Molecular Biology - Theoretical Concepts

Molecular Biology - Theoretical Section

• Importance in Pharmacy: Many drugs are synthesized through molecular biology techniques. Insulin production is a key example, where the human insulin protein gene is isolated, inserted into bacteria or fungi, and then reproduced to create insulin for use. Previously, insulin was derived from animals, but this posed allergy risks due to immune responses against animal-derived proteins.

• Understanding Drug Actions: Molecular biology helps understand how certain drugs like fluoroquinolones work on specific bodily processes. Fluoroquinolones inhibit gyrase enzymes in microbial cells.

• Forensic Applications: Molecular biology plays a vital role in forensic investigations, allowing for accurate identification in criminal cases and accident investigations. Results in forensic analysis must be 100% accurate for crucial applications and to avoid errors.

Fundamental Principle of Molecular Biology

• DNA Replication: DNA replication copies the entire DNA strand identically during cell division, transferring the complete DNA from the parent cell to the daughter cells without altering its structure.

• Transcription: Transcription of a gene happens when the body needs a particular protein. A specific gene responsible for that protein is transcribed from DNA into messenger RNA (mRNA). Further, the mRNA is translated into the protein which performs its specific function.

Prokaryotes vs. Eukaryotes

• Prokaryotes (bacteria): These primitive structures have rapid and simple biological processes and fast reproduction, leading to quicker protein production suitable for proteins with simpler structures or fewer post-translational modifications, such as insulin. However, isolating the protein from bacterial contaminants is a significant challenge in these simple organisms.

• Eukaryotes (human cells): The processes for protein synthesis in complex eukaryotes, like humans and animals, are more intricate and require post-translational modifications. These modifications may involve adding sugar molecules or other groups (such as adding a heme group to globin, a protein part of hemoglobin), as well as complex folding for functionality. Eukaryotic cells possess the necessary machinery for these processes. However prokaryotes do not because they are simpler.

Protein Structure

• Primary Structure: The amino acid sequence of a protein, as determined by mRNA after translation.

• Secondary Structure: Amino acids arrange into beta-sheets and alpha-helices.

• Tertiary Structure: The three-dimensional spatial arrangement of the polypeptide chain.

• Quaternary Structure: When two or more polypeptide chains with tertiary structures assemble.

DNA and its properties

• Individual Variation: DNA sequences differ between individuals, closely matching in family members, but distinct overall. However, the DNA sequence remains the same in all cells of an individual throughout their life. Mutations are localized in specific places.

• Common Traits: Regions of DNA code for proteins, typically shared across individuals (like genes encoding insulin). A majority (over 90%) of DNA doesn't directly translate to proteins; this is a key contributor to individual variability. DNA can be used to determine ethnic background, family relations, and identify individuals based on particular sequences. Such variations are also helpful in regulatory mechanisms for transcription and translation.

Gene Expression

• Mechanism: The process of converting genetic information encoded in genes into functional protein products. Transcription and translation are essential steps in gene expression.

• Regulation by Anti-mRNA: Anti-mRNA molecules (also called microRNAs) can bind to mRNA molecules and inhibit protein translation, influencing gene expression.

Protein Modification & Inhibition

• Phosphorylation: Some proteins become functional only after phosphorlyation by the addition a phosphate group, with the help of kinase enzymes. An inhibitor of kinase, often used as a medication to inhibit proteins, could prevent this activation process by reducing phosphorlyation.

• Dephosphorylation: Conversely, some proteins are functional in their unphosphorylated states. A phosphatase inhibitor could prevent their activation when a phosphate group needs to be removed to become functional.

• Pharmaceutical Targets: Protein modification (inhibition or modulation) is a crucial aspect of studying specific bodily processes (like insulin action) to understand target proteins and create pharmaceutical targets for therapeutic treatments.

DNA Structure

• Bases: DNA is comprised of four nitrogenous bases: adenine (A), guanine (G), cytosine (C), and thymine (T).

• Pairing Rules: A pairs with T, and C pairs with G.

• Stability: Thymine (in DNA) is more stable than uracil (in RNA) because a methyl group is attached. Methylation (adding a methyl group) to a compound (e.g. thymine) enhances its stability against nucleases, enzymes that break down nucleotides.

DNA and RNA Differences

• Structure: DNA is a large molecule composed of a considerable number of nucleotides compared to the short sequence composed of mRNA.

• Lifespan: DNA has a long lifespan, depending on the cell, compared to mRNA, which has a short lifespan, degraded after protein translation.

• Stability: The presence of hydroxyl groups (OH) on the ribose sugar in RNA makes it susceptible to degradation in alkaline conditions, in contrast to the absence of these groups in DNA.

DNA Denaturation and Renaturation

• Denaturation: This process involves breaking hydrogen bonds between DNA strands by increasing temperature( heat) or reducing salt concentration, effectively separating the strands. Denaturation is a common preparatory step for various DNA-related studies.

• Renaturation: Renaturation is the process where separated strands are cooled again (lowered temperature or increased salt concentration) allowing hydrogen bonnding to recur, and the double helix re-forms, allowing re-formation of the original DNA structure.

Restriction Enzymes

• Function: Recognizes specific sequences in DNA and cuts the DNA molecules at those sites. This is a crucial tool in genetic engineering.

• Role in protecting bacteria: Certain bacteria utilize restriction enzymes to defend against viral infections by cleaving the viral DNA.

• Methylation Protection: Bacteria protect their own DNA from cleavage by modifying the susceptible DNA strands at restriction enzyme recognition sites. Methylation modification prevents digestion of the bacteria-specific DNA. methylation is a crucial mechanism.

DNA Fingerprinting

• Uniqueness: DNA sequences and restriction enzyme patterns are unique to each individual, offering a specific identifier for individuals.

• Forensic Applications: DNA fingerprinting is used to identify individuals based on their unique DNA patterns in criminal investigation, and accident scenes. A blood stain or hair, often used for fingerprinting, may contain a DNA sample, which can be isolated.

Nucleotides

• Components: Nucleotides are composed of a nitrogenous base (purine or pyrimidine), a sugar (ribose in RNA or deoxyribose in DNA), and a phosphate group.

• Types: There are five bases in DNA and RNA.

• Importance: Nucleotides are the building blocks of nucleic acids. DNA and RNA, crucial molecules in biological processes, are composed of nucleotides.

RNA Types and Functions

• mRNA (messenger RNA): Carries genetic information from DNA to ribosomes during protein synthesis..

• rRNA (ribosomal RNA): Structural component of ribosomes, where protein synthesis occurs.

• snRNA (small nuclear RNA): Involved in gene regulation and mRNA processing.

• tRNA (transfer RNA): Transfers amino acids to ribosomes during protein synthesis. tRNA has a specific structure resembling a cloverleaf. It carries a specific amino acid and recognizes the corresponding codon of the mRNA. The structure of the tRNA provides a crucial mechanism to match the mRNA codon with the respective amino acid.

Enzymatic Reactions for Nucleic Acid Degradation

• Exonucleases: Remove nucleotides from the ends of DNA or RNA strands.

• Endonucleases: Break phosphodiester bonds within DNA or RNA strands. Both examples illustrate crucial enzymes important for DNA and RNA function, repair, or destruction.

DNA Replication Steps

• Initiation: Replication starts at specific origin sites (Ori) on the DNA molecule.

• Unwinding (Denaturation): The DNA double helix unwinds and separates into two single strands.

• Primer Synthesis: Short RNA primers are synthesized to provide a starting point for DNA polymerase.

• DNA Synthesis: DNA polymerase replicates the DNA strands, using the separated strands as templates.

• Ligation of Segments: Newly synthesized DNA segments are joined together and,

• Chromatin Reconstitution: The DNA is subsequently rewound to form the original chromatin structure.

DNA Polymerase Types and Functions

• Key Roles in DNA Replication: These enzymes are central to DNA synthesis. Different types of DNA Polymerase are responsible for the detailed tasks within the replication process. Different types of DNA polymerase are each responsible for distinct functions during replication, showcasing the intricate specialization within the process.

• Comparison: Different DNA Polymerase types exhibit differences in factors like efficiency, proofreading capabilities, and functions within replication. Different DNA polymerase types play unique roles within the replication process, reflecting the specialization within the broader process.

• Activities: Some types are more efficient at synthesizing the initial DNA while others specialized in dealing with correcting errors.