Chapter 5: Molecular Basis of Inheritance

The DNA

Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA) are the two types of nucleic acids found in living systems. DNA acts as the genetic material in most organisms. RNA mostly functions as a messenger, adapter, structural, and catalytic molecule.

Structure of Polynucleotide Chain

A nucleotide has three components: a nitrogenous base, a pentose sugar (ribose in RNA, and deoxyribose in DNA), and a phosphate group. There are two types of nitrogenous bases:

• Purines: Adenine (A) and Guanine (G).
• Pyrimidines: Cytosine (C), Thymine (T, only in DNA), and Uracil (U, only in RNA). A nitrogenous base is linked to the OH of 1’C pentose sugar through an N-glycosidic linkage to form a nucleoside. When a phosphate group is linked to the OH of 5’C of a nucleoside through a phosphoester linkage, a corresponding nucleotide is formed.

Double Helix Model

James Watson and Francis Crick (1953), based on X-ray diffraction data produced by Maurice Wilkins and Rosalind Franklin, proposed the Double Helix model for the structure of DNA.

Key features:

1. Made of two polynucleotide chains, with a sugar-phosphate backbone.
2. The two chains have anti-parallel polarity (one is \(5’ \rightarrow 3’\), the other is \(3’ \rightarrow 5’\)).
3. Bases project inside and are paired through Hydrogen bonds (A with T with two H-bonds; G with C with three H-bonds).
4. The two chains are coiled in a right-handed fashion. Erwin Chargaff’s rule states that for a double-stranded DNA, the ratios between Adenine and Thymine, and Guanine and Cytosine are constant and equal one.

DNA Packaging

In prokaryotes like E. coli, DNA is held together with some proteins in a region called the nucleoid. In eukaryotes, there is a set of positively charged, basic proteins called histones. Histones are organized to form a unit of eight molecules called a histone octamer. The negatively charged DNA is wrapped around the positively charged histone octamer to form a structure called a nucleosome. Nucleosomes constitute the repeating unit of a structure in the nucleus called chromatin.

DNA Replication

Watson and Crick suggested a semi-conservative mechanism of replication. Matthew Meselson and Franklin Stahl (1958) proved it experimentally using heavy nitrogen (\(^{15}N\)) and E. coli. In eukaryotic replication, multiple origins of replication exist. The main enzyme is DNA-dependent DNA polymerase, which catalyzes polymerization highly efficiently in the \(5’ \rightarrow 3’\) direction. Consequently, on one strand (template with polarity \(3’ \rightarrow 5’\)), replication is continuous (leading strand), while on the other (\(5’ \rightarrow 3’\)), it is discontinuous (lagging strand). The discontinuous fragments (Okazaki fragments) are joined by DNA ligase.

Central Dogma

The Central Dogma in molecular biology states the flow of genetic information: DNA \(\xrightarrow{\text{Transcription}}\) mRNA \(\xrightarrow{\text{Translation}}\) Protein

Transcription

The process of copying genetic information from one strand of the DNA into RNA is termed transcription. A transcription unit in DNA is defined primarily by three regions:

1. A Promoter
2. The Structural gene
3. A Terminator The enzyme DNA-dependent RNA polymerase catalyzes polymerization in only one direction, i.e., \(5’ \rightarrow 3’\). Therefore, the DNA strand with polarity \(3’ \rightarrow 5’\) acts as a template strand.

In eukaryotes, the primary transcript (hnRNA) undergoes processing:

• Splicing: Introns are removed and exons are joined.
• Capping: Methyl guanosine triphosphate is added to the 5’-end.
• Tailing: Polyadenylate tail (200-300 adenylate residues) is added to the 3’-end.

Genetic Code and Translation

The genetic code directs the sequence of amino acids during synthesis of proteins. Features of genetic code:

1. It is a triplet (61 codons code for amino acids, 3 are stop codons).
2. It is unambiguous and specific (one codon codes for only one amino acid).
3. It is degenerate (some amino acids are coded by more than one codon).
4. It is universal.

Translation refers to the process of polymerization of amino acids to form a polypeptide. The order and sequence of amino acids are defined by the sequence of bases in the mRNA. The cellular factory responsible for synthesizing proteins is the ribosome.

Regulation of Gene Expression

In prokaryotes, control of the rate of transcriptional initiation is the predominant site for control of gene expression. Lac Operon: The lac operon consists of one regulatory gene (the i gene, codes for the repressor) and three structural genes (z, y, and a). It is an inducible operon. Lactose acts as the inducer by binding to the repressor protein, bringing about a conformational change and preventing it from binding to the operator region, allowing RNA polymerase to transcribe the operon.

Human Genome Project (HGP) and DNA Fingerprinting

HGP (1990-2003) mapped the complete human genome (~\(3 \times 10^9\) bp). Key findings include that humans have approximately 30,000 genes, and 99.9% of DNA is exactly the same in all people. DNA Fingerprinting: Developed by Alec Jeffreys. It involves identifying differences in some specific regions in DNA sequence called repetitive DNA. It relies on Variable Number of Tandem Repeats (VNTRs) as probes that show very high degrees of polymorphism.

Competency Based Questions

Q1. Analysis of a double-stranded DNA sample shows that it contains 18% Cytosine. Calculate the percentage of Adenine in this sample based on Chargaff’s rule.

(A) 18%
(B) 32%
(C) 36%
(D) 64%

Q2. During Meselson and Stahl’s experiment, E. coli grown in \(^{15}N\) medium was shifted to a \(^{14}N\) medium. If the replication time is 20 minutes, what would be the ratio of hybrid DNA to light DNA after 60 minutes?

Q3. If the sequence of the coding strand in a transcription unit is written as follows:
5' - ATG CCT GAG CGT - 3'
Write down the sequence of the mRNA transcript.

Q4. The lac repressor is a tetrameric protein that binds to the operator sequence of the lac operon with a very high affinity (\(K_d \approx 10^{-13} M\)). Why does the addition of an inducer (like allolactose) successfully initiate transcription despite this high affinity?

Q5. A geneticist synthesizes an artificial mRNA with purely repeating dinucleotides: 5'-UGUGUGUGUGUGUGUG-3'. In a cell-free translation system, how many different types of amino acids will make up the resulting polypeptide?