Ch8: DNA/RNA and Biotechnology

Chapter 8: DNA and RNA

Fundamental Concepts and the Central Dogma

• Nature of Nucleic Acids: DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid) are long, linear biopolymers known as nucleic acids.

• Information Storage: Nucleic acids are the carriers of genetic information in a form that is passed from one generation to the next.

• The Central Dogma: This principle describes the flow of genetic information within a biological system, defined as the progression from DNA to RNA to proteins.

Chemical Composition and Nomenclature

• Biopolymer Structure: Nucleic acids consist of bases attached to a sugar-phosphate backbone.

• Pentose Sugars:   - Ribose: Found in RNA, contains hydroxyl groups ($-OH$) at both the $2'$ and $3'$ positions.   - Deoxyribose: Found in DNA, lacks an oxygen at the $2'$ position ($2'$-H instead of $2'$-OH).

• Nitrogenous Bases:   - Purines: Double-ring structures including Adenine (A) and Guanine (G).   - Pyrimidines: Single-ring structures including Cytosine (C), Uracil (U) (found in RNA), and Thymine (T) (found in DNA).

• Linkages: Bases are attached to the sugar via a $\beta$-Glycosidic linkage at the $1'$ position.

• Nucleoside vs. Nucleotide:   - Nucleoside: A base attached to a sugar (e.g., Adenosine).   - Nucleotide: A nucleoside with one or more phosphate groups attached (e.g., Adenylate/ATP).

• Naming Conventions Table:   - Adenine: Nucleoside = Adenosine (Deoxyadenosine); Nucleotide = Adenylate (Deoxyadenylate); Nucleic Acid = RNA (DNA).   - Guanine: Nucleoside = Guanosine (Deoxyguanosine); Nucleotide = Guanylate (Deoxyguanylate); Nucleic Acid = RNA (DNA).   - Cytosine: Nucleoside = Cytidine (Deoxycytidine); Nucleotide = Cytidylate (Deoxycytidylate); Nucleic Acid = RNA (DNA).   - Thymine: Nucleoside = Thymidine (Deoxythymidine); Nucleotide = Thymidylate (Deoxythymidylate); Nucleic Acid = DNA.   - Uracil: Nucleoside = Uridine; Nucleotide = Uridylate; Nucleic Acid = RNA.

Structural Characteristics of the Double Helix

• Stabilization: The double helix is stabilized by the following:   - Hydrogen bonds between specific base pairs.   - Van der Waals interactions (base stacking).

• Function: The structure facilitates the replication of genetic material.

• Grooves: DNA features two grooves, the Major groove and the Minor groove.   - These arise because glycosidic bonds of a base pair are not diametrically opposite each other.   - The Major groove is wider and deeper than the Minor groove.   - Hydrogen-bond donor and acceptor atoms within these grooves enable crucial interactions with proteins during replication and transcription.

• Base-Pair Anatomy: Guanine pairs with Cytosine ($G-C$) and Adenine pairs with Thymine ($A-T$). These pairs have essentially the same shape and are held together by weak hydrogen bonds.

Chargaff’s Rule and Statistical Ratios

• Observation: Erwin Chargaff observed in the 1940s that the ratio of A to T and G to C is nearly $1:1$ across various organisms, while the A to G ratio varies.

• Data Ratios (A:T | G:C | A:G):   - Human being: $1.00$ | $1.00$ | $1.56$   - Salmon: $1.02$ | $1.02$ | $1.43$   - Wheat: $1.00$ | $0.97$ | $1.22$   - Yeast: $1.03$ | $1.02$ | $1.67$   - Escherichia coli: $1.09$ | $0.99$ | $1.05$   - Serratia marcescens: $0.95$ | $0.86$ | $0.70$

Structural Forms and Sugar Puckering

• B-form: The "default" DNA form. It is a right-handed double helix with anti-parallel strands held together by Watson–Crick base pairs.

• A-form: Wider and shorter than B-DNA, with tilted base pairs relative to the helix axis. Seen in RNA double helices and RNA–DNA hybrid helices.

• Z-form: A left-handed double helix with zigzagged phosphoryl groups; its biological role is unknown, though Z-DNA-binding proteins exist.

• Sugar Pucker Explanations:   - A-DNA: $C-3'$ lies out of the plane ($C-3'$ endo), leading to an $11$-degree tilting of base pairs.   - B-DNA: $C-2'$ lies out of the plane ($C-2'$ endo).

• Stem-Loop Motifs: Single-stranded nucleic acids can form structures when two complementary sequences within the same strand pair to form a double helix. These may include mismatched or unmatched bases and are often stabilized by $Mg^{2+}$ ions.

DNA Replication Mechanisms

• Meselson and Stahl Experiment: Proved the semi-conservative model of replication.   - Step 1: Label parent DNA with ${}^{15}N$ growth media.   - Step 2: Shift to ${}^{14}N$ media.   - Step 3: Perform density-gradient equilibrium sedimentation to observe ${}^{14}N$ and ${}^{15}N$ distribution.

• DNA Polymerases: Enzymes that catalyze phosphodiester-bridge formation.   - Reaction: $(DNA)<em>n + dNTP \rightarrow (DNA)</em>{n+1} + PP_i$

• Klenow Fragment: Part of DNA polymerase I from E. coli, consisting of:   - The polymerase unit, shaped like a "right hand" (fingers and thumb wrap the DNA, active site in the palm).   - A $3' \rightarrow 5'$ exonuclease for proofreading and correcting products.

The Replication Fork and Priming

• Primer Requirements: Polymerases require a primer and synthesize only in the $5' \rightarrow 3'$ direction.

• Primase: An RNA polymerase that synthesizes a short RNA strand ($\sim 5$ nucleotides) complementary to the DNA template. This initiates synthesis, and the RNA is later removed by hydrolysis and replaced with DNA.

• Replication Fork Dynamics:   - Leading strand: Synthesized continuously in the $5' \rightarrow 3'$ direction.   - Lagging strand: Synthesized discontinuously in small fragments called Okazaki fragments ($\sim 1,000$ nucleotides).   - Trombone Model: The lagging strand is looped so it passes through the polymerase active site in the $3' \rightarrow 5'$ direction, allowing "backwards" synthesis. Loops are released and reformed after synthesizing fragments.

Coordination of Replication Enzymes

• Helicase: Separates double helix strands using ATP for energy. It is a ring-like structure of six subunits.   - AMP-PNP: A nonhydrolyzable ATP analog used in crystallization studies.   - Mechanism: A single strand fits through the ring; ATP binding and hydrolysis cause conformational changes that move the helicase two bases at a time.

• Supercoiling and Management:   - DNA in front of helicase becomes overwound (torsionally stressed).   - Linking Number ($Lk$): Describes DNA topology. $Lk = Tw + Wr$.   - Twist ($Tw$): Helical winding (Positive = Right-Handed).   - Writhe ($Wr$): Coiling of the axis/supercoiling (Negative = Right-handed).

• Topoisomerases:   - Type I: DNA binds in the cavity; Tyr 723 attacks a phosphoryl group to cleave one strand. The strand rotates to unwind and is kemudian resealed.   - Type II (Gyrase): Binds the G segment, utilizes ATP to trap a T segment, cleaves both strands of the G segment, passes the T segment through, and ligates the G segment.

Prokaryotic Replication Components

• DNA Polymerase III Holoenzyme:   - Alpha ($\alpha$): Polymerase activity.   - Epsilon ($\epsilon$): $3' \rightarrow 5'$ proofreading exonuclease.   - Theta ($\theta$): Accessory subunit.   - Beta-subunit ($\beta_2$): Sliding clamp that provides high processivity (ability to catalyze consecutive reactions without releasing substrate).   - Gamma-tau complex ($\gamma\tau_2\delta\delta'\chi\phi$): Serves as the clamp loader.

• Initiation and Termination:   - $oriC$: Unique start site in bacteria. DnaA proteins bind high-affinity sites to form an oligomer, exposing single strands. DnaB (Helicase) then loads.   - Ter sites: Specific termination sites bound by Tus (termination utilization substance). Tus acts as a one-way gate for replication forks.

Analytical Techniques in DNA Technology

• Restriction Enzymes: Prokaryotic endonucleases that cleave specific, usually palindromic, sequences.

• Gel Electrophoresis: Separates DNA fragments using agarose or polyacrylamide gels; visualized with ethidium bromide.

• Blotting:   - Southern blotting: Identification of DNA sequences using a DNA probe (short labeled single-strand DNA).   - Northern blotting: Analysis technique for RNA.

• Sanger Sequencing: Uses capillary electrophoresis and fluorescence to determine base sequences.

• Polymerase Chain Reaction (PCR): Amplifies target DNA exponentially ($2^n$-fold).   - Requirements: Flanking primers, dNTPs, heat-stable DNA polymerase (e.g., Taq).   - Cycle: $95^\circ\text{C}$ (separation), $54^\circ\text{C}$ (annealing), $72^\circ\text{C}$ (elongation).

Recombinant DNA and Genome Editing

• Vectors: DNA molecules (like plasmids or $\lambda$ bacteriophages) that replicate autonomously. DNA fragments are inserted using restriction enzymes and DNA ligase.

• cDNA Libraries: Double-stranded DNA generated from mRNA via Reverse Transcriptase. Allows expression of eukaryotic genes (without introns) in bacteria.

• Mutagenesis:   - Site-Directed: Introducing specific mutations using PCR and mutagenic primers.   - Cassette Mutagenesis: Swapping a gene segment for a synthetic oligonucleotide (cassette).   - Inverse PCR: Used to introduce deletions into plasmids.

• Next-Generation Sequencing (NGS):   - High-throughput parallel methods (e.g., Reversible terminator, Pyrosequencing, Ion semiconductor).   - Pyrosequencing: Nucleotide addition releases pyrophosphate ($PP_i$), converted to light via ATP sulfurylase and luciferase.

• Quantification:   - qPCR: Uses SYBR Green I dye; fluorescence threshold ($C_T$) determines copy number.   - Microarrays: Measure gene expression changes (e.g., tumor vs normal) through competitive hybridization of colored cDNA.

• Genome Editing Systems:   - ZFNs (Zinc-Finger Nucleases): Combine FokI nuclease with engineered DNA-binding fingers.   - CRISPR-Cas9: Uses a single guide RNA (sgRNA) to target DNA near a PAM (protospacer adjacent motif). Cas9 contains REC (binding) and NUC (cleaving) lobes.   - Repair follows via NHEJ (error-prone, gene knockout) or HDR (template-directed changes).

• RNA Interference (RNAi): Gene knockdown technique.   - Dicer: Cleaves dsRNA into siRNA.   - RISC: Unwinds RNA and cleaves target mRNA containing the complementary sequence.