7.2 DNA Structure, RNA & Protein Synthesis

Structure of Nucleic Acids: The DNA Double Helix

Deoxyribonucleic acid (DNA) is the macromolecule that stores the genetic code for all living organisms. DNA is a polymer composed of monomeric units called nucleotides. Each DNA nucleotide consists of three chemical sub-components:

1. A five-carbon sugar named deoxyribose.
2. A negatively charged phosphate group attached to the 5' carbon of deoxyribose.
3. A nitrogen-containing nitrogenous base attached to the 1' carbon of deoxyribose.

          DNA NUCLEOTIDE                  NITROGENOUS BASE CATEGORIES
     Phosphate                            +-----------------------------+
         |                                | PURINES (Double Ring):      |
   O = P - O - CH2    Base                |   Adenine (A), Guanine (G)  |
         |      \    /                    +-----------------------------+
         O       C4--C1                   | PYRIMIDINES (Single Ring):  |
                /      \                  |   Thymine (T), Cytosine (C) |
               C3------C2                 +-----------------------------+
               (OH at 3' position)

There are four distinct nitrogenous bases in DNA divided into two chemical categories:

• Purines (double-ring structure): Adenine (A) and Guanine (G).
• Pyrimidines (single-ring structure): Thymine (T) and Cytosine (C).

In 1953, James Watson and Francis Crick (utilizing X-ray diffraction images produced by Rosalind Franklin) determined that DNA exists as a double helix composed of two polynucleotide strands winding around a central axis. The outer edges of the helix consist of alternating sugar and phosphate groups linked by strong covalent phosphodiester bonds, forming the sugar-phosphate backbone. The nitrogenous bases project inward toward the center of the helix, perpendicular to the backbone.

Directionality and Complementary Base Pairing

DNA strands possess structural directionality: one end terminates in a free 5' phosphate group, while the opposite end terminates in a free 3' hydroxyl (-OH) group. The two strands of the DNA double helix run antiparallel to each other, meaning one strand runs in the $5' \rightarrow 3'$ direction while the complementary strand runs $3' \rightarrow 5'$.

The two strands are held together across the center by weak hydrogen bonds between complementary nitrogenous bases according to Chargaff's Rules:

• Adenine (A) always pairs specifically with Thymine (T) via 2 hydrogen bonds ($A = T$).
• Guanine (G) always pairs specifically with Cytosine (C) via 3 hydrogen bonds ($G \equiv C$).

Because purines always pair with pyrimidines, the distance between the two sugar-phosphate backbones remains constant at 2 nanometers throughout the entire double helix.

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DNA Replication

Before a cell divides in mitosis or meiosis, it must duplicate its entire genome during the S phase of interphase. DNA replication proceeds via a semiconservative model, meaning the two strands of the parent DNA molecule separate, and each original strand serves as a physical template for the synthesis of a new complementary daughter strand. Consequently, each new double-stranded DNA molecule consists of one conserved parent strand and one newly synthesized daughter strand.

                  DNA REPLICATION FORK AT A GLANCE

   5' -----------------------------------------------------> 3'  (Leading Strand)
      -----------------------[ DNA Polymerase III ]-------->
   3' ====================\ /=============================== 5'  (Parent DNA)
                           X  <-- [ DNA Helicase Unwinding ]
   5' ====================/ \=============================== 3'  (Parent DNA)
      <-- [Okazaki Frags] --[ DNA Polymerase III ]---------
   3' <----------------------------------------------------- 5'  (Lagging Strand)

Key Enzymes of DNA Replication

DNA replication is executed by a complex protein machine comprising specific enzymes:

1. DNA Helicase: Unwinds the double helix and breaks the hydrogen bonds between complementary base pairs at designated sites called origins of replication, forming a Y-shaped replication fork.
2. Topoisomerase (DNA Gyrase): Binds ahead of the replication fork to relieve excessive torsional strain and supercoiling caused by helicase unwinding.
3. Single-Stranded Binding Proteins (SSBs): Bind temporarily to single strands of unwound DNA to prevent them from re-annealing into a double helix.
4. RNA Primase: Synthesizes a short RNA primer (approximately 10–12 nucleotides long) complementary to the DNA template, providing a free 3'-OH group necessary for DNA polymerase initiation.
5. DNA Polymerase III: The main replicating enzyme. It reads the template strand in the $3' \rightarrow 5'$ direction and synthesizes new complementary DNA strictly in the $5' \rightarrow 3'$ direction by adding matching deoxyribonucleotides.
   • Leading Strand: Synthesized continuously toward the advancing replication fork in the $5' \rightarrow 3'$ direction, requiring only one RNA primer.
   • Lagging Strand: Synthesized discontinuously away from the replication fork in short segments called Okazaki fragments. Each fragment requires its own RNA primer.
6. DNA Polymerase I: Removes the RNA primers on both strands and replaces them with corresponding DNA nucleotides.
7. DNA Ligase: Catalyzes the formation of phosphodiester bonds to join adjacent Okazaki fragments on the lagging strand, creating a continuous sugar-phosphate backbone.

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RNA Structure and Functional Types

Ribonucleic acid (RNA) is a single-stranded nucleic acid involved in gene expression and protein synthesis. RNA differs structurally from DNA in three major ways:

1. Pentose Sugar: RNA contains ribose, which has a hydroxyl group (-OH) on carbon 2', whereas deoxyribose has a hydrogen atom (-H).
2. Nitrogenous Base: RNA contains Uracil (U) instead of Thymine (T). Uracil is a pyrimidine that forms two hydrogen bonds with Adenine ($A = U$).
3. Strand Architecture: RNA is typically single-stranded, allowing it to fold into complex three-dimensional structures, whereas DNA is double-stranded.

Three Primary Functional Types of RNA

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Gene Expression: Transcription

Gene expression is the process by which genetic information encoded in DNA directs the synthesis of functional proteins. The Central Dogma of Molecular Biology states that genetic information flows from DNA $\rightarrow$ RNA $\rightarrow$ Protein.

$\text{DNA} \xrightarrow{\text{Transcription}} \text{mRNA} \xrightarrow{\text{Translation}} \text{Protein}$

Transcription is the synthesis of an RNA molecule from a DNA template. In eukaryotic cells, transcription occurs within the nucleus.

Steps of Transcription

1. Initiation: RNA Polymerase recognizes and binds to a specific regulatory sequence on DNA called a promoter (often containing a consensus sequence known as the TATA box) located upstream of the target gene. RNA polymerase unwinds the DNA strands without requiring a primer.
2. Elongation: RNA polymerase moves along the template strand (antisense strand) in the $3' \rightarrow 5'$ direction, synthesizing a single-stranded messenger RNA (mRNA) molecule in the $5' \rightarrow 3'$ direction. As RNA polymerase advances, it inserts complementary ribonucleotides (A with U, G with C, C with G, and T with A).
3. Termination: RNA polymerase reaches a specific terminator sequence at the end of the gene, causing the enzyme to detach from the DNA and release the newly synthesized pre-mRNA transcript.

Eukaryotic Post-Transcriptional Processing

Before the primary transcript (pre-mRNA) can leave the nucleus for translation, it undergoes three critical modifications:

• 5' Capping: A modified guanine nucleotide (7-methylguanosine cap) is added to the 5' end to protect mRNA from enzymatic degradation and assist ribosome binding.
• 3' Polyadenylation: A chain of 100–250 adenine nucleotides (poly-A tail) is added to the 3' end to enhance mRNA stability and facilitate nuclear export.
• RNA Splicing: Large molecular complexes called spliceosomes excise non-coding intervening sequences (introns) from the pre-mRNA and splice together coding sequences (exons). The mature mRNA transcript then exits the nucleus through nuclear pores into the cytoplasm.

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Gene Expression: Translation

Translation is the process in which the genetic code carried by mature mRNA is decoded by cytoplasmic ribosomes to assemble a specific sequence of amino acids into a polypeptide chain (protein).

The Genetic Code and Codons

The genetic code is written in three-letter nucleotide sequences called codons. Because there are 4 nitrogenous bases, there are $4^3 = 64$ possible codons:

• 61 codons specify particular amino acids.
• AUG serves as the universal Start Codon, which codes for the amino acid methionine.
• 3 codons (UAA, UAG, UGA) serve as Stop Codons, which do not code for amino acids but signal the end of translation.

The genetic code is unambiguous (each codon specifies only one amino acid) and degenerate/redundant (most amino acids are specified by more than one codon, often differing only in the third position—the wobble position).

                 TRANSLATION ELONGATION AT THE RIBOSOME

                        [ Incoming tRNA ]
                                |  Amino Acid
                                v
                 +-----------------------------+
                 |     E site   P site   A site|
                 |    (Exit)   (Peptidyl)(Aminoacyl)
                 |       |        |        |   |
                 |      tRNA     tRNA    tRNA  |
                 |       |        |        |   |
  5' mRNA -------|----- [UAC] -- [AUG] -- [GCU] -|------- 3' (Codons)
                 +-----------------------------+
                           Ribosome Complex

Steps of Translation

Translation takes place on ribosomes in the cytoplasm (free-floating or attached to the rough endoplasmic reticulum) and proceeds through three steps:

1. Initiation: The small ribosomal subunit binds to the 5' cap of mature mRNA and scans until it finds the AUG start codon. An initiator tRNA carrying methionine, possessing the complementary anticodon UAC, base-pairs with the AUG codon. The large ribosomal subunit then joins the complex, positioning the initiator tRNA in the P (Peptidyl) site of the ribosome.
2. Elongation:
   • A second tRNA with an anticodon matching the codon in the adjacent A (Aminoacyl) site enters the ribosome.
   • The enzyme peptidyl transferase (part of rRNA) forms a covalent peptide bond between the amino acid in the P site and the new amino acid in the A site, transferring the growing polypeptide chain to the A-site tRNA.
   • The ribosome translocates (moves three nucleotides down mRNA in the $5' \rightarrow 3'$ direction). The uncharged tRNA moves to the E (Exit) site and exits the ribosome, while the tRNA holding the peptide chain shifts from the A site to the P site, opening the A site for the next charged tRNA.
3. Termination: Elongation continues until a stop codon (UAA, UAG, or UGA) enters the A site. A protein called a release factor binds to the stop codon, causing peptidyl transferase to hydrolyze the bond holding the polypeptide to tRNA. The completed polypeptide is released to fold into a functional protein, and the ribosomal complex disassembles.