular biology? The study of biological phenomena at the molecular level, in particular the study of: the molecular structure of DNA and the information it encodes; the biochemical basis of gene expression...

MOLECULAR BIOLOGY AND GENETICS Department of Biochemistry  What is molecular biology? The study of biological phenomena at the molecular level, in particular the study of: the molecular structure of DNA and the information it encodes; the biochemical basis of gene expression and regulation.  The last 5–10 years mark the beginning of public awareness of molecular biology. The real starting point of this field occurred half a century ago when James D. Watson and Francis Crick suggested a structure for the salt of deoxyribonucleic acid (DNA). DNA is the hereditary material: each chromosome is a single molecule of DNA, and genes are sequences of DNA.  How Molecular Biology came about? Microscopic biology began in Robert 1665 Hooke Robert Hooke (1635-1703) discovered organisms are made up of cells Matthias Schleiden (1804- 1881) and Theodor Schwann (1810-1882) further expanded the study of cells in 1830s Theodor Matthias Schleiden Schwann  Major events in the history of Molecular Biology 1800 - 1870 1865 Gregor Mendel discover the basic rules of heredity of garden pea. – An individual organism has two alternative heredity units for a given trait (dominant Mendel: The Father of Genetics trait v.s. recessive trait) 1869 Johann Friedrich Miescher discovered DNA and named it nuclein. Johann Miescher  Major events in the history of Molecular Biology 1880 - 1900 1881 Edward Zacharias showed chromosomes are composed of nuclein. 1899 Richard Altmann renamed nuclein to nucleic acid. By 1900, chemical structures of all 20 amino acids had been identified  Major events in the history of Molecular Biology 1900-1911 1902 - Emil Hermann Fischer wins Nobel prize: showed amino acids are linked and form proteins – Postulated: protein properties are defined by Emil amino acid composition and arrangement, Fischer which we nowadays know as fact 1911 – Thomas Hunt Morgan discovers genes on chromosomes are the discrete units of heredity Thomas Morgan 1911 Pheobus Aaron Theodore Lerene discovers RNA  Major events in the history of Molecular Biology 1940 - 1950 1941 – George Beadle and Edward Tatum identify that genes make proteins George Edward Beadle Tatum 1950 – Edwin Chargaff find Cytosine complements Guanine and Adenine complements Thymine Edwin Chargaff  Major events in the history of Molecular Biology 1950 - 1952 1950s – Mahlon Bush Hoagland first to isolate tRNA Mahlon Hoagland 1952 – Alfred Hershey and Martha Chase make genes from DNA Hershey Chase Experiment  Major events in the history of Molecular Biology 1952 - 1960 1952-1953 James D. Watson and Francis H. C. James Watson Crick deduced the double and Francis Crick helical structure of DNA 1956 George Emil Palade showed the site of enzymes manufacturing in the cytoplasm is made on RNA organelles called ribosomes. George Emil Palade  Major events in the history of Molecular Biology 1970 1970 Howard Temin and David Baltimore independently isolate the first restriction enzyme DNA can be cut into reproducible pieces with site-specific endonuclease called restriction enzymes; – the pieces can be linked to bacterial vectors and introduced into bacterial hosts. (gene cloning or recombinant DNA technology)  Major events in the history of Molecular Biology 1970- 1977 1977 Phillip Sharp and Richard Roberts demonstrated that pre-mRNA is processed by the excision Phillip Sharp Richard Roberts of introns and exons are spliced together. Joan Steitz determined that the 5’ end of snRNA is partially complementary to the consensus sequence of 5’ splice junctions. Joan Steitz  Major events in the history of Molecular Biology 1986 - 1995 1986 Leroy Hood: Developed automated sequencing mechanism 1986 Human Genome Initiative Leroy Hood announced 1990 The 15 year Human Genome project is launched by congress 1995 Moderate-resolution maps of chromosomes 3, 11, 12, and 22 maps published (These maps provide the locations of “markers” on each chromosome to make locating genes easier)  Major events in the history of Molecular Biology 1995-1996 1995 John Craig Venter: First bactierial genomes sequenced 1995 Automated fluorescent sequencing instruments and John Craig Venter robotic operations 1996 First eukaryotic genome- yeast-sequenced  Major events in the history of Molecular Biology 1997 - 1999 1997 E. coli sequenced 1998 PerkinsElmer, Inc.. Developed 96-capillary sequencer 1998 Complete sequence of the Caenorhabditis elegans genome 1999 First human chromosome (number 22) sequenced  Major events in the history of Molecular Biology 2000-2001 2000 Complete sequence of the euchromatic portion of the Drosophila melanogaster genome 2001 International Human Genome Sequencing:first draft of the sequence of the human genome published  Major events in the history of Molecular Biology 2003- Present April 2003 Human Genome Project Completed. Mouse genome is sequenced. April 2004 Rat genome sequenced. ……………………………… 2014: Cells that might cure diabetes (growing cells that resemble human β cells). CRISPR/Cas9 is a technique that allows for the highly specific and rapid modification of DNA in a genome, the complete set of genetic instructions in an organism. 1 Aug French scientists Bolotin, Institut National de la 2005 suggested CRISPR spacer Quinquis, Recherche sequences can provide Sorokin, Agronomique cell immunity against Ehrlich phage infection and degrade DNA 23 Jan CRISPR-Cas9 used to control Grunwald, University of California San 2019 genetic inheritance in mice Gntz, Diego Poplawski, Xu, Bier, Cooper 30 Jul World Health Organisation 2019 called on countries to ban experiments that would lead to more gene-edited babies  Gene-edited babies ????? Jiankui He presented the gene-editing project that led to the birth of two baby girls. He’s work did make the twin girls less likely to get HIV. This extremely irresponsible behavior violated the ethical consensus of scientists all over the world The transforming principle is DNA In vivo experiments 1928: Frederick Griffith described a transforming principle that transmitted the ability of bacteria to cause pneumonia in mice.  In 1928, Frederick Griffith described a transforming principle that transmitted the ability of bacteria to cause pneumonia in mice. Griffith used pathogenic and nonpathogenic strains of Streptococcus pneumoniae to infect mice. They are designated “S” to distinguish them from nonpathogenic strains. Nonpathogenic strains are designated as (R) colonies. These R strains do not cause pneumonia. When Griffith injected mice with live bacteria of an S strain, they invariably died of pneumonia. When he injected mice with live R bacteria, the mice remained healthy. Mice injected with heat-killed S bacteria also remained healthy. However, when heat-killed S bacteria and live R bacteria were injected, the mice died.  Griffith called this the “transforming principle.” He concluded there was transfer of some component of the pathogenic (S) bacteria which allowed the nonpathogenic (R) bacteria to make the polysaccharide coat and evade the mouse immune response.  The transforming principle is DNA In vitro experiments 1944: Oswald Avery, Colin MacLeod, and Maclyn McCarty demonstrated that purified DNA was sufficient to cause transformation.  Creativity in approach leads to the one gene-one enzyme hypothesis 1941: George Beadle and Edward Tatum were the first to demonstrate a link between a gene and a step in a metabolic pathway catalyzed by an enzyme.  The importance of technological advances: the Hershey-Chase experiment 1952: Alfred Hershey and Martha Chase demonstrated that the genetic material of a virus that infects bacteria, bacteriophage T2, is DNA. Their findings suggested that DNA could be the universal hereditary material. 1.3 A model for the structure of DNA: the DNA double helix  1953: James Watson and Francis Crick proposed the double helix as a model for the structure of DNA. Their discovery was based, in part, on X-ray diffraction analysis performed by Rosalind Franklin in Maurice Wilkin’s lab 1.4 The central dogma of molecular biology The central dogma  Replication: The process of making an exact copy of DNA from the original DNA. Transcription: The process of DNA being copied to generate a single-stranded RNA identical in sequence to one strand of the double-stranded DNA. Translation: The process of the RNA nucleotide sequence being converted into the amino acid sequence of a protein. Reverse transcription: the process of a single- stranded DNA copy being generated from a single-stranded RNA.  DNA: The Code of Life The structure and the four genomic letters code for all living organisms Adenine, Guanine, Thymine, and Cytosine which pair A-T and C-G on complimentary strands.  Structure of DNA the structure of DNA at three levels of increasing complexity, known as the primary, secondary, and tertiary structures of DNA.  The primary structure of DNA refers to its nucleotide structure and how the nucleotides are joined together. The secondary structure refers to DNA’s stable three-dimensional configuration, the helical structure worked out by Watson and Crick. DNA’s tertiary structures, which are the complex packing arrangements of double-stranded DNA in chromosomes. Cells Information and Machinery Cells store all information to replicate itself – Human genome is around 3 billions base pair long – Almost every cell in human body contains same set of genes – But not all genes are used or expressed by those cells Overview of organizations of life Nucleus = library Chromosomes = bookshelves Genes = books Almost every cell in an organism contains the same libraries and the same sets of books. Books represent all the information (DNA) that every cell in the body needs so it can grow and carry out its vaious functions.  More Terminology The genome is an organism’s complete set of DNA. – a bacteria contains about 600,000 DNA base pairs – human and mouse genomes have some 3 billion. human genome has 24 distinct chromosomes. – Each chromosome contains many genes. Gene – basic physical and functional units of heredity. – specific sequences of DNA bases that encode instructions on how to make proteins. Proteins – Make up the cellular structure – large, complex molecules made up of smaller subunits called amino acids.  All Life depends on 3 critical molecules DNAs – Hold information on how cell works RNAs – Act to transfer short pieces of information to different parts of cell – Provide templates to synthesize into protein Proteins – Form enzymes that send signals to other cells and regulate gene activity – Form body’s major components (e.g. hair, skin, etc.) The Structure of DNA  Primary structure: the components of nucleic acids The primary structure of DNA consists of a string of nucleotides joined together by phosphodiester linkages. Each nucleotide subunit is composed of three parts: a five-carbon sugar, a phosphate group, and a base.  Components of nucleotides Five-carbon sugars Nitrogenous bases The phosphate functional group DNA DNA has a double helix structure which composed of – sugar molecule – phosphate group – and a base (A,C,G,T) DNA always reads from 5’ end to 3’ end for transcription replication 5’ ATTTAGGCC 3’ 3’ TAAATCCGG 5’  Five-carbon sugars  The sugars of nucleic acids—called pentose sugars—have five carbon atoms.  This difference gives rise to the names ribonucleic acid (RNA)and deoxyribonucleic acid (DNA).  The additional oxygen atom in the RNA nucleotide makes it more reactive and less chemically stable than DNA. For this reason, DNA is better suited to serve as the long-term repository of genetic information.  Nitrogenous bases As their name suggests, the bases are nitrogen- containing molecules having the chemical properties of a base (a substance that accepts an H+ ion or proton in solution). Two of the bases, adenine (A) and guanine (G) have a double carbon–nitrogen ring structure; these are called purines. The other three bases, thymine (T), cytosine (C), and uracil (U) have a single ring structure; these are called pyrimidines. Thymine is found in DNA only, while uridine is specific for RNA. Nitrogenous bases  Edwin Chargaff’s “rules” [A] = [T] [G] = [C] [A] + [G] = [T] + [C] %G+C differs among species but is constant in all cells of an organism within a species. Varies from 22 to 73% The phosphate functional group  The phosphate functional group (PO4) gives DNA and RNA the property of an acid (a substance that releases an H+ ion or proton in solution) at physiological pH, hence the name “nucleic acid.” The phosphate functional group The phosphate group consists of a phosphorus atom bonded to four oxygen atoms. The phosphate group is always bonded to the 5´-carbon atom of the sugar  Nucleosides and nucleotides DNA and RNA chains are formed through a series of three steps: 1. A base attached to a sugar is a nucleoside. 2. A nucleoside with one or more phosphates attached is a nucleotide. 3. Nucleotides are linked by 5′ to 3′ phosphodiester bonds between adjacent nucleotides to form a DNA or RNA chain.  Nomenclature of nucleotides Example: the base cytosine (C) DNA deoxycytidine 5′-triphosphate (dCTP) RNA cytidine 5′-triphosphate (CTP) Generic deoxynucleoside 5′-triphosphate (dNTP) nucleoside 5′-triphosphate (NTP)  Polynucleotide strands DNA is made up of many nucleotides connected by covalent bonds, which join the 5´-phosphate group of one nucleotide to the 3´-carbon atom of the next nucleotide. These bonds, called phosphodiester linkages, are strong covalent bonds; a series of nucleotides linked in this way constitutes a polynucleotide strand. These bonds are very stable and do not break spontaneously within cells.  Significance of 5′ and 3′ An important characteristic of the polynucleotide strand is its direction, or polarity. The 5′-PO4 and 3′-OH ends of a DNA or RNA chain are distinct and have different properties. By convention, a DNA sequence is written with the 5′ end to the left and the 3′ end to the right.  The length of RNA and DNA RNA The number of nucleotides (nt) or bases is used as a measure of length. Double-stranded DNA The number of base pairs (bp) is used as a measure of length. 1000 bp = 1 kilobase pair (kb or kbp) 1,000,000 = 1 megabase pair (Mb or Mbp)  Concepts The primary structure of DNA consists of a string of nucleotides. Each nucleotide consists of a five-carbon sugar, a phosphate, and a base. There are two types of DNA bases: purines (adenine and guanine) and pyrimidines (thymine and cytosine) 2.3 Secondary structure of DNA  The secondary structure of DNA refers to its three-dimensional configuration—its fundamental helical structure.  DNA’s secondary structure can assume a variety of configurations, depending on its base sequence and the conditions in which it is placed.  The double helix  A fundamental characteristic of DNA’s secondary structure is that it consists of two polynucleotide strands wound around each other—it’s a double helix.  The sugar–phosphate linkages are on the outside of the helix, and the bases are stacked in the interior of the molecule.  The two polynucleotide strands run in opposite directions—they are antiparallel, which means that the 5´ end of one strand is opposite the 3´ end of the other strand.  The double helix  The strands are held together by two types of molecular forces.  Hydrogen bonds link the bases on opposite strands.  These bonds are relatively weak compared with the covalent phosphodiester bonds.  As we will see, several important functions of DNA require the separation of its two nucleotide strands, and this separation can be readily accomplished because of the relative ease of breaking and reestablishing the hydrogen bonds.  Hydrogen bonds form between the bases Two common “Watson-Crick” or “complementary” base pairs: Adenine (A) is joined to thymine (T) by two hydrogen bonds. Guanine (G) is joined to cytosine (C) by three hydrogen bonds.  Why aren’t there other stable base pairs present in DNA? May not be able to form two or more hydrogen bonds. Pairing of G with T produces a pair with a similar shape to Watson-Crick base pairs. Fidelity of DNA replication: proofreading and DNA repair mechanisms correct mistakes. GU base-pairing is of importance in RNA structure.  if G-C to G-T changes were to occur in DNA, the sequence of bases in the DNA could change drastically with each cell division. In fact, there are proofreading mechanisms and DNA repair mechanisms that recognize non-Watson–Crick base pairs and correct the majority of mistakes  The second force that holds the two DNA strands together is the interaction between the stacked base pairs. These stacking interactions contribute to the stability of the DNA molecule but do not require that any particular base follow another. Thus, the base sequence of the DNA molecule is free to vary, allowing DNA to carry genetic information. Base stacking provides chemical stability to the DNA double helix The hydrophobic nitrogenous bases stack onto each other without a gap by means of a helical twist. A double-stranded DNA molecule has a hydrophobic core composed of stacked bases.  base stacking Once the bases are attached to a sugar and a phosphate to form a nucleotide, they become soluble in water, but even so their insolubility still places strong constraints on the overall conformation of DNA in solution. The paired, relatively flat bases tend to stack on top of one another by means of a helical twist This feature of double-stranded DNA is known as “base stacking.”  The nitrogenous bases The molecular processes of cellular life generally take place in a watery solution, and intracellular components are largely molecules that are easily dissolved in water. The nitrogenous bases are an exception as they are nonpolar and thus hydrophobic (“water hating”). Various representations of the DNA double helix  Structure of the Watson-Crick DNA double helix Polarity in each strand: 5′ 3′ Two strands are antiparallel Major and minor grooves The two polynucleotide strands of a DNA molecule are not identical but are complementary DNA strands. Concepts  Major and minor grooves The major groove carries a “message” that can be read by DNA binding proteins.. In the major groove, the pattern of hydrogen- bonding groups is different for AT, TA, GC, and CG base pairs. In the minor groove, there is only one difference in the pattern between AT and GC base pairs.  The major groove has a significant role in sequence-specific DNA–protein interactions. The minor groove of DNA is less informative. Most transcription factors (proteins involved in regulating gene expression) bind DNA in the major groove.  Distinguishing between features of alternative double-helical structures B-DNA (Watson-Crick DNA) A-DNA Z-DNA  The three-dimensional structure of DNA described by Watson and Crick is termed the B-DNA structure. This structure exists when plenty of water surrounds the molecule and there is no unusual base sequence in the DNA—conditions that are likely to be present in cells. The B-DNA structure is the most stable configuration under physiological conditions.  Another secondary structure that DNA can assume is the A-DNA structure, which exists if less water is present. Like B-DNA, A-DNA is an alpha (right- handed) helix but it is shorter and wider than B-DNA and its bases are tilted away from the main axis of the molecule. There is little evidence that A-DNA exists under physiological conditions  Z-DNA A radically different secondary structure, called Z-DNA forms a left-handed helix. In this form, the sugar–phosphate backbone zigzags back and forth, giving rise to its name. A Z-DNA structure can result when DNA is placed in a high- salt solution. It can arise under physiological conditions if the molecule contains particular base sequences, such as stretches of alternating C and G nucleotides.  The predominant form of DNA in vivo is B-DNA. But, there is evidence for a role of Z-DNA in vivo: – Z-DNA binding proteins. – Short sections of Z-DNA within a cell are energetically favorable and stable. – Role in regulating gene expression? DNA can undergo reversible strand separation  Denaturation or “melting” of DNA The hydrogen bonds can be broken and the DNA strands separated by heating the DNA molecule, whereas the phosphodiester bonds remain intact. Base stacking in duplex DNA quenches the capacity of bases to absorb UV light. Hyperchromicity: As DNA “melts” its absorption of UV light increases. Tm (melting temperature): The temperature at which half of the bases in a dsDNA sample have denatured.  Renaturation or “reannealing” of DNA The capacity to renature denatured DNA permits hybridization. Hybridization is the complementary base pairing of strands from two different sources. The rate at which DNA reanneals is a function of the length of the DNA and the initial concentration in the sample.  Denaturation of DNA Lowering the salt concentration of a DNA solution promotes denaturation by removing the cations that shield the negative charges on the two strands from each other. At low ionic strength, the mutually repulsive forces of these negative charges from the backbone phosphoryl groups are enough to denature the DNA, even at a relatively low temperature. In addition, high pH or organic solvents such as formamide disrupt the hydrogen bonding between DNA strands and promote denaturation.  Concepts DNA can assume different secondary structures, depending on the conditions in which it is placed and on its base sequence. B-DNA is thought to be the most common configuration in the cell. Watson-Crick base pairs.  Two DNA strands associate via non- covalent hydrogen bonds to form double- stranded DNA  Bases pair precisely with their complementary base: A pairs with T with 2 H-bonds C pairs with G with 3 H-bonds  These are called Watson-Crick base pairs  The sequence of one strand dictates the sequence of the other strand – thus the strands are complementary to one another  The two strands are antiparallel – the 5′ end of one strand pairs with the 3′ end of the other 2.5: The Structure of DNA Figure 02-24  RNA molecules often have some base pairs that are not Watson-Crick interactions  These non-canonical base pairs often feature chemically modified bases, such as methylation (addition of a CH3)  Pairing with modified bases can introduce structural differences such as kinks 2.7: RNA Folding and Structure Figure 02-38  Hoogsteen base pairing Monomeric A and T derivatives form A-T base pairs with adenine N7 as the hydrogen bonding acceptor (normally N1). This is Hoogsteen base pairing Hoogsteen base pairs are biologically relevant. They help stabilize tRNA tertiary structure. G-C always form Watson-Crick base pairs in crystallized structures because of their 3 hydrogen bonds.  Wobble U-G pair Watson-Crick C-G pair  Another non-canonical pair is G-U, which has two hydrogen bonds (like A-U) - This is known as a “wobble” base pair 2.7: RNA Folding and Structure Figure 02-39 2.4 Unusual DNA secondary structures  Slipped structures Occur at tandem repeats Found upstream of regulatory regions  A tandem repeat (sometimes called a direct repeat) in DNA is two or more adjacent, approximate copies of a pattern of nucleotides, arranged in a head to tail fashion. For example, the sequence 5′-TACGTACGTACGTACG-3′ contains four tandem repeats of “TACG”  Slipped structures are found upstream of regulatory sequences (e.g. for gene transcription) in vitro. It is possible that they have importance for DNA–protein interactions.  Formation of DNA slipped structures can lead to repeat expansion during DNA replication. A number of hereditary neurological diseases are caused by the expansion of simple triplet repeat sequences.  Cruciform structures Paired stem-loop formations Characterized in vitro for many inverted repeats in plasmids and phage Role in vivo?  Triple helix DNA A third strand of DNA joins the first two to form triplex DNA (intra- or inter-molecular). Favored by purine-pyrimidine stretches with mirror repeat symmetry. The Watson-Crick duplex associates with the third strand through Hoogsteen hydrogen bonds. 2.5 Tertiary structure of DNA  Supercoiling of DNA Supercoils form a twisted, 3-D structure which is more favorable energetically. Less stable than relaxed DNA. Negative (left-handed) supercoil: underwound Positive (right-handed) supercoil: overwound  Closed circular DNA can be supercoiled  Supercoiled DNA is under tension and twists in on itself  Open, uncoiled circular DNA is said to be relaxed 2.5: The Structure of DNA Figure 02-27  What is the significance of supercoiling in vivo? Virtually all DNA within prokaryotes and eukaryotes is negatively supercoiled. Some architectural proteins, induce DNA negative supercoiling upon binding. DNA is restrained when it is supercoiled around DNA-binding proteins, such as in nucleosomes. Unrestrained supercoiled domains are in equilibrium between tension and unwinding of the helix. DNA supercoiling plays an important role in many processes, such as replication, transcription, and recombination Genome of some viruses: small circle Relaxed circle: reduced activity Negatively supercoiled circle: increased activity Bacterial genome: very large circle Form independent DNA loop domains Eukaryotic genomes: linear Form independent DNA loop domains  Negative supercoiling makes it easier to separate the DNA strands during replication and transcription. The DNA of thermophilic Archaea exists in a positive supercoiled state that protects the DNA from denaturation at high temperatures.  B-DNA→ Z-DNA transitions may be triggered by negative supercoiling. Topoisomerases are enzymes that introduce transient breaks in DNA strands and release the strain of supercoiling.  Topoisomerases play important roles in many cellular processes, including chromosome condensation and segregation, DNA replication, gene transcription, and recombination  Mechanism of action of a type I topoisomerase The enzyme binds to a circular DNA molecule with one negative supercoil and unwinds thedouble helix.  What is the significance of supercoiling in vivo? Virtually all DNA within both prokaryotic and eukaryotic cells exists in the negative supercoiled state. DNA supercoiling plays an important role in many genetic processes, such as replication, transcription, and recombination. Genome Organization  DNA is associated with architectural proteins and packaged into chromosomes. But, genetic information has to be accessible for processes such as replication and transcription. 5.2 Genome organization varies in different organisms Diversity in the number of chromosomes that make up eukaryotic genomes Butterflies: >200 chromosomes Kangaroos: 12 Humans: 46 Adder tongue fern: 1260 Male jack jumper ant: 1 Viruses with DNA genomes Not considered organisms because they are not made of cells. But, viruses have a genome and they evolve.  Two classes of genomes Small genomes of viruses, archaea, bacteria (1000 tandem repeats of GC rich sequence. Telomeric DNA synthesized and maintained by Telomerase – Adds tandem repeats of TTGGG – Is a ribonucleoprotein, uses internal ribonucleotide sequences as a template Eukaryotic Replication Enzymes 1. Enzymes of eukaryotic DNA replication aren’t as well characterized as their prokaryotic counterparts. The replication process is similar in both groups—DNA denatures, replication is semiconservative and semidiscontinuous and primers are required. 2. Fifteen DNA polymerases are known in mammalian cells: a. Three DNA polymerases are used to replicate nuclear DNA. Pol α (alpha) extends the 10-nt RNA primer by about 30nt. Pol δ(delta) and Pol ε(epsilon) extend the RNA/DNA primers, one on the leading strand and the other on the lagging (it is not clear which synthesizes which). b. Other DNA pols replicate mitochondrial or chloroplast DNA, or are used in DNA repair.  In linear chromosomes with multiple origins, the elongation of DNA in adjacent replicons also provides a 3´-OH group preceding each primer. At the very end of a linear chromosome, however, there is no adjacent stretch of replicated DNA to provide this crucial 3´-OH group. When the primer at the end of the chromosome has been removed, it cannot be replaced with DNA nucleotides, which produces a gap at the end of the chromosome, suggesting that the chromosome should become progressively shorter with each round of replication.  Fig. 3.15 Synthesis of telomeric DNA by telomerase Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Chapter 3 slide 338  Telomeres Eukaryotic chromosomes end with tandem repeats of a simple G-rich sequence. Humans: TTAGGG Tetrahymena: TTGGGG Seal the ends of chromosomes. Confer stability by keeping the chromosomes from ligating together. The ends of chromosomes— the telomeres—possess several unique features, one of which is the presence of many copies of a short repeated sequence. Solution to the end replication problem The chromosome would be shortened with each successive generation of an organism, leading to the eventual elimination of the entire telomere, destabilization of the chromosome, and cell death. But chromosomes don’t normally become shorter each generation and destabilize. Solution reported by Carol Greider and Elizabeth Blackburn in 1985. Studied Tetrahymena thermophila, a single-celled eukaryote with over 40,000 telomeres. Discovered the enzyme telomerase.  Telomerase is a ribonucleoprotein (RNP) complex with reverse transcriptase activity. Contains an essential RNA component that provides the template for telomere repeat synthesis. – RNA: Telomerase RNA component (TERC) – Protein: Telomerase reverse transcriptase (TERT)  Reverse transcriptases are DNA polymerases that copy RNA into DNA  Reverse transcriptases use single-stranded RNA as a template, but are structurally and catalytically similar to DNA polymerases  Reverse transcriptases are encoded by viruses and by DNA elements in eukaryotes called retrotransposons  Telomerase, which is needed to synthesize chromosome ends, is a specialized reverse transcriptase 6.4: Specialized Polymerases  Maintenance of telomeres by telomerase Telomerase elongates the 3′ end of the template for the lagging strand (G-rich overhang).  Telomeres Ends of linear chromosomes Centromere Telomere Telomere Repetitive DNA sequence (TTAGGG in vertebrates) Specialized proteins Form a 'capped' end structure  What do telomeres do? They protect the chromosomes. They separate one chromosome from another in the DNA sequence Without telomeres, the ends of the chromosomes would be "repaired", leading to chromosome fusion and massive genomic instability. Telomeres are also thought to be the "clock" that regulates how many times an individual cell can divide. Telomeric sequences shorten each time the DNA replicates.  What is telomerase Telomerase is a ribonucleoprotein enzyme complex (a cellular reverse transcriptase) that has been referred to as a cellular immortalizing enzyme. It stabilizes telomere length by adding hexameric (TTAGGG) repeats onto the telomeric ends of the chromosomes, thus compensating for the erosion of telomeres that occurs in its absence.  How Does Telomerase Work? Telomerase works by adding back telomeric DNA to the ends of chromosomes, thus compensating for the loss of telomeres that normally occurs as cells divide. Most normal cells do not have this enzyme and thus they lose telomeres with each division. In humans, telomerase is active in germ cells, in vitro immortalized cells, the vast majority of cancer cells and, possibly, in some stem cells. High telomerase activity exists in germ cells,  Telomerase, aging, and cancer In most unicellular organisms, telomerase has a “housekeeping function.” In most human somatic cells, not enough telomerase is expressed to maintain a constant telomere length: Progressive shortening of telomeres. High levels of telomerase activity in ovaries, testes, rapidly dividing somatic cells, and cancer cells.  Telomerase and aging: the Hayflick limit The Hayflick limit is the point at which cultured cells stop dividing and enter an irreversible state of cellular aging (senescence). Proposed to be a consequence of telomere shortening.  Telomere shortening: a molecular clock for aging? Telomerase: A target for anti-aging therapy or anti-cancer therapy? Cellular senescence may be a mechanism to protect multicellular organisms from cancer. Cancer cells become immortalized and thus can grow uncontrolled. In most cancer cells, telomerase has been reactivated. Direct evidence for a relationship between telomere shortening and aging Evidence from experiments in human cells in culture and in transgenic mice. The telomeres of genetically engineered mice that lack a functional telomerase gene undergo progressive shortening in successive generations. After several generations, these mice show some signs of premature aging, such as graying, hair loss, and delayed wound healing However, there are reports of instances where short telomere length does not correlate with entry into cellular senescence. Transcription in Bacteria  A central event in gene expression is the copying of the sequence of the template strand of a gene into a complementary RNA transcript. The biochemistry of transcript formation is straightforward. The regulatory mechanisms that have been developed by bacteria to control transcription are complex and highly variable. 10.2 Mechanism of transcription  RNA polymerase is the enzyme that catalyzes RNA synthesis. Using DNA as a template, RNA polymerase joins, or “polymerizes,” nucleoside triphosphates (NTPs) by phosphodiester bonds from 5' to 3'.  In bacteria, transcription and translation are coupled―they occur within a single cellular compartment. As soon as transcription of the mRNA begins, ribosomes attach and initiate protein synthesis. The whole process occurs within minutes.  Transcription and translation are uncoupled in eukaryotes The primary transcript (pre- mRNA) is a precursor to the mRNA. The pre-mRNA is modified at both ends, and introns are removed to produce the mRNA. 3 After processing, the mRNA is exported to the cytoplasm for translation by ribosomes. © John Wiley & Sons, Inc.  Minimal requirements for gene transcription. – Gene promoter – RNA polymerase Additional factors are required for the regulation of transcription.  Bacterial promoter structure RNA polymerase binds to a region of DNA called a promoter. Bacterial promoters are not absolutely conserved but they do have a consensus sequence.  Conserved sequence: When nucleotide sequences of DNA are aligned with each other, each has exactly the same series of nucleotides in a given region. Consensus sequence: there is some variation in the sequence but certain nucleotides are present at high frequency.  the consensus sequence TATAAT, which is also known as the TATA box or Pribnow box after one of its discoverers, David Pribnow Now, it is usually called the −10 sequence (or −10 region or element).  Another region with similar sequences among many promoters is centered at −35. The consensus sequence at −35 is TTGACA. The spacing between the −10 and −35 sequences and the start point for transcription is important. Promoter strength The relative frequency of transcription initiation. Related to the affinity of RNA polymerase for the promoter region. The more closely regions within the promoter resemble the consensus sequences, the greater the strength of the promoter.  Structure of bacterial RNA polymerase Comprised of a core enzyme plus a transcription factor called the sigma factor (). Together they form the complete, fully functional enzyme complex called the holoenzyme.  The core enzyme The core enzyme catalyzes polymerization. High affinity for most DNA.  The σ factor is primarily involved in recognition of gene promoters. The −35 and the −10 sequences are necessary for recognition by the σ70 factor, while the −10 sequence is the region of contact for the core enzyme.  Sigma factor The sigma () factor decreases the nonspecific binding affinity of the core enzyme. Binding results in closing of the core enzyme “pincers.” Primarily involved in recognition of gene promoters.  In E. coli the most abundant  factor is 70. For expression of some genes, bacterial cells use alternative  factors. For expression of some genes, bacterial cells use alternative σ factors, specific to different subsets of promoters. The sigma factor stimulates tight binding of RNA polymerase to the promoter The holoenzyme containing  dissociates more slowly from template DNA compared with the core polymerase alone.  Initiation of transcription Initiation consists of three stages: 1. Formation of a closed promoter complex. 2. Formation of an open promoter complex. 3. Promoter clearance.  Stages of Transcription --DNA dependent RNA polymerase --5’ to 3’ direction --Walk (literally) on the DNA --Upstream and downstream regions  Initiation Closed promoter complex RNA polymerase holoenzyme binds to the promoter at nucleotide positions 35 and 10. The DNA remains double-stranded. The complex is reversible. The term “closed” indicates that the DNA remains double- stranded and the complex is reversible. Open promoter complex ~18 bp around the transcription start site are melted to expose the template strand DNA. AT rich promoters require less energy to melt. Transcription is aided by negative supercoiling of the promoter region of some genes. The open complex is generally irreversible.  Transcription is initiated in the presence of NTPs. No primer is required for initiation by RNA polymerase. In contrast to most DNA polymerases, no primer is required for initiation by RNA polymerase. RNA polymerase can initiate RNA synthesis de novo. Conformational changes during the steps of transcription initiation Promoter clearance During the “promoter clearance” step, there is a staged disruption of σ factor–core enzyme interaction. Older “classic” model   factor release. Current model   factor does not completely dissociate; some domains are displaced. The displaced domains allow the nascent RNA to emerge from the RNA exit channel. Electron micrograph of RNA polymerase molecules from E. coli bound to several promoter sites of phage T7 DNA  Elongation After about 9-12 nt of RNA have been synthesized, the initiation complex enters the elongation stage. Direction of transcription around the E. coli chromosome Of the 50 operons or genes whose transcription direction is known, 27 are transcribed clockwise and 23 in the counterclockwise direction around the circle, using the opposite strand as a template. Only one strand of a given operon’s DNA is used as a template for transcription.  The origin and terminus of replication divide the genome into oppositely replicated halves or “replichores.” Most operons or genes are transcribed in the direction of replication. This may lead to fewer collisions of DNA and RNA polymerase and less topological strain from opposing supercoils.  As RNA polymerase moves during elongation, it holds the DNA strands apart, forming a transcription “bubble.” The moving polymerase protects a “footprint” of ~30 bp along the DNA against nuclease digestion.  One strand of DNA acts as the template for RNA synthesis by complementary base pairing. The catalytic site has both a substrate-binding and a product-binding site. Transcription always proceeds in the 5′→3′ direction.  Completion of the single nucleotide addition cycle is accompanied by a shift of the active site of the RNA polymerase forward by one position along the DNA template. As a result, the 9–12 bp RNA–DNA hybrid retains a constant length but becomes one base pair longer at the downstream end and one base pair shorter at the upstream end.  Transcription continues in a processive manner as nucleotides are added to the growing RNA strand by RNA polymerase according to the rules of complementary base pairing. Which moves – the RNA polymerase or the DNA? Whether it is the RNA polymerase that moves along the DNA or vice versa remains a subject of debate Two models Model 1: RNA polymerase moves along and the DNA rotates. – This is the more widely accepted model. Model 2: RNA polymerase remains stationary, and the DNA moves along and rotates. The overall process of transcription has a significant local effect on DNA structure The DNA ahead of the RNA polymerase is wound more tightly; positive supercoils form. Behind the polymerase, DNA becomes less tightly wound; negative supercoils form. Topoisomerase I and gyrase (bacterial topoisomerase II) resolve this supercoiling and restore the DNA to its relaxed form.  Proofreading Proofreading by RNA polymerase Backtracks 3′→5′ Pauses Nucleolytic cleavage  Termination of transcription In most bacteria, there are two types of terminators: Rho-independent Rho-dependent  Rho-independent termination Terminator is characterized by an inverted repeat consensus sequence. Formation of a stem-loop in the exit channel. Less stable U-A hybrid helix. Polymerase pauses, resulting in transcript release. Rho-independent terminators—do not require  intrinsic termination). RNA transcription stops --when the newly synthesized RNA molecule forms a G-C-rich hairpin loop followed by a run of As --Create a mechanical stress --Pulls the poly-U transcript out of the active site of the RNA polymerase --A-U has very weak interaction  Rho-dependent termination Terminator is an inverted repeat with no simple consensus sequence. Controlled by the ability of the Rho protein to gain access to the mRNA. Because ribosomes translate mRNA at the same rate as the mRNA is transcribed, Rho is prevented from loading onto the newly formed RNA until the end of a gene or operon.  Rho - the termination factor protein – rho is an ATP-dependent helicase – it moves along RNA transcript, finds the "bubble", unwinds it and releases RNA chain Rho binds specifically to a C-rich site called a Rho utilization or rut site at the 5′ end of the newly formed RNA, as it emerges from the exit site of RNA polymerase. Rho binds specifically to a C-rich site called a Rho utilization or rut site at the 5′ end of the newly formed RNA, as it emerges from the exit site of RNA polymerase  Termination Signals in E. coli Rho-dependent terminators (non-intrinsic) — require a protein factor () and rut site Rut proteins bind specific RNA sequences (>>Cs and No Lactose - expression 5’ 3’ Activator protein Glucose – No cAMP P Lac rep X => expression Lactose - 5’ 3’  The lac promoter and lacZ structural gene are widely used in molecular biology research Commonly used reporter gene. Expression of heterologous proteins in bacteria. In the lab IPTG is used as an inducer; it interacts with the Lac repressor but is not metabolized by -galactosidase.  10.4 Mode of action of transcriptional regulators The lac operon and other operons illustrate fundamental principles of gene regulation that are universal. Constitutively active RNA polymerase that alone works with a certain frequency. Transcriptional activators increase the frequency of initiation. Transcriptional repressors decrease the frequency of initiation. 10.5 Control of gene expression by RNA  Differential folding of RNA: transcriptional attenuation of the tryptophan operon Regulation of the tryptophan operon occurs by two mechanisms: Transcriptional attenuation. Conventional protein-mediated repression.  Newly synthesized RNA can fold to form either of two competing hairpin structures: – antiterminator or terminator The leader RNA preceding the antiterminator contains a 14 nt coding region, trpL, which includes two tryptophan codons. When bacterial cells have adequate levels of tryptophan-charged tRNATrp The leader peptide (trpL) is synthesized. The terminator forms in the leader transcript. Transcription is terminated. When cells are deficient in charged tRNATrp The ribosome translating trpL stalls at one of the tryptophan codons. The antiterminator forms and termination is blocked. The structural genes involved in tryptophan biosynthesis are transcribed. Conventional protein-mediated repression of the trp operon In the absence of tryptophan, the genes encoding enzymes for the biosynthesis of tryptophan are transcribed and translated. When enough tryptophan has been produced: Tryptophan binds to the dimeric Trp repressor protein. The Trp repressor binds the trp operator and blocks access of RNA polymerase to the trp promoter. Transcription in Eukaryotes Many eukaryotes are estimated to have 20,000–25,000 genes. Some of these are expressed (transcribed) in all cells all of the time, while others are expressed as cells enter a particular pathway of differentiation or as conditions in and around the cells change. DNA–protein interactions, protein–protein interactions are of critical importance for regulating gene transcription. The most important and widely used strategy for regulating gene expression is altering the rate of transcription of a gene. However, the control of gene expression can be exerted at many other levels, including processing of the RNA transcript, transport of RNA to the cytoplasm, translation of mRNA, and mRNA and protein stability. Eukaryotic gene regulation involves: DNA-protein interactions Protein-protein interactions Chromatin structure Nuclear architecture Cellular compartmentalization 11.2 Overview of transcriptional regulation Transcription and translation are uncoupled in eukaryotes Transcription takes place in the nucleus and translation takes place in the cytoplasm. The whole process may take hours, or in some cases, months for developmentally regulated genes. Gene expression can be controlled at many different levels. Transcription is mediated by: Sequence-specific DNA-binding transcription factors. The general RNA polymerase II (RNA pol II) transcriptional machinery. Coactivators and corepressors. Elongation factors.  Eukaryotes have different types of RNA polymerase Bacteria have one type of RNA polymerase that is responsible for transcription of all genes. Eukaryotes have multiple nuclear DNA- dependent RNA polymerases and organelle- specific polymerases. Focus here on regulation of transcription of protein-coding genes by RNA polymerase II. 11.3 Protein-coding gene regulatory elements The big picture: Transcription factors interpret the information present in gene promoters and other regulatory elements and transmit the appropriate response to the RNA pol II transcriptional machinery. What turns on a particular gene in a particular cell is the unique combination of regulatory elements and the transcription factors that bind them.  Gene regulatory elements are specific cis-acting DNA sequences that are recognized by trans- acting transcription factors. Two broad categories of cis-acting regulatory elements. – Promoter elements. – Long-range regulatory elements. Regulatory elements that map far from a gene are trans-acting DNA sequences because they encode transcription factors Genes that encode proteins that interact directly or indirectly with target genes cis-acting elements – Known genetically as transcription factors  Regulatory elements that map near a gene are cis-acting DNA sequences cis-acting elements – Promoter – very close to gene’s initiation site – Enhancer can lie far way from gene Can be reversed Augment or repress basal levels of transcription  Structure and function of promoter elements The gene promoter is the collection of cis- regulatory elements that are: Required for the initiation of transcription. Increase the frequency of initiation only when positioned near the transcriptional start site. The recognition site for RNA pol II general transcription factors. The gene promoter region Core promoter elements. Proximal promoter elements.  Core promoter elements Approximately 60 bp DNA sequence overlapping the transcription start site. Serves as the recognition site for RNA pol II and the general transcription factors. A particular core promoter many contain some, all, or none of the common motifs. Promoter elements become nonfunctional when moved even a short distance from the start of transcription or if their orientation is altered.  Some of the known core promoter elements are the TATA box, the initiator element (Inr), the TFIIB recognition element (BRE), the downstream promoter element (DPE), and the motif ten element (MTE). The general transcription factor TFIID is responsible for the recognition of all known core promoter elements, with the exception of the BRE which is recognized by TFIIB.  Promoter proximal elements Regulation of TFIID binding to the core promoter in yeast depends on an upstream activating sequence (UAS). Multicellular eukaryotic genes are likely to contain several promoter proximal elements. e.g. CAAT box and the GC box  Promoter proximal elements Transcription factors that bind promoter proximal elements do not always directly activate or repress transcription. Promoter proximal elements increase the frequency of initiation of transcription, but only when positioned near the transcriptional start site. Transcription factors may serve as “tethering elements.”  Structure and function of long-range regulatory elements Additional regulatory elements in multicellular eukaryotes that can work over distances of 100 kb or more from the gene promoter. Long-range regulatory elements in multicellular eukaryotes include enhancers and silencers, insulators, locus control regions (LCRs), and matrix attachment regions (MARs).  Enhancers and silencers Insulators Locus control regions (LCRs) Matrix attachment regions (MARs) Enhancers and silencers Insulators  Locus control regions (LCRs) Organize and maintain a functional domain of active chromatin. Although sometimes referred to as “enhancers” of transcription, LCRs, unlike classic enhancer elements, operate in an orientation-dependent manner. Matrix attachment regions (MARs)  Gene Regulation by RNA Processing and Degradation In bacteria, most gene regulation is at the level of transcription, because transcription and translation take place simultaneously, leaving little opportunity to control gene expression after transcription In eukaryotes, transcription takes place in the nucleus and the pre-mRNAs are then processed before moving to the cytoplasm for translation, and so there are more opportunities for gene control after transcription. 11.4 The general transcriptional machinery  General, but diverse, components of large multi-protein RNA polymerase machines required for promoter recognition and the catalysis of RNA synthesis. Three major classes of proteins that regulate transcription The general (basal) transcription machinery Transcription factors Transcriptional coactivators and corepressors  Components of the general transcription machinery RNA polymerase II General transcription factors: TFIIB, TFIID, TFIIE, TFIIF, and TFIIH Mediator Four major steps of transcription initiation 1.Preinitiation complex assembly 2.Initiation 3.Promoter clearance and elongation 4.Reinitiation  General transcription factors and preinitiation complex formation A set of five general transcription factors, denoted TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Responsible for promoter recognition and unwinding of promoter DNA. Nomenclature denotes “transcription factor for RNA polymerase II.”  RNA polymerase II is absolutely dependent on these auxiliary transcription factors for the initiation of transcription. TFIIA and its subunit TFIIJ are not absolutely required for transcription initiation in vitro, so are not considered general transcription factors.  TFIID recruits the rest of the transcriptional machinery Binding of TFIID to the core promoter is a critical rate limiting step. TFIID is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFs)  TFIIB orients the complex on the promoter TFIIB binds to one end of TBP and to a GC-rich DNA sequence after the TATA motif. The TFIID-TBP-DNA complex “signposts” the direction for the start of transcription. The complex indicates which strand acts as the template.  TFIIE, TFIIF, and TFIIH binding completes the preinitiation complex formation RNA polymerase II joins the assemblage in association with TFIIF and Mediator. TFIIE binds and recruits TFIIH. Promoter melting is mediated by the helicase activity of TFIIH.  Unwinding is followed by “capture” of the nontemplate strand by TFIIF. The template strand descends into the active site of RNA polymerase II. Because of the conserved spacing from the TATA box to the transcription start site, the start site is positioned in the polymerase active site.  Mediator: a molecular bridge A 20-subunit complex which transduces regulatory information from the activator and repressor proteins to RNA pol II. Mediator serves as a molecular bridge between the transactivation domain of various transcription factors and RNA pol II.  Initiation of transcription Assembly of the preinitiation complex. A period of abortive initiation. Promoter clearance. Elongation. Abortive initiation RNA polymerase II synthesizes a series of short transcripts As it moves, the polymerase holds the DNA strands apart forming a transcription bubble. A transcript of >10 nucleotides and bubble collapse lead to promoter clearance. Promoter clearance Requires phosphorylation of the C-terminal domain (CTD) of RNA pol II. Phosphorylation helps RNA pol II to leave behind most of the general transcription factors. TFIID remains bound at the promoter and allows the rapid formation of a new preinitiation complex. Once phosphorylated, RNA polymerase II can: Unwind DNA. Polymerize RNA. Proofread.  11.5 The role of specific transcription factors in gene regulation Transcription factors influence that rate of transcription of specific genes either positively or negatively by: Specific interactions with DNA regulatory elements. Interaction with other proteins.  Gene regulation at the transcriptional level generally occurs via changes in the amounts or activities of transcription factors. The genes encoding transcription factors may be activated or repressed by other regulatory proteins. Transcription factors themselves may be activated or deactivated by proteolysis, covalent modifications, ligand binding, etc.  Transcription factors mediate gene- specific transcriptional activation or repression Transcription factors that serve as repressors block the general transcription machinery. Transcription factors that serve as activators increase the rate of transcription by several mechanism.  Transcription factors are modular proteins Composed of separable, functional domains. The three major domains are a DNA- binding domain, a transactivation domain, and a dimerization domain. Some of the most common DNA-binding domain motifs: Helix-turn-helix Zinc finger Basic leucine zipper Basic helix-loop-helix 11.6 Transcriptional coactivators and corepressors Increase or decrease transcriptional activity without binding DNA directly by: Serving as scaffolds for recruitment of proteins with enzymatic activity. Having enzymatic activity themselves for altering chromatin activity. Two main classes of coactivators Chromatin modification complexes. Chromatin remodeling complexes.  Chromatin modification complexes Multiprotein complexes that modify histones post-translationally, in ways that allow greater access of other proteins to DNA. Post-translational modification of histone N- terminal tails Changes in Chromatin Structure One type of gene control in eukaryotic cells is accomplished through the modification of gene structure.  DNA methylation Another change in chromatin structure associated with transcription is the methylation of cytosine bases, which yields 5-methylcytosine. The methylation of cytosine in DNA is distinct from the methylation of histone proteins of transcription in vertebrates and plants, whereas transcriptionally active DNA is usually unmethylated in these organisms. Abnormal patterns of methylation are also associated with some types of cancer. TRANSLATION  Translation is the production of a protein from the information in an mRNA  Proteins are made from 20 different amino acids  The information in the mRNA (nucleic acid) is translated into protein (amino acids) via transfer RNA molecules (tRNA)  tRNAs read the mRNA by base-pairing 3 nucleotides – the region on the tRNA is the anticodon, and on the mRNA it is the codon  tRNAs each carry a specific amino acid at the 3′ end. Amino acids are attached to tRNAs by aminoacyl-tRNA synthetases  Amino acids attached to tRNAs are joined together in a chain 11.1: Overview of Translation Figure 11-01  Ribosomes carry out translation  Ribosomes have small and large subunits. The small subunit deciphers the mRNA and the large mediates the chemical bond formations  Ribosomes are about 2/3 RNA and 1/3 protein  Ribosomes move 5′ to 3′ along an mRNA molecule  Proteins are synthesized at a rate of about 15 amino acids per second, with an error rate of about 11-3 to 11-4 per acid (much more error-prone than DNA replication)  Translation factors (often GTPases) associate with ribosomes and help with translation  Translation has four main stages: initiation, elongation, termination and ribosome recycling 11.1: Overview of Translation Figure 11-02  Transfer RNAs (tRNAs) decipher mRNAs and carry amino acids  tRNAs are small RNAs of ~75-94 nucleotides. Each tRNA is specific to a certain amino acid  tRNA structure has four regions of double-stranded RNA, including 3 stem-loops  The 5′ and 3′ ends base-pair and form the acceptor stem, with a conserved 3′ CCA tail. This binds the amino acid  The anticodon loop has three nucleotides that base pair with the codon in mRNA  Thus, the tRNA forms a physical link between the mRNA and the correct amino acid 11.2: tRNA and the Genetic Code Figure 11-03  tRNA has modified nucleotides – for example, the DHU (D) loop is named after dihydrouridine (D)  The TpsiC (T) loop has ribothymidine (T) and pseudouridine (ψ)  tRNA is often drawn as a clover-leaf structure (a), but a folded tRNA has an L-shape (b). The “bend” has some unusual interactions, like base-triple interactions  The bases in a tRNA anticodon are typically stacked on top of each other in a structure called a U-turn  A hypermodified purine occurs just after the anticodon, to prevent this from base-pairing with the codon in mRNA. This helps align the codon and anticodon properly 11.2: tRNA and the Genetic Code Figure 11-03  Each triplet codon specifies a single amino acid (a sense codon) or no amino acid (stop codon; nonsense codon). Stop codons signal the end of the protein coding region of the mRNA  A triplet code is the simplest that can specify all 20 amino acids (a doublet code could only specify 42 (i.e. 16)  Every possible codon is used, so some amino acids are encoded by more than one codon. In many cases, the first two nucleotides are the same and only the third differs (e.g. CU-x for leucine and GC-x for alanine)  A single tRNA doesn’t recognize all the codons for a specific amino acid. Different tRNAs that carry the same amino acids are called isoacceptors 11.2: tRNA and the Genetic Code Figure 11-05  The codon interacts with the anticodon  The first two positions on the mRNA (reading 5′ to 3′) are read by strict Watson-Crick base-pairing with positions 2 and 3 of the anticodon  In the third position of the codon, which interacts with position 1 of the anticodon, pairing deviations are allowed – this is called wobble pairing  Wobble pairing allows some non-Watson-Crick interactions, like G-U. Sometimes, inosine is present in the anticodon, and this can pair with U, C or A  Wobble pairing means that the same tRNA can interpret both UUU and UUC (a)  Wobble pairing means that each codon does not need its own tRNA – there are around 40 tRNAs for the 61 codons 11.2: tRNA and the Genetic Code Figure 11-06   Aminoacylation attaches amino acids to tRNAs. This is done by aminoacyl-tRNA transferases in a two-step process requiring ATP. The enzyme for a certain amino acid is denoted aaRS, e.g. GlyRS  First, the amino acid is activated by attachment of AMP. This releases pyrophosphate and provides energy  The activated aminoacyl- adenylate remains attached to the enzyme  The enzyme then transfers the amino acid to the 2′ or 3′ OH of the ribose of the terminal adenosine on the tRNA 3′ CCA tail 11.3: Aminoacyl-tRNA Synthetases Figure 11-07  Each amino acid has its own aminoacyl-tRNA synthetase  The specific amino acid with which a tRNA is loaded is indicated with a three letter superscript, such as tRNAMet  The correct amino acid for a tRNA is referred to as cognate  Loading is accurate – fewer than one error per 104 aminoacylation events  Aminoacyl-tRNA synthetases recognize tRNAs by sequence and structural features (called identity elements)  The correct amino acids are chosen in a two- step process. Most aminoacyl-tRNA synthetases have an aminoacylation site and an editing site 11.3: Aminoacyl-tRNA Synthetases Figure 11-08a  Ribosomes catalyze the formation of peptide bonds between amino acids  Ribosomes are large – 2.5mDa to 4mDa – ~2/3 of which is ribosomal RNA (rRNA) and 1/3 protein  Ribosomes have a large and a small subunit, each containing ribosomal proteins (r- proteins) and RNA  The small subunit mediates interactions between mRNA and tRNA  The large subunit catalyzes bond formation and has an exit tunnel through which the growing polypeptide emerges – this is often a target for antibiotics  The interface between the subunits is important for movement of tRNAs and mRNA 11.4: Structure of the Ribosome  Eukaryotic and bacterial ribosomes are generally conserved, but differ in their composition (also see Fig 11-14) 11.4: Structure of the Ribosome Figure 11-11  The rRNAs in the ribosomal subunits are divided into domains  The 16S (18S in eukaryotes) has 3 major and one minor domain (a). The 23S (or 28S) has six domains (b)  In the small subunit, the domains are discrete (c; left), but they are interwoven in the large subunit (c; right)  The large subunit in bacteria also has a second RNA – the 5S RNA. Eukaryotes have the 5.8S RNA  Ribosomal RNAs and proteins are extremely highly conserved across species 11.4: Structure of the Ribosome Figure 11-15  tRNAs bind successively at three sites within the ribosome – the aminoacyl (A) site, the peptidyl (P) site, and the exit (E) site  These sites are shown in the model above with tRNAs bound in the three positions 11.5: The Translation Cycle: The Ribosome in Action Figure 11-16  Translation involves four main steps:  Initiation  Elongation  Termination  Ribosome recycling 11.5: The Translation Cycle: The Ribosome in Action Figure 11-17 Initiation  The AUG at the start of an open reading frame is identified by initiation factors (IFs), the ribosome, and a special initiator methionine tRNA  Early initiation involves the small ribosomal subunit, and then the large subunit joins the complex  This results in a ribosome with a methionine-loaded tRNA bound in the P site  The ribosome is now ready to move along the mRNA 11.5: The Translation Cycle: The Ribosome in Action Figure 11-17  Elongation  Elongation factor Tu (in bacteria; eEF1A in eukaryotes) loads the next charged tRNA into the A-site, according to the codon in the mRNA  Peptide bond formation is catalyzed between the amino acid in the P site and the amino acid in the A site  This transfers the growing polypeptide to the tRNA in the A site  EFG (in bacteria; EF2 in eukaryotes) then promotes movement of the mRNA-tRNA through the ribosome (translocation)  This moves the peptidyl-tRNA that was in the A site into the P site and brings a new codon into the A site  The tRNA in the E site leaves the ribosome 11.5: The Translation Cycle: The Ribosome in Action Figure 11-17  Termination and ribosome recycling  Termination occurs when the ribosome reaches a stop codon (UAG, UAA or UGA)  Stop codons are recognized by class I release factors, not tRNAs  Bacterial RF1 recognizes UAA and UGA, RF2 - UAA and UGA  eRF1 (eukaryotes) recognizes all three stop codons  This interaction promotes release of the polypeptide from the ribosome  Class 2 release factors are also involved  The large and small subunits of the ribosome dissociate and release the remaining tRNA and mRNA  Recycling factor RRF and EFG help dissociation in bacteria 11.5: The Translation Cycle: The Ribosome in Action Figure 11-17  As elongation proceeds, the initiation codon (AUG) is freed  This allows further rounds of initiation to occur, so many ribosomes pile up on mRNA  These structures – polysomes – are easily visualized on mRNA by electron microscopy (shown) 11.5: The Translation Cycle: The Ribosome in Action Figure 11-18  Translation factors generally work in two ways  GTPase factors catalyze GTP hydrolysis, providing energy and undergoing conformational changes  The conformational changes are linked with progression of the ribosome though translation - EFTu depends on GTP hydrolysis in order to deliver aminoacyl-tRNAs  Other translation factors bind to the ribosome and stop inappropriate interactions – IF1 and IF3 in bacteria prevent initiator tRNA from binding to the A site and stop the large and small subunits associating too early 11.6: Protein Factors Critical to the Translation Cycle  There are three steps to translation initiation, achieved differently in eukaryotes and in bacteria - The small ribosomal subunit identifies the start codon in an mRNA - A methionyl-tRNA is loaded into the P site of the ribosome, and base- pairs with the start codon - The large ribosomal subunit joins the complex 11.7: Translation Initiation - Shared Features in Bacteria and Eukaryotes   The initiator codon is usually AUG, decoded by the initiator tRNA  This differs in eukaryotes (tRNAiMet; a) and bacteria (tRNAfMet; f denotes a formyl group; a)  Different GTPases are involved in binding of methionyl-tRNA to the P site in eukaryotes and bacteria  The bacterial initiator tRNA has a C-A wobble mismatch in the acceptor stem. The eukaryotic initiator has an A-U pair in the acceptor stem  Both have three G-C pairs in the anticodon stem; these are important for binding to EFTu 11.7: Translation Initiation - Shared Figure 11-22 Features in Bacteria and Eukaryotes  Bacterial mRNAs are often polycistronic – having several open reading frames  Each open reading frame has its own start and stop codon  Initiation codons usually have a Shine-Dalgarno sequence – this is a polypurine tract 6-8 bases upstream of the initiator AUG  Shine-Dalgarno pairs with a polypyrimidine region in the 3′ end of the bacterial 16S rRNA (the anti-Shine-Dalgarno sequence)  The pairing interaction guides the initiator AUG into the ribosomal P site 11.8: Bacterial Translation Initiation Figure 11-23  The Shine-Dalgarno sequence has the consensus AGGAGGU, and it pairs with a polypyrimidine region in the 3′ end of the bacterial 16S rRNA (the anti-Shine- Dalgarno sequence)  Sequence deviations from the consensus control strength of translation 11.8: Bacterial Translation Initiation Figure 11-24  Three initiation factors help guide f-Met-tRNAMet to the P site: IF1, IF2 and IF3  IF1 and IF3 bind in the A and E sites in the small ribosomal subunit in the absence of mRNA or f-Met- tRNAMet (a)  This directs the initiator tRNA to the P site, and also stops the large ribosomal subunit binding inappropriately (b)  IF2 is a GTPase and is involved in hydrolyzing GTP to provide energy for joining the large and small ribosomal subunits (b)  All three initiation factors are displaced when the subunits combine – initiation is complete (b) 11.8: Bacterial Translation Initiation Figure 11-25  Initiation is very different in eukaryotes in comparison with bacteria  There are no eukaryotic equivalents of the Shine-Dalgarno and anti-Shine-Dalgarno sequences – in any case, the small ribosomal subunit does not directly bind to mRNA (so more initiation factors are needed)  Eukaryotic mRNAs only usually encode one protein – they are monocistronic  Initiation usually occurs at the first AUG in the mRNA, but this is sometimes inefficient and more initiation occurs at the second or third AUG  Recognition of the AUG is sensitive to the sequence context – the Kozak sequence (consensus: (A/GXXAUGG) 11.9: Eukaryotic Translation Initiation Figure 11-26  Elongation consists of decoding, peptide bond formation and translocation  An aminoacyl-tRNA with an anti-codon that is complementary to the mRNA codon is chosen by the ribosome (this is decoding)  The codon/anti-codon interaction is cognate (fully accurate), near-cognate (single mismatch) and non-cognate (more than one mismatch) 11.10: Translation Elongation: Decoding, Peptide Bond Formation, and Translocation Figure 11-28  Cognate aminoacyl-tRNAs bind more strongly with the ribosome than non- cognate and near-cognate ones (thermodynamic contributions to fidelity; red arrows)  Cognate helix recognition stimulates conformational changes in the ribosome - this leads the ribosome to act as a GAP on EFTu, and promotes acceptance of the aminoacyl-tRNA in the A site (kinetic contributions to fidelity; green arrows) 11.10: Translation Elongation: Decoding, Peptide Bond Formation, and Translocation  Peptide bond formation involves transfer of the polypeptide chain to the aminoacyl-tRNA in the A site (a)  The peptidyl transferase active site has a highly conserved rRNA element  Binding of an aminoacyl-tRNA in the A site allows the peptidyl-tRNA to be positioned in the active site  The 2′ OH of the peptidyl-tRNA catalyzes the transfer (b) 11.10: Translation Elongation: Decoding, Figure 11-30 Peptide Bond Formation, and Translocation  In translocation, the mRNA bound to tRNA in the A and P sites moves through the ribosome  This opens the A site for a new aminoacyl-tRNA (a)  Structural rearrangements are involved in translocation  EFG can bind in the A site and seems to promote the structural rearrangements (b) 11.10: Translation Elongation: Decoding, Figure 11-31 Peptide Bond Formation, and Translocation  Translation continues until the ribosome encounters a stop codon in the mRNA (UAA, UAG or UGA)  Class 1 release factors, not tRNAs, recognize stop codons  Release factors (RFs) also promote hydrolytic release of the finished peptide  RFs and tRNAs are similar in structure (a)  RFs position in the A site (b) 11.11: Translation Termination and Reinitiation Figure 11-32  Stop codon recognition is highly accurate – premature peptide release happens very rarely (1/100000 events)  However, decoding errors are more likely during stressful conditions, when there may be a shortage of some of the aminoacyl-tRNAs  A mismatch in the codon/anti-codon helix at the P site triggers conformational changes that decrease the fidelity in the A site – this leads to lots of errors and probably early termination  The faulty peptide is detected, and then degraded, likely by the cellular peptidases and proteases. This acts as a quality control mechanism 11.11: Translation Termination and Reinitiation Figure 11-35  After translation is finished, the ribosome must be released so it can be reused  The recycling substrate is a complex of the ribosome, mRNA and tRNA  In bacteria, the ribosome recycling factor (RRF) acts with EFG and promotes disassembly  tRNA and mRNA fall off the small subunit, which is stabilized by IF3 11.11: Translation Termination and Reinitiation Figure 11-36a  Bacterial mRNAs are often polycistronic (have more than one open reading frame)  Sometimes, the small ribosomal subunit remains associated with mRNA and scans in both directions for another AUG  Most eukaryotic mRNAs are monocistronic – the few polycistrons are often very small, encoding a probably non-functional product  In this case, upstream, open reading frames (uORFs) often regulate gene expression, via reinitiation of translation from a not fully recycled ribosome – in the 5′ to 3′ direction only (unlike bacteria) 11.11: Translation Termination and Reinitiation  Rarely, the normal situation, where a codon defines a specific amino acid, does not apply  Recoding is where the readout is reprogrammed in an mRNA-specific fashion – i.e., a codon is interpreted differently in a specific mRNA, and in competition with the normal reading of the codon  Recoding can produce several proteins from a single gene  Nonsense suppression (a) is where stop codons are misread and termination fails to occur  Frameshifting is where the mRNA shifts so that peptide synthesis proceeds in a different reading frame (b) 11.13: Recoding: Programmed Stop Codon Read-Through and Frameshifting Figure 11-43  Ribosomes move along mRNA, reading codons (3 nucleotides) sequentially  If the ribosome moves by a different number of nucleotides (a number that is not a multiple of three), the reading frame is shifted  If the frame is shifted, different codons are read by the ribosome and a different peptide is produced  Frameshifting occurs most often +1 (blue) or -1 (pink) nucleotides from the original  Programmed frameshifting can be involved in gene regulation 11.13: Recoding: Programmed Stop Codon Read-Through and Frameshifting Figure 11-47  A programmed frameshifting example is production of bacterial termination factor RF2  RF2 has 2 open reading frames – a short one followed by a longer one in +1 frame The frameshift occurs at the sequence CUU UGA C  When there is lots of RF2 in the cell, it recognizes the stop codon (UGA)  When there is insufficient RF2, binding to UGA is slower and a +1 frameshift occurs because Leu-tRNALeu recognizes near-cognate UUU in the +1 frame as well as CUU 11.13: Recoding: Programmed Stop Codon Read-Through and Frameshifting Figure 11-48  Antibiotics are small molecules that kill or disrupt organism growth  The most effective antibiotics to use as therapeutic drugs are those that can target a bacterial or fungal process but not disrupt the same process in mammals  Small differences in translation between bacteria and eukaryotes mean that some good antibiotics are those that target the ribosome and other translation proteins  Some examples of antibiotics and their target processes are shown 11.14: Antibiotics that Target the Ribosome Figure 11-50  Antibiotics are typically small (about 1/1000 the size of the ribosome)  Thus, antibiotics tend to bind to critical regions of the ribosome, or to translation factors 11.14: Antibiotics that Target the Ribosome Figure 11-51   Erythromycin and similar drugs block the tunnel in the ribosome that the peptide exits from  This stops translation by preventing it proceeding 11.14: Antibiotics that Target the Ribosome Figure 11-52 RNA PROCESSING  RNAs are synthesized from DNA templates, but the molecules produced are often not functional. These are precursor RNAs (pre-RNAs), and need to be modified to make the mature, functional RNA  This is known as RNA processing – examples of processing are shown 10.1: Overview of RNA Processing Figure 10-01  Ribonucleases cleave the RNAs into smaller parts  Exonucleases successively remove nucleotides from the end of a transcript, most often in the 3′ to 5′ direction but sometimes 5′ to 3′  Exonucleases are not usually sequence specific  Endonucleases cleave the DNA within the strand. Some are specific for double- stranded RNA, some for single-stranded  RNase III and RNase P are examples of endonucleases 10.2: tRNA and rRNA Processing Figure 10-05  RNA processing has three main benefits - Contribution to regulation of gene activity - Diversity – many different RNAs can be produced from one gene via alternative splicing (by removal of different combinations of introns; figure) - Quality control - defective mRNAs are detected and degraded 10.1: Overview of RNA Processing Figure 10-02  Gene regulation through RNA splicing An example of alternative mRNA splicing that regulates gene expression is the control of whether a fruit fly develops as male or female  tRNA and rRNA transcripts are made as long precursors that must be processed  An E. coli precursor encodes three rRNAs and several tRNAs  The S. cerevisiae precursor encodes three rRNAs  Encoding several RNAs in one precursor ensures that similar amounts of each RNA are made 10.2: tRNA and rRNA Processing Figure 10-04  Both ends of eukaryotic RNAs are modified during transcription  The end modifications protect the mRNAs from nuclease degradation and help with protein interactions  The 5′ ends are capped with a 7-methylguanine nucleotide via a 5′ -5′ triphosphate linkage (this is the 5′ cap; pink). This guanine is then methylated at N7 (pink arrow)  The 5′ cap is needed for efficient elongation and termination of the transcript, for mRNA processing and export from the nucleus, and for directing translation  In more complex eukaryotes, the 2′ O of the second and sometimes third base are methylated (green arrows) 10.4: mRNA Capping and Polyadenylation Figure 10-10  The 3′ end of most eukaryotic mRNAs have about 200 adenosines added – this is a polyadenosine, or poly(A) tail  mRNAs have polyadenylation sites, where pre-mRNAs are cleaved and the poly(A) tail added  Multiple polyadenylation sites are found in some mRNAs, such as cyclin D1 mRNA above, and these can participate in regulation  Polyadenylation at the distal site (a) retains multiple regulatory sequences  Polyadenylation at the proximal site (b) eliminates the regulatory sequences 10.4: mRNA Capping and Polyadenylation Figure 10-12   Polyadenylation at the 3′ end of eukaryotic mRNAs starts with an initial cleavage  This cleavage usually occurs after a CA that lies between a conserved AAUAAA hexamer and a U or GU-rich region  After cleavage, ~200 adenosines are added by poly(A) polymerase  A larger protein complex is required for polyadenylation than for 5′ capping, probably because it is more complex to recognize the different polyadenylation sites in different mRNAs 10.4: mRNA Capping and Polyadenylation Figure 10-13  The final RNA is made up of sequences from discrete exons, separated by introns, from the precursor RNA  There are many different classes of intron, with different abundance and position depending on the organism. For all types, introns need to be removed from precursor RNA  Most introns do not contain genes and are excised and degraded (as above). Exceptions are snoRNAs and certain miRNAs  Some introns are removed by proteins, some by RNPs, and some excise themselves. All mechanisms use transesterification reactions  Introns are far more prevalent in eukaryotes than in bacteria. Introns allow for exon shuffling, where exons are exchanged and reordered via recombination, allowing evolution of different genes. In addition, differential removal of introns gives different transcripts from the same gene 10.5: RNA Splicing Figure 10-15  Eukaryote genes often have several introns, which can be very long (thousands of bases) and account for up to ~90% of a pre-mRNA  The human dystrophin gene (above) has 78 introns (gray) and exons are sometimes many thousands of bases apart  Some eukaryotes, like yeast, have fewer, shorter introns  Most eukaryotic introns are not self-splicing, so splicing is mediated by the spliceosome, which is made of several small nuclear ribonucleoproteins (snRNP)  Spliceosome catalyzed splicing is similar to that of Group II introns 10.6: Eukaryotic mRNA Splicing by the Spliceosome Figure 10-18  Additional modifications of mRNA (RNA editing) further enhances the range of molecules that can be produced  Specific nucleotides can be modified, inserted or deleted  Insertions or deletions can be one or two nucleotides, or can be more extensive  The Trypanosoma brucei NADH dehydrogenase 7 gene (left) undergoes extensive editing - Black nucleotides are encoded in mitochondrial DNA - Blue asterisks show where uridines have been deleted - Red shows uridines that have been inserted  For such modified genes, it is difficult to predict the protein sequence from the DNA sequence alone 10.8: RNA Editing Figure 10-27  There are two main common RNA edits: - Deamination of adenosine to inosine (the most common edit in more complex eukaryotes). Inosine is interpreted as guanosine, so changes in the coding region can change the final protein sequence - Deamination of cytidine to uridine, which has been found to date mainly, but not exclusively, in plant mitochondrial and chloroplast mRNAs  Nucleotide conversions like these are widespread throughout life  Uridine insertions and deletions have only been found so far in mitochondrial genes of single-celled eukaryotes like trypanosomes  Cytidine insertions have only been observed in slime molds 10.8: RNA Editing Figure 10-28  Cytidine to uridine deamination is also observed in the mRNA that makes human apoliprotein B  The deamination of a particular cytidine results in formation of a stop codon, resulting in a shorter version of the protein (APOB48) – this occurs in the small intestine, and is needed for lipid absorption from food  The long version of the protein (APOB100) is made in the liver, and is involved in cholesterol transport 10.8: RNA Editing Figure 10-29  RNAs need to be degraded at some point, removing RNAs that are no longer needed and recycling the nucleotides  “Normal” RNAs (those the cell produces) are degraded in a different way to foreign and defective RNAs  Some RNAs, like rRNAs, are needed a lot, and are fairly stable. Others, like some mRNAs, are only required for short periods of time and so are rapidly degraded  RNA stability is described as RNA half-life: the time in which the amount of RNA is reduced by half  RNA half-lives range from 24 hours in vertebrates 10.9: Degradation of Normal RNAs  RNA stability is affected by several factors  The structures at the 5′ and 3′ ends are important. The 5′ cap in eukaryotic mRNAs protect against exonuclease digestion. Bacterial RNAs with a 5′ - triphosphate are more stable than those with a monophosphate  Stem loop structures at the 5′ and 3′ ends also contribute to stability. 3′ end stem-loops in bacteria, including those formed in Rho-independent termination, protect against 3′ to 5′ exonuclease activity  In bacteria, a 3′ poly(A) tail decreases stability. This structure increases stability in eukaryotic mRNA, and elements that remove the tails contribute to decreased stability  Other RNA processes, like splicing, transport and translation, can impact half- lives by blocking or allowing access to degrading enzymes 10.9: Degradation of Normal RNAs  AU-rich elements (AREs) are important for mRNA stability  AREs are found in the 3′ UTRs of some mRNAs  ARE presence directs poly(A) removal and increases mRNA instability  c-fos in vertebrates encodes an ARE- containing short-lived transcription factor that promotes cellular growth  Some tumor-causing viruses express v- fos, which does not have the ARE  The v-fos transcript is not degraded properly, and this leads to excessive cellular growth 10.9: Degradation of Normal RNAs Figure 10-31  Some types of molecules can be harmful to the cell – such as foreign RNA or RNA from viruses or defective endogenous RNAs  Foreign nucleic acids are removed by RNA interference (RNAi) in eukaryotes and CRISPR interference in bacteria  In RNAi, foreign dsRNAs are chopped into 20-30 nt fragments  The short fragments are loaded onto Argonaute proteins that have nuclease domains  The RNA fragment guides the protein to the foreign RNA and the nuclease degrades it 10.10: Degradation of Foreign and Defective RNAs Figure 10-35a  RNA Interference and Gene Regulation The expression of a number of eukaryotic genes is controlled through RNA interference, also known as RNA silencing and posttranscriptional gene silencing. Recent research suggests that as much as 30% of human genes are regulated by RNA interference. RNA interference is triggered by small RNA molecules know as microRNAs (miRNAs) and small interfering RNAs (siRNAs), depending on their origin and mode of action DNA Repair Pathways  DNA damage poses a continuous threat to genomic integrity. Cells have evolved a range of DNA repair enzymes and repair polymerases as complex as the DNA replication apparatus itself. DNA replication, repair, and recombination share many common features. 7.2 Mutations and DNA damage Mutation: Source of the Genetic Variability Required for Evolution  Mutation --A change in the genetic material (molecular level)  Mutant  --an organism that exhibits a novel phenotype Spontaneous mutations Occur as a result of natural processes in cells. e.g. DNA replication errors Induced mutations Occur as a result of interaction of DNA with an outside agent that causes DNA damage.  Spontaneous replication errors arise from altered base structures and from wobble base pairing. Small insertions and deletions can occur through strand slippage in replication and through unequal crossing over.  The simplest type of mutation is a nucleotide substitution. Mutations that alter a single nucleotide are called point mutations.  Transitions and transversions can lead to silent, missense, or nonsense mutations Transition mutations replace one pyrimidine base with another, or one purine base with another. Transversion mutations replace a pyrimidine with a purine or vice versa. In humans, the ratio of transitions to transversions is approximately 2:1 Whether or not nucleotide substitutions have a phenotypic effect depends on: Do they alter a critical nucleotide in a gene regulatory region? Do they alter a critical nucleotide in the template for a functional RNA molecule? Are they silent, missense, or nonsense mutations in a protein-coding gene?  Silent mutations Mutations that change the nucleotide sequence without changing the amino acid sequence are called synonymous mutations or silent mutations.  Missense mutations Nucleotide substitutions in protein-coding regions that do result in changed amino acids are called nonsynonymous mutations or missense mutations. May alter the biological properties of the protein. Sickle cell anemia is an AT→TA transversion: – Glutamic acid codon in the -globin gene replaced by a valine codon  Nonsense mutations A nucleotide substitution that creates a new stop codon is called a nonsense mutation. Causes premature chain termination during protein synthesis. Nearly always a nonfunctional product. Types of Mutations – Changes in chromosome number and structure – Point mutation--changes at specific nucleotide in a gene (A,T,C,G) – Insertion mutations--insert fragment of DNA – Deletion mutations--delete fragment of DNA  Insertions or deletions can cause frameshift mutations If the length of an insertion or deletion is not an exact multiple of three nucleotides, this results in a shift in the reading frame of the resulting mRNA. Usually leads to production of a nonfunctional protein. Frameshift Mutations: alteration of the open reading frame (ORF) © John Wiley & Sons, Inc.  Induced Mutations Induced mutations occur upon exposure to physical (energy) or chemical (reaction) mutagens.  The Electromagnetic Spectrum X-rays induce mutations through ionization. (DNA ionization--radical anions and cations-- G.+) Ultraviolet light induces mutations through excitation. © John Wiley & Sons, Inc.  Ionizing Radiation Causes Changes in Chromosome Structure Ionizing radiation breaks chromosomes and can cause deletions, duplications, inversions, and translocations Mutagenesis by Ultraviolet Irradiation Hydrolysis of cytosine to a hydrate may cause mis-pairing during replication Cross-linking of adjacent thymine forms thymidine dimers, which block DNA replication and activate DNA repair mechanisms.. UV-A 320 to 400 nm UV-B/C