Unit 3: Ultrastructure and function of Nucleus

Composition of Nucleus

The nucleus is the "control center" of the eukaryotic cell. It contains the cell's genetic material and controls its growth, metabolism, and reproduction.

Key Components:

1. Nuclear Envelope: A double membrane (two phospholipid bilayers) that surrounds the nucleus. It is continuous with the Endoplasmic Reticulum.
2. Nuclear Pores: Thousands of small channels in the nuclear envelope. They are complex structures (Nuclear Pore Complex) that regulate the passage of molecules (like mRNA out, proteins in) between the nucleus and cytoplasm.
3. Nucleoplasm: The jelly-like fluid inside the nucleus, similar to cytosol.
4. Nucleolus: A dense, non-membranous structure inside the nucleus. Its primary function is to synthesize ribosomal RNA (rRNA) and assemble ribosomes.
5. Chromatin: The complex of DNA and proteins (mainly histones) that forms chromosomes.
   • Euchromatin: Loosely packed chromatin, genetically active (genes are being transcribed).
   • Heterochromatin: Tightly packed chromatin, genetically inactive.

Nucleic Acids: DNA and RNA Composition

Nucleic acids are polymers made of monomers called nucleotides.

A nucleotide consists of three parts:

1. A Phosphate Group
2. A 5-Carbon (Pentose) Sugar:
   • Deoxyribose in DNA (lacks an oxygen at the 2' carbon).
   • Ribose in RNA (has an oxygen at the 2' carbon).
3. A Nitrogenous Base:
   • Purines (double-ring): Adenine (A), Guanine (G)
   • Pyrimidines (single-ring): Cytosine (C), Thymine (T) [Only in DNA], Uracil (U) [Only in RNA]

Structure of DNA (A, B, and Z forms)

The structure of DNA was famously discovered by James Watson and Francis Crick (with crucial data from Rosalind Franklin). It is a double helix with two antiparallel strands.

Key features of the (B-DNA) double helix:

• Two Strands: Held together by hydrogen bonds between the bases.
• Complementary Base Pairing: Adenine (A) pairs with Thymine (T) via 2 H-bonds. Guanine (G) pairs with Cytosine (C) via 3 H-bonds.
• Antiparallel: The two strands run in opposite directions. One strand is 5' (phosphate) to 3' (hydroxyl), and the other is 3' to 5'.

DNA can exist in different structural forms (conformations):

Replication of DNA

DNA replication is the process of making an exact copy of a DNA molecule. It occurs during the S (Synthesis) phase of the cell cycle. The process is semiconservative (each new DNA molecule has one old "parent" strand and one new "daughter" strand).

Step-by-step mechanism:

1. Initiation: Replication begins at specific sites called Origins of Replication (ori).
2. Unwinding: The enzyme Helicase unwinds and separates the two DNA strands, creating a replication fork.
3. Priming: The enzyme Primase adds a short RNA primer to each strand, as DNA polymerase can only add to an existing 3' end.
4. Elongation: The enzyme DNA Polymerase III adds new, complementary DNA nucleotides to the 3' end of the primer, synthesizing the new strand in the 5' to 3' direction.
   • Leading Strand: Synthesized continuously towards the replication fork.
   • Lagging Strand: Synthesized discontinuously (away from the fork) in short pieces called Okazaki fragments.
5. Ligation: DNA Polymerase I removes the RNA primers and replaces them with DNA. The enzyme DNA Ligase joins the Okazaki fragments together.

Denaturation of DNA

Denaturation is the separation of the two strands of the DNA double helix by breaking the hydrogen bonds between the base pairs.

• How it's done:
  • Heat: Heating a DNA solution (e.g., to 95°C) will cause the strands to separate ("melt").
  • Chemicals: High pH (alkaline solutions) or chemicals like urea.
• Melting Temperature (Tm): The temperature at which 50% of the DNA strands are denatured. The Tm is higher for DNA with more G-C pairs (since they have 3 H-bonds) than A-T pairs (2 H-bonds).
• Renaturation (Annealing): If cooled slowly, the complementary single strands will find each other and re-form the double helix. This is the principle behind techniques like PCR and DNA hybridization.

DNA Polymerases

DNA Polymerases are the main enzymes of DNA replication. Their primary job is to synthesize new DNA strands by adding nucleotides one by one, complementary to the template strand.

Key Properties:

• They always synthesize the new strand in the 5' to 3' direction.
• They require a template strand to read.
• They require a primer (a short RNA sequence) to start; they cannot start a new strand from scratch.
• Many also have proofreading ability (3' to 5' exonuclease activity), allowing them to remove and correct mistakes, which ensures high fidelity of replication.

Different Types of RNA and their Role

RNA (Ribonucleic acid) is the other major nucleic acid. It is typically single-stranded, uses ribose sugar, and uses Uracil (U) instead of Thymine (T).

There are three main types of RNA involved in protein synthesis:

1. Messenger RNA (mRNA)

Role: The "Messenger" or "Code"

mRNA carries the genetic instructions (the "message") from the DNA in the nucleus to the ribosomes in the cytoplasm. It acts as the template for protein synthesis. Each set of three bases on mRNA is called a codon, which specifies one amino acid.

2. Transfer RNA (tRNA)

Role: The "Translator" or "Adapter"

tRNA's job is to read the codons on the mRNA and bring the correct amino acid to the ribosome. It has a specific 3D "cloverleaf" structure with two key sites:

• An anticodon loop that base-pairs with the mRNA codon.
• An acceptor stem (3' end) that attaches to a specific amino acid.

3. Ribosomal RNA (rRNA)

Role: The "Factory" or "Enzyme"

rRNA is the most abundant type of RNA. It is the main structural and functional component of ribosomes (along with proteins). rRNA acts as a ribozyme (an RNA enzyme), catalyzing the formation of peptide bonds between amino acids to build the protein chain.