NUCLEIC ACIDS History of Nucleic Acids Friedrich Miescher in 1869  isolated what he called nuclein from the nuclei

of pus cells Richard Altmann in 1889  Nuclein was shown to have acidic properties, hence it became called nucleic acid 1920s  the tetranucleotide hypothesis was introduced The Tetranucleotide hypothesis  Up to 1940 researchers were convinced that hydrolysis of nucleic acids yielded the four bases in equal amounts.  Nucleic acid was postulated to contain one of each of the four nucleotides, the tetranucleotide hypothesis.  Takahashi (1932) proposed a structure of nucleotide bases connected by phosphodiester linkages.

Astbury and Bell in 1938  First X-ray diffraction pattern of DNA is published.  The pattern indicates a helical structure, indicated periodicity. X-ray diffraction of DNA Wilkins & Franklin (1952): X-ray crystallography

Avery, MacLeod, and Mc Carty in 1944  demonstrate DNA could “transform” cells.  Supporters of the tetranucleotide hypothesis did not believe nucleic acid was variable enough to be a molecule of heredity and store genetic information.

DNA is Genetic Material

Erwin Chargaff in late 1940s  used paper chromatography for separation of DNA hydrolysates.  Amount of adenine is equal to amount of thymine and amount of guanine is equal to amount of cytosine. Hershey and Chase in 1952  confirm DNA is a molecule of heredity. The Hershey-Chase Experiment

Watson and Crick in 1953  determine the structure of DNA Watson & Crick Base pairing

Francis Crick in 1958  proposes the “central dogma of molecular biology” .  Kornberg purifies DNA polymerase I

1969  Entire genetic code determined

Nucleic Acids • Nucleic Acids are very long, thread-like polymers, made up of a linear array of monomers called nucleotides. • Nucleic acids vary in size in nature • tRNA molecules contain as few as 80 nucleotides • Eukaryotic chromosomes contain as many as 100,000,000 nucleotides. Two types of nucleic acid are found  Deoxyribonucleic acid (DNA)  Ribonucleic acid (RNA) DNA and RNA DNA deoxyribonucleic acid nucleic acid that stores genetic information found in the nucleus of a mammalian cell. RNA ribonucleic acid 3 types of RNA in a cell Ribosomal RNAs (rRNA) are components of ribosomes Messenger RNAs (mRNA) carry genetic information Transfer RNAs (tRNA) are adapter molecules in translation The distribution of nucleic acids in the eukaryotic cell  DNA is found in the nucleus with small amounts in mitochondria and chloroplasts  RNA is found throughout the cell The nucleus contains the cell’s DNA (genome) RNA is synthesized in the nucleus and exported to the cytoplasm

DNA as genetic material: The circumstantial evidence 1. Present in all cells and virtually restricted to the nucleus 2. The amount of DNA in somatic cells (body cells) of any given species is constant (like the number of chromosomes) 3. The DNA content of gametes (sex cells) is half that of somatic cells. In cases of polyploidy (multiple sets of chromosomes) the DNA content increases by a proportional factor 4. The mutagenic effect of UV light peaks at 253.7nm. The peak for the absorption of UV light by DNA

NUCLEIC ACID STRUCTURE  Nucleic acids are polynucleotides  Their building blocks are nucleotides NUCLEOTIDE STRUCTURE

All 1. 2. 3.

nucleotides contain three components: A nitrogen heterocyclic base A pentose sugar A phosphate residue Spot the difference

Ribose is a pentose

Chemical Structure of DNA vs RNA Ribonucleotides have a 2’-OH Deoxyribonucleotides have a 2’-H

THE SUGAR-PHOSPHATE BACKBONE  The nucleotides are all orientated in the same direction  The phosphate group joins the 3rd Carbon of one sugar to the 5th Carbon of the next in line. ADDING IN THE BASES  The bases are attached to the 1st Carbon  Their order is important  It determines the genetic information of the molecule DNA IS MADE OF TWO STRANDS OF POLYNUCLEOTIDE

DNA IS MADE OF TWO STRANDS OF POLYNUCLEOTIDE  The sister strands of the DNA molecule run in opposite directions (antiparallel)  They are joined by the bases  Each base is paired with a specific partner: A is always paired with T G is always paired with C “Purine with Pyrimidine”  The sister strands are complementary but not identical  The bases are joined by hydrogen bonds, individually weak but collectively strong  There are 10 base pairs per turn

Purines & Pyrimidines

Nucleotides are linked by phosphodiester bonds

Central Dogma of Life From DNA to Protein DNA to Protein  DNA acts as a “manager” in the process of making proteins  DNA is the template or starting sequence that is copied into RNA that is then used to make the protein Central Dogma  One gene – one protein

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This is the same for bacteria to humans DNA is the genetic instruction or gene DNA → RNA is called Transcription  RNA chain is called a transcript RNA → Protein is called Translation

Expression of Genes  Some genes are transcribed in large quantities because we need large amount of this protein  Some genes are transcribed in small quantities because we need only a small amount of this protein

Nucleotides as Language  We must start to think of the nucleotides – A, G, C and T as part of a special language – the language of genes that we will see translated to the language of amino acids in proteins Genes as Information Transfer

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A gene is the sequence of nucleotides within a portion of DNA that codes for a peptide or a functional RNA Sum of all genes = genome

STEP 1 – DNA REPLICATION DNA Replication  Semiconservative  Daughter DNA is a double helix with 1 parent strand and 1 new strand  Found that 1 strand serves as the template for new strand DNA Template

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Each strand of the parent DNA is used as a template to make the new daughter strand DNA replication makes 2 new complete double helices each with 1 old and 1 new strand

Replication Origin  Site where replication begins  1 in E. coli  1,000s in human  Strands are separated to allow replication machinery contact with the DNA  Many A-T base pairs because easier to break 2 H-bonds that 3 H-bonds  Note anti-parallel chains Replication Fork  Bidirectional movement of the DNA replication machinery

THE REPLICATION FACTORY  DNA replication is an intricate process requiring the concerted action of many different proteins.  The replication proteins are clustered together in particular locations in the cell and may therefore be regarded as a small “Replication Factory” that manufactures DNA copies.  The DNA to be copied is fed through the factory, much as a reel of film is fed through a movie projector.  The incoming DNA double helix is split into two single strands and each original single strand becomes half of a new DNA double helix. Because each resulting DNA double helix retains one strand of the original DNA, DNA replication is said to be semi-conservative. DNA REPLICATION PROTEINS  DNA replication requires a variety of proteins.  Each protein performs a specific function in the production of the new DNA strands.  Helicase, made of six proteins arranged in a ring shape, unwinds the DNA double helix into two individual strands.  Single-strand binding proteins, or SSBs, are tetramers that coat the single-stranded DNA.  This prevents the DNA strands from reannealing to form double-stranded DNA.  Primase is an RNA polymerase that synthesizes the short RNA primers needed to start the strand replication process.  DNA polymerase is a hand-shaped enzyme that strings nucleotides together to form a DNA strand.  The sliding clamp is an accessory protein that helps hold the DNA polymerase onto the DNA strand during replication.  RNAse H removes the RNA primers that previously began the DNA strand synthesis.  DNA ligase links short stretches of DNA together to create one long continuous DNA strand.

Components of the DNA Replication

Polymerase & Proteins Coordinated  One polymerase complex apparently synthesizes leading/lagging strands simultaneously  Even more complicated in eukaryotes

STRAND SEPARATION  To begin the process of DNA replication, the two double helix strands are unwound and separated from each other by the helicase enzyme.

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The point where the DNA is separated into single strands, and where new DNA will be synthesized, is known as the replication fork. Single-strand binding proteins, or SSBs, quickly coat the newly exposed single strands. SSBs maintain the separated strands during DNA replication. Without the SSBs, the complementary DNA strands could easily snap back together. SSBs bind loosely to the DNA, and are displaced when the polymerase enzymes begin synthesizing the new DNA strands.

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NEW STRAND SYNTHESIS  Now that they are separated, the two single DNA strands can act as templates for the production of two new, complementary DNA strands.  Remember that the double helix consists of two antiparallel DNA strands with complementary 5’ to 3’ strands running in opposite directions.  Polymerase enzymes can synthesize nucleic acid strands only in the 5’ to 3’ direction, hooking the 5’ phosphate group of an incoming nucleotide onto the 3’ hydroxyl group at the end of the growing nucleic acid chain.  Because the chain grows by extension off the 3’ hydroxyl group, strand synthesis is said to proceed in a 5’ to 3’ direction.  Even when the strands are separated, however, DNA polymerase cannot simply begin copying the DNA.  DNA polymerase can only extend a nucleic acid chain but cannot start one from scratch.  To give the DNA polymerase a place to start, an RNA polymerase called primase first copies a short stretch of the DNA strand.  This creates a complementary RNA segment, up to 60 nucleotides long that is called a primer.  Now DNA polymerase can copy the DNA strand.  The DNA polymerase starts at the 3’ end of the RNA primer, and, using the original DNA strand as a guide, begins to synthesize a new complementary DNA strand.  Two polymerase enzymes are required, one for each parental DNA strand.  Due to the antiparallel nature of the DNA strands, however, the polymerase enzymes on the two strands start to move in opposite directions.  One polymerase can remain on its DNA template and copy the DNA in one continuous strand.  However, the other polymerase can only copy a short stretch of DNA before it runs into the primer of the previously sequenced fragment.  It is therefore forced to repeatedly release the DNA strand and slide further upstream to begin extension from another RNA primer.  The sliding clamp helps hold this DNA polymerase onto the DNA as the DNA moves through the replication machinery. The sliding clamp makes the polymerase processive.  The continuously synthesized strand is known as the leading strand, while the strand that is synthesized in short pieces is known as the lagging strand.  The short stretches of DNA that make up the lagging strand are known as Okazaki fragments. THE LAGGING STRAND  Before the lagging-strand DNA exits the replication factory, its RNA primers must be removed and the Okazaki fragments must be joined together to create a continuous DNA strand.  The first step is the removal of the RNA primer.  RNAse H, which recognizes RNA-DNA hybrid helices, degrades the RNA by hydrolyzing its phosphodiester bonds. Next, the sequence gap created by RNAse H is then filled in by DNA polymerase which extends the 3’ end of the neighboring Okazaki fragment.

Finally, the Okazaki fragments are joined together by DNA ligase that hooks together the 3’ end of one fragment to the 5’ phosphate group of the neighboring fragment in an ATPor NAD+-dependent reaction.

REPLICATION IN ACTION  The process begins when the helicase enzyme unwinds the double helix to expose two single DNA strands and create two replication forks.  DNA replication takes place simultaneously at each fork. The mechanism of replication is identical at each fork.

How is DNA Synthesized?  Original theory  Begin adding nucleotides at origin  Add subsequent bases following pairing rules  Expect both strands to be synthesized simultaneously  This is NOT how it is accomplished  Actually how DNA is synthesized  Simple addition of nucleotides along one strand, as expected Called the leading strand DNA polymerase reads 3’ → 5’ along the leading strand from the RNA primer Synthesis proceeds 5’ à 3’ with respect to the new daughter strand Remember how the nucleotides are added!!!!! 5’ à 3’

Mistakes during Replication  Base pairing rules must be maintained

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 Mistake = genome mutation, may have consequence on daughter cells Only correct pairings fit in the polymerase active site If wrong nucleotide is included  Polymerase uses its proofreading ability to cleave the phosphodiester bond of improper nucleotide Activity 3’ à 5’  And then adds correct nucleotide and proceeds down the chain again in the 5’ à 3’ direction


DNA Repair  For the rare mutations occurring during replication that isn’t caught by DNA polymerase proofreading  For mutations occurring with daily assault  If no repair  In germ (sex) cells à inherited diseases  In somatic (regular) cells à cancer CONSEQUENCES OF GENETIC ERRORS :SOURCES OF GENETIC VARIATION  Mutation - any novel genetic change in the gene complement or genotype relative to the parental genotypes, beyond that achieved by genetic recombination during meiosis.  Mutations are changes in DNA structure, and therefore changes in protein and phenotype.  Mutations are rare! For every 100 million nucleotides added to a developing DNA strand only one mistake occurs on average.  Mutations are heritable; and may be beneficial, neutral, lethal, detrimental or harmful to the organism. Types of Mutation 1. Induced  viruses, UV radiation, some chemicals (nitric acid changes cytosine to uracil) or mutagens (or carcinogens - benzene, cigarette smoke).

2. Spontaneous  Proofreading mistakes during DNA replication (Base substitutions) - not necessarily a serious change.  Frame shift mutation (Addition or deletion of a base) - serious change!  A 3 letter code or codon is analogous to three letter words in a sentence.  Original sequence THE CAT SAW THE DOG  Base or letter substitutions THE BAT SAW THE DOG THE CAT SAW THE HOG THE CAB SAW THE DOG THE CAT SAW SHE DOG THE CAT SAD THE DOG THE CAT SAW THE DOC  Deletions THE CAT SAW TED OG THE ATS AWT HED OG Additions THE CAT SAW THE ZDO G THE CMA TAS WTH EDO G

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Jumping genes, transposable elements, or transposons. Discovered by Barbara McClintok (1956) while studying color variation in Indian corn. Won Nobel prize in 1983. Patches of yellow sometimes occur among the purple grains of Indian corn. She explain this by assuming that the gene was being interrupted by a foreign sequence of DNA.  These foreign bits of DNA could insert or remove themselves from a stretch of DNA causing the genes that they affected to be turned on or off. Such "jumping genes" could copy themselves and move about within the genome of the organism they occupied. 4. Chromosomal mutations (disruption in chromosomal morphology - inversions and translocations). 5. Homeotic genes  master genes that regulate suites of other genes and may affect developmental pathways especially during embryogenesis. Mutations in these master genes can cause genetic anomalies. For example, a fruit fly that possesses legs where antennae should be, or a mosquito that has its mouth parts transformed into legs. Effect of Mutation

Uncorrected Replication Errors

Mismatch repair  Enzyme complex recognizes mistake and excises newly-synthesized strand and fills in the correct pairing

Eukaryotes “label” the daughter strand with nicks to recognize the new strand  Separates new from old

Chemical Modifications

Thymine Dimers

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Caused by exposure to UV light 2 adjacent thymine residues become covalently linked

Repair Mechanisms  Different enzymes recognize, excise different mistakes  DNA polymerase synthesizes proper strand  DNA ligase joins new fragment with the polymer

STEP 2 - TRANSCRIPTION Transcription  The region of the double-stranded DNA corresponding to a specific gene is copied into an RNA molecule, called messenger RNA (mRNA).  RNA differs from DNA

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Ribose is the sugar rather than deoxyribose – ribonucleotides U instead of T; A, G and C the same Single stranded Can fold into a variety of shapes that allows RNA to have structural and catalytic functions

RNA Differences

Transcription  Similarities to DNA replication  Open and unwind a portion of the DNA  1 strand of the DNA acts as a template  Complementary base-pairing with DNA  Differences  RNA strand does not stay paired with DNA DNA re-coils and RNA is single stranded  RNA is shorter than DNA RNA is several 1000 bp or shorter whereas DNA is 250 million bp long

RNA Polymerase  Catalyzes the formation of the phosphodiester bonds between the nucleotides (sugar to phosphate)  Uncoils the DNA, adds the nucleotide one at a time in the 5’ to 3’ fashion  Uses the energy trapped in the nucleotides themselves to form the new bonds

Template to Transcripts  The RNA transcript is identical to the NON-template strand with the exception of the T’s becoming U’s RNA Elongation  Reads template 3’ to 5’  Adds nucleotides 5’ to 3’ (5’ phosphate to 3’ hydroxyl)  Synthesis is the same as the leading strand of DNA

Differences in DNA and RNA Polymerases  RNA polymerase adds ribonucleotides not deoxynucleotides  RNA polymerase does not have the ability to proofread what they transcribe  RNA polymerase can work without a primer  RNA will have an error 1 in every 10,000 nucleotides (DNA is 1 in 100,000,000 nucleotides) Types of RNA  messenger RNA (mRNA) – codes for proteins  ribosomal RNA (rRNA) – forms the core of the ribosomes, machinery for making proteins  transfer RNA (tRNA) – matches code for amino acid on mRNA and positions the right amino acid in place during protein synthesis How does the process of transcription begin?  The DNA serves as the template for producing an RNA transcript or copy of information stored on the DNA molecule.  The DNA molecule must open up and allow an enzyme called RNA polymerase read and connect together the sequence of nucleotides in the proper order. STEP 3 – TRANSLATION RNA to Protein  Translation is the process of turning mRNA into protein  Translate from one “language” (mRNA nucleotides) to a second “language” (amino acids)  Genetic code – nucleotide sequence that is translated to amino acids of the protein DNA Code

Nucleotides read 3 at a time meaning that there are 64 combinations for a codon (set of 3 nucleotides) Only 20 amino acids  More than 1 codon per AA – degenerate code with the exception of Met and Trp (least abundant AAs in proteins)

Reading Frames

Translation can occur in 1 of 3 possible reading frames, dependent on where decoding starts in the mRNA

Transfer RNA Molecules  Translation requires an adaptor molecule that recognizes the codon on mRNA and at a distant site carries the appropriate amino acid  Intra-strand base pairing allows for this characteristic shape  Anticodon is opposite from where the amino acid is attached

Wobble Base Pairing  Due to amino acids recognize because the 3rd mismatched  Allows for there only being 31 tRNA for the 61 codons

degenerate code for some tRNA can several codons spot can wobble or be

Attachment of AA to tRNA  Aminoacyl-tRNA synthase is the enzyme responsible for linking the amino acid to the tRNA  A specific enzyme for each amino acid and not for the tRNA 2 ‘Adaptors’ Translate Genetic Code to Protein

Ribosomes  Complex machinery that controls protein synthesis

2 subunits  1 large – catalyzes the peptide bond formation  1 small – binds mRNA and tRNA Contains protein and RNA  rRNA central to the catalytic activity Folded structure is highly conserved  Protein has less homology and may not be as important

Ribosome Structures May be free in cytoplasm or attached to the ER Subunits made in the nucleus in the nucleolus and transported to the cytoplasm

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Ribosomal Subunits

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1 large subunit – catalyzes the formation of the peptide bond 1 small subunit – matches the tRNA to the mRNA Moves along the mRNA adding amino acids to growing protein chain

Ribosomal Movement

4 binding sites  mRNA binding site  Peptidyl-tRNA binding site (P-site) Holds tRNA attached to growing end of the peptide  Aminoacyl-tRNA binding site (A-site) Holds the incoming AA  Exit site (E-site)


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