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MOLECULAR BIOLOGY LECTURE - BSMT 3A INTRODUCTION TO MOLECULAR BIOLOGY WHAT IS MOLECULAR BIOLOGY? Following the work of Gregor Mendel on patterns of transmission of inheritab...

MOLECULAR BIOLOGY LECTURE - BSMT 3A INTRODUCTION TO MOLECULAR BIOLOGY WHAT IS MOLECULAR BIOLOGY? Following the work of Gregor Mendel on patterns of transmission of inheritable traits of It is the study of gene structure and function at garden peas and Friedrich Miescher’s molecular level. discovery of “nuclein” (now nucleic acids) in When James Watson coined the term molecular 1869. biology, he was referring to the biology of The 1940s and 1950s saw the work of many deoxyribonucleic acid (DNA). groups in the identification of DNA as the The term, however, is still used to describe the genetic material until the elucidation of its study of nucleic acids. biochemical structure in 1953 by James D. Molecular biology is the study of the Watson and Francis H. C. Crick. biomolecules and biomolecular mechanisms that occur in living organisms. “TRANSFORMING PRINCIPLE” Molecular biology’s focus from the beginning was the structure and function of the gene: the In 1944, Oswald Avery, Colin MacLeod, and molecular nature of the gene, gene replication, Maclyn McCarty reported that it is mutation, repair, and gene expression. deoxyribonucleic acid, rather than protein, that served as the carrier of the hereditary material. IMPORTANCE OF MOLECULAR BIOLOGY DNA was described earlier in 1928 by British bacteriologist, Frederick Griffith only as the The development of molecular biology led to “transforming principle” responsible for the different technologies that have been transformation phenomenon: killed established, including in the fields of agriculture, Streptococcus pneumoniae of the virulent strain medicine, forensic sciences, etc. type III-S, when injected in mice along with Molecular biology also plays an important role living but non-virulent type II-R pneumococci, in diagnostics, in developing tests that help in which resulted in a deadly infection of type III-S diagnosing certain diseases, specifically cancer pneumococci. which involves the concept of genetics, and Avery and companions made use of different other certain conditions. enzymes - proteases that digest proteins, It also developed other therapeutic treatments ribonucleases that break apart RNA, and and other methods to contain or control the deoxyribonucleases that act on DNA - to treat spread of a virus. the “transforming principle” in Griffith’s experiment. HISTORY The transforming power was lost only with the ORIGIN OF MOLECULAR BIOLOGY deoxyribonuclease treatment, hence, attributing the transformation trait to DNA and not to Molecular biology finds its origin in the 1930s proteins nor RNA. and 1940s. As a science, it is a result of the inevitable convergence of distinct disciplines DNA AS THE GENETIC MATERIAL from both the physical and biological sciences: biochemistry, genetics, microbiology, physics, In 1952, Alfred Hershey and Martha Chase structural, and colloid chemistry, and later on confirmed that DNA is the genetic material in virology. a series of experiments. They used It picked up momentum in the 1950s and 1960s bacteriophages or viruses that infect and when it was institutionalized (Tabery, replicate in bacteria. Piotrowska, & Darden, 2019). Phages are structurally known to consist of a The term “molecular biology” was first used in protein coat and DNA as its genetic material 1938 by Warren Weaver, head of the Natural found in its interior. Science Division of the Rockefeller Foundation To detect the different parts of the phage upon in 1931. infection, radioactive elemental isotopes, Phosphorus-32 (32P) and Sulfur-35 (35S) HAYDEE RIZMA A. ABDULKAHAL 1  TRANS: PRE-MIDTERM were used to label the DNA and proteins, Watson and Crick also made the important respectively. observation that because of the specific pairing When the phages were allowed to infect of the bases, when the sequence of bases in bacteria, phage DNA was inserted into the host one chain is given, then the sequence on the bacteria, separating it from the “ghost” phage other chain can be automatically determined. (without genetic material). This strongly suggested the copying Following centrifugation and purification, 32P mechanism of the genetic material which is now was detected with bacterial cells, and radioactive the molecular basis of the semi-conservative 35S remained in the solution outside the cells DNA replication. where the protein-coated ghosts are. Further The elucidation of the DNA structure has results showed that the DNA that was internalized revolutionized molecular biology and gave way in the bacteria also conferred its ability to produce to cutting-edge biotechnologies, such as phage progeny inside the host. sequencing, recombinant DNA technology, Hershey and Chase concluded that protein was cloning, development of transgenic crops and not likely to be the genetic material, although they animals, gene amplification, gene silence, and did not specify the function of DNA. editing. Confirmation of the DNA function was later The X-ray crystallography work that produced made clear when Watson and Crick showed the the famous “photo 51” - an X-ray diffraction double-helical structure of the DNA in their 1953 article in Nature (Watson & Crick, 1953), which image by Rosalind Franklin and Raymon suggested a mechanism for storing and Gosling - was crucial to the publication of the expressing genetic information. DNA structure by Watson and Crick. Franklin’s colleague, Maurice Wilkins, showed WATSON AND CRICK MODEL OF THE DNA this photo to Watson and Crick, and it became strong evidence of the DNA double-helical In their 1953 paper, Watson and Crick structure. described the DNA as consisting of two In 1950, Austrian biochemist Erwin Chargaff, helical chains coiled around the same axis, also gave experimental evidence on the with the sugar and phosphates outside, forming nucleotide composition of the DNA of different the backbone of each chain and the bases on species that supported the complementarity of the inside linked together by hydrogen bonds. the DNA strands in the Watson and Crick model Both chains follow right-handed helices, and (Chargaff, 1950). His work reached two major each consists of phosphate diester groups conclusions: joining β-D-deoxyribofuranose residues with o First, he showed that different species have 3’-5’ linkages. varying nucleotide composition in their DNA. Furthermore, the residue on each chain is There seemed to be no pattern observed in found every 34 Å and at an angle of 36 degrees the sequence or order of nucleotides. between adjacent residues. The helical o Second, Chargaff deduced that the DNA structure turns or repeats after 34 Å. from different species or from varying tissue Watson and Crick emphasized that the novel sources maintain certain properties even if feature of the structure is the manner in which the composition varies. In particular, the two chains are being held together by purine total amount of purine (A + G) and the total and pyrimidine bases. amount of pyrimidine (C + T) are nearly A single purine in one chain is equal. hydrogen-bonded to a single pyrimidine in The second conclusion established what is the other so that the two are side by side known as the “Chargaff’s rule.” This perpendicular to the axis of the helix. information, plus the image in “photo 51” It was assumed that the bases only occur in the contributed to the derivation of the most plausible keto tautomeric form so that only double-helical model of the DNA by Watson and specific bases can bond together: adenine with Crick. thymine, and guanine with cytosine. They added the sequence by which these bases SEMI-CONSERVATIVE DNA REPLICATION appear along the chains were not restricted in any way. In 1958, Matthew Meselson and Franklin Stahl, American geneticists, provided evidence HAYDEE RIZMA A. ABDULKAHAL 2  TRANS: PRE-MIDTERM on the semiconservative nature of DNA 1961 Francois Jacob Bacterial operons replication. Jacques Monod and messenger According to this model, the old DNA strands RNA. separate and each one becomes a template for the synthesis of a new DNA strand. 1963, 1966 Marshall Nirenberg Deciphered the Two double-helices result in one round of H. Gobind Khorana genetic code. replication and each double helix is a hybrid of Philip Leder one old strand bound to a newly synthesized Heinrich Matthaei strand. 1965 Robert Holley Transfer RNA NOTABLE CONTRIBUTIONS TO THE FIELD structure OF MOLECULAR BIOLOGY 1969, 1970 Werner Arber Restriction of Year of Name Scientific Matthew Meselson endonucleases Publicatio Investigation and Daniel Nathans technology n of Contribution Hamilton Smith Notable Work 1973 Paul Berg Recombinant DNA Stanley Cohen technology; 1926 Thomas Hunt Morgan Used fruit flies, Herbert Boyer manipulation of Drosophila genes. melanogaster, as a model organism 1975 Frederick Sanger DNA sequencing to study the relationship 1976 David Baltimore Reverse between the gene Renato Dulbecco transcriptase and and chromosomes Howard Temin tumor virus activity in the hereditary process. 1977 Carl Woese Phylogenetic taxonomy of 16s 1926 Hermann J. Muller Recognized the rRNA, resulting in gene as the “basis the “tree of life” of life,” and discovered the 1977, Walter Gilbert 1977 - Walter mutagenic effect of 1978, 1986 Allan Maxam Gilbert and Allan x-rays on Maxam developed Drosophila, and a DNA sequencing utilized this technology. phenomenon as a tool to explore the 1978 - Walter size and nature of Gilbert proposed the gene. the existence of introns and exons. 1953 Barbara McClintock Mobile genetic elements 1986 - Walter (transposons). Gilbert proposed the RNA world 1956, 1961 Arthur Kornberg Discovery of DNA hypothesis as the Severo Ochoa polymerase I and origin of life. mechanism of DNA synthesis. HAYDEE RIZMA A. ABDULKAHAL 3  TRANS: PRE-MIDTERM 1969, 1970 Werner Arber Restriction of HISTORY (FROM PPT) Matthew Meselson endonucleases Daniel Nathans technology 1934 Symposium on Calvin Bridges Hamilton Smith Aspects of - part of T.H. Growth Morgan’s famous 1981 Aaron Klug Nucleic “fly group” that acid-proteins pioneered the complexes development of the fruit fly Drosophila 1986 Thomas Cech RNA as enzymes as a model genetic Sidney Altmad (ribozymes) organism 1989 James Watson Watson John T. Buchholtz spearheaded the - a plant geneticist, Human Genome 1941, became Project - a 13 year President of the international effort Botanical Society of to discover the America. 20,000 to 25,000 human genes and 1947 Symposium on Edwin Chargaff determine the Nucleic Acids - an eminent sequence of the 3 and biochemist, became billion DNA Nucleoproteins a bitter critic of subunits contained molecular biology, in human an occupation he chromosomes. described as “essentially the 1989 Mario Capecchi Knockout mice practice of Martin Evans techniques biochemistry without Oliver Smithies a license”. 1993 Kary Mullis Polymerase chain 1953 Symposium on Raymond Appleyard & reaction (PCR) Viruses George Bowen - Both phage 1996 Keith Campbell Cloning of “Dolly” geneticists. Ian Wilmut the sheep by nuclear transfer Alfred Hershey and Martha from cultured cell Chase line. - Did the simple 1998 Craig Mello RNA interference experiment that Andrew Fire (RNAi) finally convinced most people that the 2000 Thomas Steitz Crystal structure of genetic material is the ribosomes DNA. 2011 Emmanuelle Charpentier CRISPR/Cas9 1963 Symposium on Vernon Ingram Jennifer Anne Doudna gene editing Synthesis and - Demonstrated that mechanism Structure of genes control the Macromolecule amino acid s sequence of proteins; the mutation causing HAYDEE RIZMA A. ABDULKAHAL 4  TRANS: PRE-MIDTERM sickle-cell anemia amino acids in the produces a single protein product. amino acid change in the hemoglobin 1971 Symposium on Max Perutz & John protein. Structure and Kendrew Marshall W. Nirenberg Function of - Received the 1962 - Was key in Proteins at the Nobel Prize for unraveling the Three Chemistry; using genetic code, using Dimensional X-ray protein synthesis Lev crystallography, and directed by artificial after 25 years of RNA templates in effort, they were the vitro. first to solve the Matthias Staehelin atomic structures of - Worked on the small proteins—hemoglob RNA molecules, in and myoglobin, tRNAs, which respectively. translate the genetic code into amino 1975 Symposium on Sydney Brenner acid sequences of The Synapse - Contributed to the proteins. discoveries of Melvin Calvin mRNA and the - Won the 1961 Nobel nature of the genetic Prize in Chemistry code; his share of a for his work on CO2 Nobel Prize, in assimilation by 2002, however, was plants. for establishing the Francis Crick & James worm model system Watson for the study of - For their proposed developmental structure of DNA, biology. they shared in the Seymour Benzer 1962 Nobel Prize in - Using phage Physiology and genetics, defined Medicine. the smallest unit of George Gamow mutation, which - A physicist attracted turned out later to to the problem of be a single genetic code, nucleotide. *This founded an informal same work also group of like-minded provided an scientists called the experimental RNA Tie Club. definition of the gene—which he 1966 Symposium on Charles Yanofsky called a The Genetic - Proved collinearity cistron—using Code of the gene—that is, functional that successive complementation groups of tests. Later, his nucleotides studies focused on encoded successive HAYDEE RIZMA A. ABDULKAHAL 5  TRANS: PRE-MIDTERM behavior, using the Mendel’s model: It started with a 3:1 ratio. fruit fly as a model. MENDEL’S LAW OF INHERITANCE Gregor Mendel experimented with pea plants, with a 3:1 ratio. In 1865, Mendel published his findings on the inheritance of seven different traits in the garden pea. He concluded that inheritance is particulate, in which each parent contributes particles or genetic units (genes) to the offspring. Made an important generalization of phenotyping. Genes o Can exist in different forms called alleles. o One allele can be dominant over the other, which makes it recessive. Genotype o Genetic composition or allele combination. Phenotype o Greek root as phenomenon, meaning Mendel’s Law of Inheritance appearance. o Physical structure of an individual. o Observable traits. Allele - version of DNA sequence Homozygous o Organism has two copies of the same allele o Refers to a gene pair in which both the maternal and paternal genes are identical (e.g., RR or rr). o Can produce sex cells or gametes that have only one allele. o Homo = same Heterozygous o Mendel demonstrated that the allele for the o Organism has two different copies. yellow seed was dominant when he mated a o Those gene pairs in which paternal and green-seeded pea with a yellow-seeded maternal genes are different (e.g., Rr). pea. o Having two different alleles of one gene will o All of the progeny (children or descendant) exhibit the characteristic dictated by the in the first filial generation (F1) had yellow dominant allele. seeds, when these yellow peas were o Recessive allele is not lost, it can still exert allowed to self-fertilize, some green-seeded its influence when paired with another peas reappeared. recessive allele in a homozygote. ▪ Ratio of yellow to green seeds in the o Result F1 peas = heterozygous. o Hetero = different 2nd filial generation (F2) was very close Sex cells - contains only one copy of the gene to 3:1. (haploid). o 1st filial generation – contains the offspring of original parents o 2nd filial generation – is the offspring of the 1st filial individuals (F1) Mendel’s Discoveries HAYDEE RIZMA A. ABDULKAHAL 6  TRANS: PRE-MIDTERM o The Principle of Independent Segregation o Mendel also found that genes for the seven o Principle of Independent Assortment different characteristics he chose to study operate independently from one another. THE PRINCIPLE OF INDEPENDENT o Combinations of alleles of 2 different genes SEGREGATION gave ratios of 9:3:3:1 for yellow/round, Mendel's law of segregation states that only yellow/wrinkled, green/round, one of the two gene copies present in an green/wrinkled. organism is distributed to each gamete (egg or sperm cell) that it makes, and the allocation of Chromosomal Theory of Heredity the gene copies is random. o States that chromosomes carry genes. Law of Segregation: o In 1900, three botanists arrived at similar o Punnett square - can be used to predict conclusions independently and genotypes (allele combinations) and rediscovered Mendel’s work. phenotypes (observable traits) of offspring o Mendel predicted that gametes would from genetic crosses. contain only one allele of each gene instead o Test cross - can be used to determine of two. whether an organism with a dominant o If chromosomes carry the genes, their phenotype is homozygous or heterozygous. numbers should also be reduced by half in the gametes. o Chromosomes – appeared to be the discrete physical entities that carry the genes. o Autosomes – chromosomes occur in pairs in a given individual. o Sex chromosome – X chromosome is an example, where in the female fly has 2 copies and the has one (XX – female, XY – male). LAW OF INDEPENDENT ASSORTMENT Mendel's law of independent assortment states that the alleles of two (or more) different genes Crossing Over get sorted into gametes independently of one another. Dihybrid Cross: HAYDEE RIZMA A. ABDULKAHAL 7  TRANS: PRE-MIDTERM The Chromosome Theory of Inheritance 1902 Archibald Garrod first suggested a o Sex-linked genes in the fruit fly reveal that genetic cause for a human disease. genes are on chromosomes. o Thomas Hunt Morgan - established the 1902 Walter Sutton, Theodor Boveri studies of fruit flies. proposed the chromosome theory. o Worked with fruit flies (Drosophila 1910, 1916 Thomas Hunt Morgan, Calvin melanogaster). Bridges demonstrated that genes o A more convenient organism than the are on chromosomes. garden pea because of its small size, short generation time, and large number of 1913 A.H. Sturtevant constructed a offspring. genetic map. o Red-eyed flies – dominant o White-eyed flies – recessive 1927 H.J. Muller induced mutation by x-rays. o When mated red -eyed males of the F1 generation with their red- eyed sisters – 1931 Harriet Creighton, Barbara produced one quarter white – eyed males, McClintock obtained physical no females (means that eye color evidence for recombination. phenotype is sex – linked). o Sex and eye color – transmitted together 1941 George Beadle, E.L. Tatum as these characteristics are located on the proposed the one-gene/one-enzyme same chromosome (X chromosome) hypothesis 1944 Oswald Avery, Colin McLeod, Maclyn McCarty identified DNA as the material genes are made of. 1953 James Watson, Francis Crick, Rosalind Franklin & Maurice Wilkins determined the structure of DNA. 1958 Matthew Meselson, Franklin Stahl demonstrated the semiconservative replication of DNA. 1961 Sydney Brenner, François Jacob discovered messenger RNA. 1966 Marshall Nirenberg, Gobind MOLECULAR BIOLOGY TIMELINE Khorana finished unraveling the genetic code. Year Contribution 1970 Hamilton Smith discovered restriction enzymes that cut DNA at 1859 Charles Darwin published on the specific sites, which made cutting and Origin of Species. pasting DNA easy, thus facilitating DNA cloning. 1865 Gregor Mendel advanced the principles of segregation and 1972 Paul Berg made the first independent assortment. recombinant DNA in vitro. 1869 Friedrich Miescher discovered DNA. 1973 Herb Boyer, Stanley Cohen first used a plasmid to clone DNA. 1900 Hugo de Vries, Carl Correns, Erich von Tschermak rediscovered 1977 Frederick Sanger worked out Mendel’s principles. methods to determine the sequence of bases in DNA and determined the HAYDEE RIZMA A. ABDULKAHAL 8  TRANS: PRE-MIDTERM base sequence of an entire viral genome. 1977 Phillip Sharp, Richard Roberts discovered interruptions (introns) in genes and others. 1993 Victor Ambros and colleagues discovered that a cellular microRNA can decrease gene expression by base-pairing to an mRNA. 1995 Craig Venter, Hamilton Smith determined the base sequences of the genomes of two bacteria: - Haemophilus influenzae and Mycoplasma genitalium, the first genomes of free-living organisms to be sequenced. 1996 Many investigators determined the base sequence of the genome of brewer’s yeast, Saccharomyces cerevisiae, the first eukaryotic genome to be sequenced. 1997 Ian Wilmut and colleagues cloned a sheep (Dolly) from an adult sheep udder cell. 1998 Andrew Fire and colleagues discovered that RNAi works by degrading mRNAs containing the same sequence as an invading double-stranded RNA. 2003 Many investigators reported a finished sequence of the human genome. 2005 Many investigators reported the rough draft of the genome of the chimpanzee, our closest relative. 2007 Craig Venter and colleagues used traditional sequencing to obtain the first sequence of an individual human (Craig Venter). 2008 Jian Wang and colleagues used “next generation” sequencing to obtain the first sequence of an Asian (Han Chinese) human. 2008 David Bentley and colleagues used single molecule sequencing to obtain the first sequence of an African (Nigerian) human. HAYDEE RIZMA A. ABDULKAHAL 9 MOLECULAR BIOLOGY LECTURE - BSMT 3A NUCLEIC ACID AND PROTEINS INTRODUCTION on the order or sequence of nucleotides in the nucleic acid polymer. When James Watson coined the term molecular biology, he was referring to the biology of DNA STRUCTURE deoxyribonucleic acid (DNA). The term, however, is still used to describe the The double helical structure of DNA was first study of nucleic acids. described by James Watson and Francis Crick. Nucleic acids offer several characteristics that Their molecular model was founded on support their use for clinical purposes. previous observations of the chemical nature of Highly specific analyses can be carried out DNA and physical evidence including diffraction without the requirement for extensive physical analyses performed by Rosalind Franklin. or chemical selection of target molecules or The helical structure of DNA results from the organisms, allowing specific and rapid analysis physicochemical demands of the linear array of from limited specimens. nucleotides. Both the specific sequence (order) Information carried in the order or sequence of of nucleotides in the strand, as well as the the nucleotides that make up the nucleic acids surrounding chemical microenvironment, can is the basis for normal and pathological traits affect the nature of the DNA helix. from microorganisms to humans and provides a powerful means of predictive analysis. DNA Deoxyribonucleic acid (DNA) is a macromolecule of carbon, nitrogen, oxygen, phosphorous, and hydrogen atoms. It is assembled in units of nucleotides that are composed of a phosphorylated ribose sugar and a nitrogen base. There are four nitrogen bases that make up the majority of DNA found in all organisms, namely adenine, cytosine, guanine, and thymine. Nitrogen bases are attached to a deoxyribose sugar, forming a polymer with the deoxyribose NUCLEOTIDES sugars of other nucleotides through a phosphodiester bond. Each nucleotide consists of a five-carbon sugar, Linear assembly of the nucleotides makes up the first carbon of which is covalently joined to a one strand of DNA. Two strands of DNA nitrogen base and the fifth carbon to a comprise the DNA double helix. phosphate moiety. In 1871, Johann Friedrich Miescher published A nitrogen base bound to an unphosphorylated a paper on nuclein, the viscous substance sugar is a nucleoside. Adenosine (A), extracted from cell nuclei. In his writings, he guanosine (G), cytidine (C), and thymidine (T) made no mention of the function of nuclein. are nucleosides. Walther Flemming, a leading cell biologist, If the ribose sugar is phosphorylated, the describing his work on the nucleus in 1882 molecule is a nucleoside mono-, di-, or admitted that the biological significance of the triphosphate or a nucleotide. substance was unknown. o For example, adenosine with one The purpose of DNA, contained in the nucleus phosphate is adenosine monophosphate of the cell, is to store information. The (AMP). Adenosine with three phosphates is information in the DNA storage system is based adenosine triphosphate (ATP). HAYDEE RIZMA A. ABDULKAHAL 1  TRANS: PRE-MIDTERM Free nucleotides are deoxyribonucleoside acid–based tests used in the molecular triphosphates (e.g., dATP). They are routinely laboratory. designated as A, C, G, and T in the DNA As DNA is polymerized, each nucleotide to be molecule. Nucleotides can be converted to added to the new DNA strand hydrogen bonds nucleosides by hydrolysis. with the complementary nucleotide on the The five-carbon sugar of DNA is deoxyribose, parental strand (A:T, G:C). In this way the which is ribose with the number-two carbon of parental DNA strand can be replicated without deoxyribose linked to a hydrogen atom rather loss of the nucleotide order. than a hydroxyl group. NUCLEIC ACID Nucleic acid is a macromolecule made of nucleotides bound together by the phosphate and hydroxyl groups on their sugars. A nucleic acid chain grows by the attachment of the 5 ′ phosphate group of an incoming nucleotide to the 3 ′ hydroxyl group of the last nucleotide on the growing chain. Addition of nucleotides in this way gives the The hydroxyl group on the third carbon is DNA chain a polarity; that is, it has a 5 ′ important for forming the phosphodiester bond phosphate end and a 3 ′ hydroxyl end. that is the backbone of the DNA strand. DNA is double-stranded where two strands Nitrogen bases with a single-ring structure exist in opposite 5’ to 3’ or 3’ to 5’ orientations. (thymine, cytosine) are pyrimidines. Bases They are being held together by hydrogen with a double-ring structure (guanine, adenine) bonds between their respective bases (A w/ T, are purines. G w/ C). The numbering of the positions in the The bases are positioned such that the nucleotide molecule starts with the ring sugar-phosphate chain that connects them is positions of the nitrogen base, designated C or oriented in a spiral or helix around the nitrogen N 1, 2, 3, and so on. bases. The carbons of the ribose sugar are numbered The DNA double helix represents two versions 1 ′ to 5 ′ , distinguishing the positions of the of the information stored in the form of the order sugar rings from those of the nitrogen base or sequence of the nucleotides on each chain. rings. The sequences of the two strands that form the double helix are complementary, not identical. They are in antiparallel orientation, with the 5′ end of one strand at the 3′ end of the other. The formation of hydrogen bonds between two complementary strands of DNA is called The carbons of the ribose sugar are numbered hybridization. Single strands of DNA with 1 ′ to 5 ′ , distinguishing the positions of the identical sequences will not hybridize with each sugar rings from those of the nitrogen base other. rings. Two DNA chains form hydrogen bonds with DNA REPLICATION each other in a specific way. Guanines in one chain form three hydrogen bonds with cytosines The two DNA strands of a double helix have an in the opposite chain, and adenines form two antiparallel orientation because of the way DNA hydrogen bonds with thymines. is replicated. Hydrogen bonds between nucleotides are the As DNA synthesis proceeds in the 5 ′ to 3 ′ key to the specificity of most nucleic direction, DNA polymerase, the enzyme responsible for polymerizing the nucleotide HAYDEE RIZMA A. ABDULKAHAL 2  TRANS: PRE-MIDTERM chains, uses a guide, or template, to determine o Primase is an RNA synthesizing enzyme which nucleotides to add to the chain. that catalyzes the synthesis of short RNA DNA polymerase reads the template in the 3′ to primers required for priming DNA synthesis. 5′ direction. o Primase must work repeatedly on the The resulting double strand will have a parent lagging strand to prime the synthesis of strand in one orientation and a newly each Okazaki fragment. synthesized strand arranged in the opposite Once both the continuous and discontinuous orientation. strands are formed, one of the catalytic Before the double helix was determined, Erwin domains of Escherichia coli DNA polymerase I Chargaff made the observation that the amount has a small fragment with exonuclease of adenine in DNA corresponded to the amount activity. of thymine and the amount of cytosine to the The enzyme exonuclease protects the amount of guanine. sequence of nucleotides and removes In the process of replication, DNA is first mismatch since it has the capacity to proofread unwound from the helical duplex so that each newly synthesized DNA. single strand may serve as a template for the addition of nucleotides to the new strand POLYMERASES enzyme helicase is involved. Once the strands have been separated, a short Nucleic acid is a macromolecule made of piece of RNA (primer) binds to the 3’ end of the nucleotides bound together by the phosphate strand, this process involves the enzyme and hydroxyl groups on their sugars. primase. There are different types of DNA The new strand is elongated by hydrogen polymerases: bonding of the complementary incoming o DNA polymerase I was shown to catalyze nucleotide to the nitrogen base on the template DNA replication in prokaryotes. strand (elongation). o DNA polymerase I and II are also This process involves DNA polymerase III responsible for the repair of gaps. which is a polymerizing enzyme that catalyzes o DNA polymerase III is the main the formation of the phosphodiester bond. This polymerizing enzyme during bacterial enzyme can only continue synthesis and replication. lengthen the DNA strand in the presence of an Most DNA polymerases have more than one RNA primer. function, including, in addition to polymerization, A question arises as to how one of the strands pyrophosphorolysis and pyrophosphate of the duplex can be copied in the same exchange, the latter two activities being a direction as its complementary strand that runs reversal of the polymerization process. antiparallel to it. DNA polymerase enzymes thus have the Okazaki and Okazaki addressed this problem in capacity to synthesize DNA in a 5 ′ to 3 ′ 1968 when studying DNA replication in direction and degrade DNA in both a 5 ′ to 3 ′ Escherichia coli. The fragments changed into and 3 ′ to 5 ′ direction. larger pieces with time, showing that they were The catalytic domain of E. coli DNA pol I has covalently linked together shortly after two fragments carrying the two functions, a synthesis. These fragments are called Okazaki large fragment with the polymerase activity and fragments and were the key to explaining how a small fragment with the exonuclease activity. both strands were copied. The large fragment without the exonuclease While DNA replication proceeds in a continuous activity (Klenow fragment) has been used manner on the 3′ to 5′ strand, or the leading extensively in the laboratory for in vitro DNA strand, the replication apparatus jumps ahead synthesis. a short distance on the 5′ to 3′ strand and then One purpose of the exonuclease function in the copies backward toward the replication fork. various DNA polymerases is to protect the The 5 ′ to 3 ′ strand copied in a discontinuous sequence of nucleotides, which must be manner is the lagging strand. faithfully copied. Copying errors will result in The lagging strands begin to bind with an base changes or mutations in the DNA. enzyme known as primase. HAYDEE RIZMA A. ABDULKAHAL 3  TRANS: PRE-MIDTERM The 3 ′ to 5 ′ exonuclease function is required to DNA LIGASE ensure that replication begins or continues with a correctly base-paired nucleotide. DNA ligase catalyzes the formation of a During DNA synthesis, this exonuclease phosphodiester bond between adjacent 3′ function gives the enzyme the capacity to -hydroxyl and 5 ′ -phosphoryl nucleotide ends. proofread newly synthesized DNA, that is, to Its existence was predicted by the observation remove a misincorporated nucleotide by of replication, recombination, and repair breaking the phosphodiester bond and replace activities in vivo. it with the correct one. DNA ligase was discovered in five different The breaks or gaps present in the daughter laboratories. strand are called nicks. To complete the strand The isolated enzyme was found to catalyze the formation, the Okazaki fragments and nicks are end-to-end joining of separated strands of DNA. replaced or closed again by DNA ligase. One type of DNA polymerase, terminal OTHER DNA METABOLIZING ENZYMES transferase, can synthesize polynucleotide NUCLEASES chains without a template. This enzyme will add nucleotides to the end of a DNA strand in the Two types: absence of hydrogen base pairing with a o Endonucleases template. o Exonucleases In contrast to endonucleases, exonucleases ENZYMES THAT METABOLIZE DNA degrade DNA from free 3 ′ -hydroxyl or 5 ′ -phosphate ends. Consequently, they will not Once DNA is polymerized, it is not static. The work on closed circular DNA. information stored in the DNA must be tapped These enzymes are used, under controlled selectively to make RNA and, at the same time, conditions, to manipulate DNA in vitro, 28 for protected from damage. instance, to make step-wise deletions in Enzymes that modify and digest foreign DNA linearized DNA or to modify DNA ends after prevent infection. cutting with restriction enzymes. They are key tools of recombinant DNA Exonucleases have different substrate technology, the basis for commonly used requirements and will therefore degrade molecular techniques. specific types of DNA ends. RESTRICTION ENZYMES HELICASES Genetic engineering was stimulated by the The release of DNA for transcription, discovery of deoxyriboendonucleases, or replication, and recombination without tangling endonucleases which break the is brought about through cutting and re-closing sugar-phosphate backbone of DNA. of the DNA sugar-phosphate backbone. Restriction enzymes are endonucleases that These functions are carried out by a series of recognize specific base sequences and break enzymes called helicases. or restrict the DNA polymer at the Helicases are of two types: sugar-phosphate backbone. o Topoisomerases Restriction enzymes are named for the o Gyrases organism from which they were isolated. Topoisomerases interconvert topological isomers or relax supertwisted DNA. Gyrases (type II topoisomerases) untangle DNA through double-strand breaks. They also separate linked rings of DNA (concatemers). METHYLTRANSFERASES DNA methyltransferases catalyze the addition of methyl groups to nitrogen bases, usually adenines and cytosines in DNA strands. HAYDEE RIZMA A. ABDULKAHAL 4  TRANS: PRE-MIDTERM Unlike prokaryotic DNA, eukaryotic DNA is or recombination of the genes of both methylated in specific regions. In eukaryotes, parents. DNA-binding proteins may limit accessibility or The nature of this recombination is manifested guide methyltransferases to specific regions of in the combinations of inherited traits of the DNA. subsequent generations. Recombinant DNA technology is a controlled RECOMBINATION IN SEXUALLY mixing of genes. REPRODUCING ORGANISMS RECOMBINATION IN ASEXUAL Recombination is the mixture and assembly of PRODUCTION new genetic combinations. It occurs through the molecular process of crossing over or physical Movement and manipulation of genes in the exchange between molecules. laboratory began with the study of natural A recombinant molecule or organism is one that recombination in asexually reproducing holds a new combination of DNA sequences. bacteria. The beneficial effect of natural recombination is Genetic information in asexually reproducing observed in the heterosis, or hybrid vigor, organisms can be recombined in three ways: observed in genetically mixed or hybrid conjugation, transduction, and transformation. individuals compared with purebred organisms. Sexually reproducing organisms mix genes in three ways. o First, at the beginning of meiosis, duplicated chromosomes line up and recombine by crossing over or breakage and reunion of the four DNA duplexes. CONJUGATION The F factor was shown to be an o This generates newly recombined duplexes extrachromosomal circle of double-stranded with genes from each duplicate. Then, the DNA or plasmid carrying the genes coding for recombined duplexes are randomly construction of the mating bridge. assorted into gametes so that each gamete Genes carried on the F factor are transferred contains one set of each of the recombined across the bridge and simultaneously replicated parental chromosomes. so that one copy of the F factor remains in the F + bacteria, and the other is sent into the F − bacteria. TRANSDUCTION Just as animal and plant viruses infect eukaryotic cells, bacterial viruses, or bacteriophages, infect bacterial cells. Alfred Hershey and Martha Chase confirmed that the DNA of a bacterial virus was the carrier o Finally, the gamete will merge with a of its genetic determination in the transduction gamete from the other parent carrying its process. own set of recombined chromosomes. The resulting offspring will contain a new set HAYDEE RIZMA A. ABDULKAHAL 5  TRANS: PRE-MIDTERM TRANSFORMATION There are several types of RNAs found in the cell. This is the basis for modern-day recombinant o Ribosomal RNA, messenger RNA, transfer techniques. RNA, and small nuclear RNAs have distinct Frederick Griffith in 1928 first observed this type cellular functions. of recombinant technique, wherein he RNA is copied, or transcribed, from DNA. concluded that something from the dead smooth-type bacteria had “transformed” the TRANSCRIPTION rough-type bacteria into the virulent smooth-type bacteria. Transcription is the copying of one strand of They concluded that the “transforming factor” DNA into RNA by a process similar to that of that Griffith had first proposed was DNA. DNA replication. This activity, catalyzed by RNA polymerase, PLASMIDS occurs mostly in interphase. Whereas a single type of RNA polymerase catalyzes the A bacterial cell can contain, in addition to its synthesis of all RNA in most prokaryotes, there own chromosome complement, are three types of RNA polymerases in extrachromosomal entities, or plasmids. eukaryotes: Most plasmids are double-stranded circles of o RNA polymerase pol I, pol II, and pol III. 2,000 to 100,000 bp (2 to 100 kilobase pairs) in o Pol I and III synthesize noncoding RNA. size. o Pol II is responsible for the synthesis oF Plasmids can carry genetic information. Due to messenger RNA (mRNA), the type of RNA their size and effect on the host cell, plasmids that carries genetic information to be carry only a limited amount of information. translated into protein. The plasmid DNA duplex is compacted, or super-coiled. Breaking one strand of the plasmid duplex, or nicking, will relax the supercoil, whereas breaking both strands will linearize the plasmid. Different physical states of the plasmid DNA can be resolved by distinct migration characteristics during gel electrophoresis. Plasmids were initially classified into two general types: large plasmids and small plasmids. o Large plasmids include the F factor and some of the R plasmids. Large plasmids INITIATION carry genes for their own transfer and propagation and are self-transmissible. RNA polymerase and its supporting accessory o Small plasmids are more numerous in the proteins assemble on DNA at a specific site cell, about 20 copies per chromosomal called the promoter. equivalent; however, they do not carry Initiation sites of transcription (RNA synthesis) genes directing their maintenance. greatly outnumber DNA initiation sites in both prokaryotes and eukaryotes. RNA In prokaryotes, a basal transcription complex comprised of the large and small subunits of Ribonucleic acid (RNA) is a polymer of RNA polymerase and additional sigma factors nucleotides similar to DNA. It differs from DNA assembles at the start site. in the sugar moieties, having ribose instead of The eukaryotic transcription complex requires deoxyribose and, in one nitrogen base RNA polymerase and up to 20 additional factors component, having uracil instead of thymine. for accurate initiation. RNA is synthesized as a single strand rather than as a double helix. HAYDEE RIZMA A. ABDULKAHAL 6  TRANS: PRE-MIDTERM ELONGATION RNA synthesized beyond the site trails out of the polymerase and is bound by another RNA polymerases in both eukaryotes and exonuclease that begins to degrade the RNA 5 ′ prokaryotes synthesize RNA using the base to 3 ′ toward the RNA polymerase. sequence of one strand of the double helix as a When the exonuclease catches up with the guide. polymerase, transcription stops. The complementary strand of the DNA template The pol III termination signal is a run of adenine (that is not copied) has a sequence identical to residues in the template. Pol III transcription that of the RNA product (except for the U for T termination requires a termination factor. substitution in RNA). Unlike DNA synthesis, RNA synthesis does not TYPES AND STRUCTURES OF RNA require priming. RIBOSOMAL RNA The first ribonucleoside triphosphate retains all of its phosphate groups as the RNA is A structural component of ribosomes, the polymerized in the 5 ′ to 3 ′ direction. cellular organelles where protein synthesis occurs. It helps catalyze the formation of peptide bonds between amino acids during translation. The largest component of cellular RNA comprising 80% to 90% of the total cellular RNA. In prokaryotes, there are three rRNA species: o 16S – small subunit o 23S and 5S – large subunit TERMINATION Termination is accomplished in some genes by interactions between RNA polymerase and nucleotide signals in the DNA template. In other genes, an additional factor, rho, is required for termination. Rho is a helicase enzyme that associates with RNA polymerase and inactivates the elongation complex at a cytosine-rich termination site in In Eukaryotes, rRNA is synthesized from highly the DNA. repeated gene clusters. Rho-independent termination occurs at G:C-rich o 45S – precursor RNA regions in the DNA, followed by A:T-rich o 18S – small subunit regions. The G:C bases are transcribed into o 5.8S and 28S – large subunit RNA and fold into a short double-stranded o 5S – large subunit hairpin, which slows the elongation complex. The elongation complex then dissociates as it reaches the A:T-rich area. In eukaryotes, mRNA synthesis, catalyzed by pol II, proceeds along the DNA template until a polyadenyla- tion signal (polyA site) is encountered. At this point, the process of termination of transcription is activated. Pol I in eukaryotes terminates transcription just prior to a site in the DNA (Sal box) with the cooperation of a termination factor, TTF1. HAYDEE RIZMA A. ABDULKAHAL 7  TRANS: PRE-MIDTERM splicing and the remaining sequences MESSENGER RNA that code for the protein product are exons. Carries genetic information from the DNA in the nucleus to the ribosomes in the cytoplasm. It serves as a template for protein synthesis during translation. Prokaryotic mRNA is sometimes polycistronic; that is, one mRNA codes for more than one protein. Eukaryotic mRNA, in contrast, is monocistronic, having only one protein per mRNA. o Eukaryotic mRNA undergoes a series of post-transcriptional processing events before it is translated into protein. Some messages are transcribed constantly and are relatively abundant in the cell (constitutive transcription), whereas others are transcribed Why are eukaryotic genes interrupted by only at certain times during the cell cycle or introns? under particular conditions (inducible, or o Splicing may be important for the timing of regulatory, transcription). the translation of mRNA in the cytoplasm. Messenger RNA Processing: Alternative splicing modified products of o Polyadenylation genes by alternate insertion of different exons. For example, the production of calcitonin in the ▪ a polyA tail (polyadenylic acid at the 3’ thyroid or calcitonin gene-related peptide in the terminus); not coded in genomic DNA. It brain depends on the exons included in the is added to the RNA after synthesis of mature mRNA in these tissues. the pre-mRNA. ▪ Polyuridine or polythymine residues SMALL NUCLEAR RNA covalently attached to cellulose or Small nuclear RNA (snRNA) functions in sepharose substrates are often used to splicing in eukaryotes. specifically isolate mRNA in the Small nuclear RNA stays in the nucleus after its laboratory. transcription by RNA polymerase I or III. ▪ The enzyme polyadenylate polymerase is responsible for adding the adenines to TRANSFER RNA the end of the transcript. A run of up to 200 nucleotides of polyA is typically Transports amino acids to the ribosome during found on mRNA in mammalian cells. protein synthesis. Each tRNA molecule carries a specific amino o Capping acid and recognizes the corresponding codon on the mRNA through its anticodon region. ▪ Eukaryotic mRNA is blocked at the 5 ′ terminus by a 5′-5′ pyrophosphate OTHER RNAS bridge to a methylated guanosine. The structure is called a cap. Since the late 1990s, increasing varieties of o Splicing RNA species have been described in prokaryotes and eukaryotes. ▪ Eukaryotic coding regions are In addition to RNA synthesis and processing, interrupted with long stretches of these molecules influence numerous cellular noncoding DNA (intervening) sequences processes, including plasmid replication, called introns. bacteriophage development, chromosome ▪ Introns are removed from hnRNA by structure, and development. HAYDEE RIZMA A. ABDULKAHAL 8  TRANS: PRE-MIDTERM RNA POLYMERASES RNA HELICASES RNA synthesis is catalyzed by RNA polymerase RNA synthesis and processing require the enzymes. activity of helicases to catalyze the unwinding of One multisubunit prokaryotic enzyme is double-stranded RNA. responsible for the synthesis of all types of RNA These enzymes have been characterized in in the prokaryotic cell. prokaryotic and eukaryotic organisms. Eukaryotes have three different RNA Another activity of these enzymes is in the polymerase enzymes. removal of proteins from RNA–protein All of these enzymes are DNA-dependent RNA complexes. polymerases; that is, they require a DNA template. PROTEINS AND THE GENETIC CODE o pol I (rRNA), pol II (mRNA), and pol III (tRNA) Proteins are the products of transcription and translation of the nucleic acids. OTHER RNA-METABOLIZING ENZYMES Even though nucleic acids are most often the RIBONUCLEASES focus of “molecular analysis,” the ultimate effect of the information stored and delivered by the Ribonucleases degrade RNA in a manner nucleic acid is manifested in proteins. similar to the degradation of DNA by Even if proteins are not being tested directly, deoxyribonucleases. they manifest the phenotype directed by the There are several types of ribonucleases, nucleic acid information. generally classified as endoribonucleases and exoribonucleases. AMINO ACIDS Endonucleases include RNase H, which digests the RNA strand in a DNA–RNA hybrid; RNase I, Proteins are polymers of amino acids. Proteins which cleaves single-stranded RNA; and are polypeptides that can reach sizes of more RNase III, which digests double-stranded RNA. than a thousand amino acids in length. RNase P removes precursor nucleotides from Each amino acid has characteristic biochemical tRNA molecules. RNase A, RNase T1, and properties determined by the nature of its amino RNase T2 cleave single-stranded RNA at acid side chain. specific residues. Amino acids are grouped according to their A combination of RNase A, T1, and T2 is used polarity (tendency to interact with water at pH 7) in some laboratory procedures investigating as follows: nonpolar, uncharged polar, gene expression and transcript structure. negatively charged polar, and positively charged polar. Proline differs from the rest of the amino acids in that its side chain is cyclic, with the amino group attached to the end carbon of the side chain making a five-carbon ring. Histidine has a unique synthetic pathway using metabolites common to purine nucleotide biosynthesis, which affords the connection of amino acid synthesis to nucleotide synthesis. The amino and carboxyl terminal groups of the amino acids are joined in a carbon-carbon-nitrogen (–C–C–N–) substituted RNases are ubiquitous, stable enzymes that amide linkage (peptide bond) to form the degrade all types of RNA. Some RNases are protein backbone. secreted by higher eukaryotes, possibly as an At one end of the peptide will be an amino antimicrobial defense mechanism. group (the amino-terminal, or NH 2 end), and at the opposite terminus of the peptide will be a carboxyl group (the carboxy-terminal, or COOH end). HAYDEE RIZMA A. ABDULKAHAL 9  TRANS: PRE-MIDTERM The sequence of amino acids in a protein THE GENETIC CODE determines the nature and activity of that protein. The nature of a gene was further clarified with This sequence is the primary structure of the the deciphering of the genetic code by Francis protein and is read by convention from the Crick, Marshall Nirenberg, Philip Leder, Gobind amino terminal end to the carboxy terminal end. Khorana, and Sydney Brenner. Minor changes in primary structure can have The genetic code is not information in itself but such drastic effects because the amino acids is a dictionary to translate the 4-nucleotide must often cooperate with one another to bring sequence information in DNA to the 20–amino about protein structure and function. acid sequence information in proteins. Structures of Protein: Codon selection may be important for the o Primary Structure - read by convention differentiation of human tissues and may also from the amino terminal end to the carboxy have a role in the development of diseases, terminal end. such as cancer, where differentiation pathways o Secondary Structure - alpha-helix and are altered. beta-sheet structures, where some proteins CA begins the code for both histidine and consist almost entirely of alpha helices or glutamine. Three codons—UAG, UAA, and beta sheets. UGA—that terminate protein synthesis are o Tertiary Structure - If a protein loses its termed nonsense codons. tertiary structure, it is denatured where The characteristics of the genetic code have folded proteins are not functional if it is consequences for molecular analysis. denatured improperly. Proteins can also be Mutations or changes in the DNA sequence will denatured by heat (e.g., the albumin in egg have different effects on phenotype, depending white) or by conformations forced on on the resultant changes in the amino acid innocuous peptides by infectious prions. sequence. o Quaternary Structure - Proteins that work together in this way are called oligomers, each component protein being a monomer. Proteins are classified according to function as enzymes and as transport, storage, motility, structural, defense, or regulatory proteins. In contrast to simple proteins that have no other components except amino acids, conjugated proteins do have components other than amino acids. The nonprotein component of a conjugated protein is the nonprotein prosthetic group. GENES TRANSLATION AMINO ACID CHARGING A gene is defined as the ordered sequence of nucleotides on a chromosome that encodes a The nature of a gene was further clarified with specific functional product. the deciphering of the genetic code by Francis A gene is the fundamental physical and Crick, Marshall Nirenberg, Philip Leder, Gobind functional unit of inheritance. Khorana, and Sydney Brenner. A gene contains not only structural Proteins are the products of transcription and sequences that code for an amino acid translation of the nucleic acids. sequence but also regulatory sequences that After transcription of the sequence information are important for the regulated expression of in DNA to RNA, the transcribed sequence must the gene be transferred into proteins. Through the genetic code, a specific nucleic acid sequence is translated to an amino acid sequence and, ultimately, to a phenotype. HAYDEE RIZMA A. ABDULKAHAL 10  TRANS: PRE-MIDTERM Protein synthesis starts with activation of the acid, generating a dipeptidyl-tRNA in the A site, amino acids by covalent attachment to tRNA, or catalyzed by peptidyl transferase. tRNA charging, a reaction catalyzed by 20 After formation of the peptide bond, the aminoacyl tRNA synthetases. ribosome moves, shifting the dipeptidyl-tRNA The process of attaching an amino acid to its from the A site to the P site with the release of corresponding transfer RNA (tRNA) is called the “empty” tRNA from a third position, the E amino acid activation, also known as tRNA site, of the ribosome. charging or aminoacylation. During translation, the growing polypeptide The product of the reaction is an ester bond begins to fold into its mature conformation. This between the 3 ′ hydroxyl of the terminal adenine process is assisted by molecular chaperones. of the tRNA and the carboxyl group of the In bacteria, translation and transcription occur amino acid. simultaneously. In nucleated cells, the majority of translation occurs in the cytoplasm. PROTEIN SYNTHESIS A cellular surveillance system for RNA with premature termination codons, nonsense-mediated decay (NMD),121 a degradation of messenger RNAs with premature termination codons, supposedly occurred in mammalian nuclei. Translation takes place on ribosomes, ribonucleoprotein particles first observed by electron microscopy of animal cells. Protein synthesis in the ribosome almost always starts with the amino acid methionine in eukaryotes and N-formylmethionine in bacteria, mitochondria, and chloroplasts. Initiating factors that participate in the formation of the ribosome complex differentiate the initiating methionyl tRNAs from those that add methionine internally to the protein. In protein translation, the small ribosomal subunit first binds to initiation factor 3 (IF-3) and then to specific sequences near the 5 ′ end of the mRNA, the ribosomal binding site. Another initiation factor, IF-2 bound to GTP and the initiating tRNA Met or tRNA fMet, then joins the complex. In this complex, the tRNA Met or tRNA fMet is situated in the peptidyl site (P site) of the functional ribosome. In contrast, all other tRNAs bind to an adjacent site, the aminoacyl site (A site) of the ribosome. The first peptide bond is formed between the amino acids in the A and P sites by transfer of the N -formylmethionyl group of the first amino acid to the amino group of the second amino HAYDEE RIZMA A. ABDULKAHAL 11