bio aos 1

Nucleic Acids: Structure and Function

Overview of Nucleic Acids

• Nucleic acids are essential biomolecules that store and transmit genetic information necessary for protein synthesis.

• There are two primary types of nucleic acids: DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid).

• DNA serves as the genetic blueprint, while RNA plays various roles in translating that information into proteins.
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Structure of DNA and RNA

• Both DNA and RNA are macromolecules composed of smaller units called nucleotides, which consist of a sugar, a phosphate group, and a nitrogenous base.

• DNA contains four nitrogenous bases: Adenine (A), Thymine (T), Guanine (G), and Cytosine (C).

• RNA contains the same bases except Thymine is replaced by Uracil (U).

• The structure of DNA is a double helix, while RNA is typically single-stranded.

Nucleotide Comparison

• DNA nucleotides: Deoxyribose sugar, phosphate group, and one of four nitrogenous bases (A, T, G, C).

• RNA nucleotides: Ribose sugar, phosphate group, and one of four nitrogenous bases (A, U, G, C).

• The 5' and 3' designations refer to the carbon atoms in the sugar molecule, indicating the directionality of the nucleic acid strand.

Polynucleotide Formation and Base Pairing

• Nucleotides link together through phosphodiester bonds to form polynucleotide chains, a process known as polymerization.

• DNA strands are held together by hydrogen bonds between complementary bases: A pairs with T, and C pairs with G.

• The double helix structure of DNA allows for efficient storage and replication of genetic information.
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This diagram shows a segment of DNA with base pairs cytosine (C) and guanine (G), and thymine (T) and adenine (A) connected by hydrogen bonds

Understanding the Genetic Code

• The genetic code is a set of rules that defines how sequences of nucleotides correspond to amino acids in proteins.

• It is described as a triplet code, where three nucleotides (a triplet) correspond to one amino acid, known as a codon in RNA.

• The genetic code is universal, meaning it is the same across all living organisms, and degenerate, as multiple codons can encode the same amino acid.

Steps in Gene Expression

• Gene expression involves the process of converting genetic information from DNA into functional proteins.

• The main steps include transcription (copying DNA to mRNA), RNA processing (modifying mRNA), and translation (synthesizing proteins from mRNA).

• Transcription occurs in the nucleus, where RNA polymerase synthesizes mRNA from the DNA template.

Transcription Process

• RNA polymerase binds to the promoter region of the gene, unwinding the DNA strands.

• It reads the template strand and assembles a complementary pre-mRNA strand.

• Transcription concludes when a termination sequence is reached, resulting in a pre-mRNA molecule.

RNA Processing and Translation

• In eukaryotic cells, pre-mRNA undergoes processing where introns are removed and exons are spliced together by spliceosomes.

• A methyl cap is added to the 5' end and a poly-A tail to the 3' end of the mRNA for stability and export from the nucleus.

• During translation, ribosomes read the mRNA sequence to synthesize proteins, starting from the 5' end.

Gene Expression Overview

Key Processes in Gene Expression

• Transcription: The process where RNA polymerase binds to the promoter region of DNA, unwinds the DNA, and synthesizes pre-mRNA by adding complementary nucleotides to the template strand. This process ends when a termination sequence is reached.

• Translation: The ribosome reads the mRNA sequence, and tRNA molecules bring the corresponding amino acids. The amino acids are linked together through condensation reactions to form a polypeptide chain. Translation concludes when a stop codon (UAA, UAG, UGA) is encountered.

• Post-Transcriptional Modifications: Introns are removed from the pre-mRNA, and exons are spliced together. A methyl cap is added to the 5' end, and a poly-A tail is added to the 3' end to stabilize the mRNA and facilitate its export from the nucleus.

• Role of tRNA: tRNA molecules have anticodons that are complementary to mRNA codons, ensuring the correct amino acids are delivered during translation.

• Ribosome Function: The ribosome is composed of two subunits and is essential for translating mRNA into a polypeptide chain, facilitating the binding of tRNA and catalyzing peptide bond formation.

• Gene Expression Regulation: The expression of genes can be continuously active or regulated based on cellular needs, with only specific genes being expressed in different cell types or developmental stages.

Components of Gene Expression

• RNA Polymerase: An enzyme that synthesizes RNA from a DNA template, crucial for initiating transcription at the promoter region.

• Promoter Region: A specific DNA sequence where RNA polymerase binds to initiate transcription, determining the start point of gene expression.

• Introns and Exons: Introns are non-coding regions that are removed during RNA processing, while exons are coding sequences that remain in the mature mRNA.

• Spliceosome: A complex responsible for the splicing of introns from pre-mRNA, ensuring only exons are joined to form mature mRNA.

• Stop Codons: Specific codons (UAA, UAG, UGA) that signal the termination of translation, leading to the release of the newly synthesized polypeptide.

• Polypeptide Formation: The process of linking amino acids through peptide bonds, forming a functional protein based on the mRNA sequence.

Gene Structure

Basic Structure of a Gene

• Definition of a Gene: A gene is a segment of DNA that encodes for a polypeptide, consisting of coding (exons) and non-coding (introns) regions.

• Exons: Segments of DNA that contain the information necessary for coding a polypeptide, contributing to the final protein product.

• Introns: Non-coding sections of a gene that interrupt the coding sequence and are removed during RNA processing.

• Promoter and Operator Regions: The promoter is where RNA polymerase binds to initiate transcription, while the operator is a regulatory sequence that can inhibit transcription when bound by a repressor protein.

• Regulatory Genes: Genes that produce proteins (like repressor proteins) that regulate the expression of other genes, often located distantly from the genes they control.

• Gene Orientation: Genes are oriented in a 5' to 3' direction, with the promoter located upstream of the coding region.

Types of Genes

• Structural Genes: These genes code for proteins, tRNA, or rRNA that are essential for cellular functions. Examples include keratin, insulin, and hemoglobin.

• Regulatory Genes: Genes that produce proteins that regulate the expression of other genes, controlling when and how much of a protein is made.

• Gene Expression Variability: Different genes are expressed in different cell types or developmental stages, despite all cells containing the same genetic information.

• Gene Regulation Mechanisms: The regulation of gene expression can occur at multiple levels, including transcriptional, post-transcriptional, and translational levels.

• Eukaryotic vs. Prokaryotic Genes: Eukaryotic genes often contain introns and require extensive processing, while prokaryotic genes are typically organized in operons and lack introns.

• Example of a Structural Gene: Insulin, a small protein composed of 51 amino acids, illustrates the concept of structural genes coding for essential proteins.

Gene Regulation

Understanding Gene Regulation

• Definition of Gene Regulation: The process by which cells control the expression of genes, determining which proteins are produced and in what quantities.

• Importance of Gene Regulation: Essential for cellular differentiation, adaptation to environmental changes, and maintaining homeostasis within an organism.

• Gene Expression vs. Gene Regulation: Gene expression refers to the process of synthesizing proteins from genes, while gene regulation involves the mechanisms that control this expression.

• Continuous vs. Regulated Gene Expression: Some genes are expressed continuously (e.g., housekeeping genes), while others are regulated based on cellular needs or environmental conditions.

• Prokaryotic Gene Regulation Example: The trp operon in E. coli demonstrates how gene regulation allows bacteria to conserve resources by only producing enzymes for tryptophan synthesis when it is not available in the environment.

• Repressor Proteins: Proteins that bind to operator regions to inhibit transcription, preventing unnecessary production of enzymes when the corresponding substrate (e.g., tryptophan) is present.

The trp Operon

• Background of the trp Operon: E. coli requires tryptophan for protein synthesis and can either obtain it from the environment or synthesize it when not available.

• Function of the trp Operon: The operon consists of genes that encode enzymes necessary for tryptophan synthesis, regulated by the availability of tryptophan.

• Mechanism of Regulation: When tryptophan is absent, the repressor protein does not bind to the operator, allowing transcription of the operon. When tryptophan is present, it binds to the repressor, enabling it to bind to the operator and block transcription.

• Components of the trp Operon: Includes the promoter region, operator, and structural genes (trpE, trpD, trpC, trpB, trpA) that encode the enzymes for tryptophan synthesis.

• Significance of the trp Operon: Serves as a classic example of gene regulation in prokaryotes, illustrating how organisms adapt to nutrient availability.

• Visual Representation: Diagrams of the trp operon can help illustrate the binding sites and the flow of transcription and translation processes.

Amino Acids and Protein Structure

Amino Acids as Building Blocks

• Definition of Amino Acids: Amino acids are organic compounds that serve as the monomers of proteins, with 20 different amino acids commonly found in living organisms.

• Basic Structure of Amino Acids: Each amino acid consists of an amino group (-NH2), a carboxyl group (-COOH), and a unique side chain (R-group) that determines its properties.

• Diversity of Proteins: Proteins vary in size and function, with small proteins like insulin consisting of 51 amino acids, while larger proteins like titin can have over 33,000 amino acids.

• Peptide Bond Formation: When two amino acids join, the amino group of one reacts with the carboxyl group of another, forming a peptide bond and releasing water (condensation reaction).

• Levels of Protein Structure: Proteins have four hierarchical levels of structure: primary (amino acid sequence), secondary (alpha helices and beta sheets), tertiary (3D folding), and quaternary (multiple polypeptide chains).

• Functional Importance: The specific sequence and structure of amino acids in a protein determine its function and role in biological processes.

Overview of Proteins

Introduction to Proteins

• Proteins are essential macromolecules composed of amino acids, with varying sizes and functions. For example, insulin is a small protein consisting of 51 amino acids, while titin, a muscle protein, is significantly larger with 33,000 amino acids.

• Proteins play critical roles in biological processes, including catalysis, structure, transport, and signaling.

Chemical Structure of Amino Acids

• All 20 amino acids share a common structure, which includes an amino group (-NH2), a carboxyl group (-COOH), and a unique side chain known as the R-group, which determines the properties of each amino acid.

• The R-group can vary widely, influencing the amino acid's characteristics and its role in protein structure.

Peptide Bond Formation

• Peptide bonds form when the amino group of one amino acid reacts with the carboxyl group of another, releasing a water molecule in a condensation reaction.

• This process of linking amino acids continues, resulting in a polypeptide chain, where each amino acid is referred to as an amino acid residue.

Hierarchical Levels of Protein Structure

• Proteins are analyzed at four levels of structure: primary, secondary, tertiary, and quaternary, each representing a different aspect of protein complexity.

• The primary structure is the linear sequence of amino acids, while secondary structures include local folding patterns like alpha-helices and beta-pleated sheets, stabilized by hydrogen bonds.

Protein Structure and Function

Primary Structure

• The primary structure is the specific sequence of amino acids in a polypeptide chain, which determines the protein's unique characteristics and function.

• A disulfide bridge, a type of covalent bond, can form between cysteine residues, contributing to the protein's stability.

Secondary Structure

• Secondary structures arise from hydrogen bonding between amino acids, leading to formations such as alpha-helices and beta-pleated sheets.

• These structures are crucial for the protein's overall shape and function, particularly in enzyme active sites.

Tertiary Structure

• The tertiary structure refers to the overall three-dimensional shape of a protein, resulting from interactions among R-groups, including hydrogen bonds, ionic bonds, and hydrophobic interactions.

• This structure is vital for the protein's biological activity and functionality.

Quaternary Structure

• Quaternary structures occur when multiple polypeptide chains come together to form a functional protein, as seen in hemoglobin, which consists of four polypeptide chains.

• The stability and functionality of quaternary structures are maintained by various types of bonds, including hydrogen, ionic, and covalent bonds.

The Diversity of Proteins and Enzymes

Understanding the Proteome

• The proteome is the complete set of proteins expressed by a cell, tissue, or organism, and varies between different cell types due to differential gene expression.

• Humans are estimated to have around 100,000 different proteins, highlighting the complexity and diversity of protein functions.

Enzymes as Biological Catalysts

• Enzymes are specialized proteins that accelerate biochemical reactions by lowering the activation energy required for the reaction to occur.

• They are not consumed in the reactions they catalyze, allowing them to be reused multiple times.

Metabolic Pathways

• Metabolism encompasses all chemical reactions in an organism, divided into anabolism (building up molecules) and catabolism (breaking down molecules).

• Anabolic processes, such as protein synthesis and photosynthesis, require energy, while catabolic processes, like cellular respiration, release energy.

Properties of Enzymes

• Enzymes exhibit specificity, typically catalyzing only one type of reaction, and are sensitive to environmental conditions such as temperature and pH.

• Optimal conditions for enzyme activity vary, with most enzymes functioning best at neutral pH and specific temperature ranges.

Protein Secretory Pathway

Overview of the Secretory Pathway

• The protein secretory pathway is the process by which cells package proteins into vesicles for export outside the cell.

• This pathway involves the rough endoplasmic reticulum (RER), Golgi apparatus, and vesicles, which work together to ensure proper protein processing and transport.

Steps in the Protein Secretory Pathway

• Step 1: The rough ER synthesizes and folds proteins into transport vesicles.

• Step 2: Transport vesicles carry proteins to the Golgi apparatus for further modification and packaging.

• Step 3: The Golgi apparatus processes proteins and packages them into secretory vesicles for exocytosis.

• Step 4: Secretory vesicles fuse with the cell membrane, releasing proteins into the extracellular environment.

Understanding Recombinant Plasmids

Definition and Importance

• A recombinant plasmid is a plasmid that has been genetically engineered to contain foreign DNA, allowing for the expression of specific proteins in host organisms.

• Plasmids are small, circular DNA molecules found in bacteria, which can replicate independently of chromosomal DNA.

• They are not essential for bacterial survival, making them ideal for genetic manipulation.

• Recombinant plasmids serve as vectors to transfer genetic material into host cells, crucial for biotechnology applications such as insulin production.

• The universal nature of the genetic code allows for proteins to be synthesized in different organisms, facilitating cross-species gene expression.

Key Components of Recombinant Plasmids

• Gene of Interest: The specific gene that researchers want to express, often coding for a protein of commercial value, such as insulin or growth hormones.

• Plasmid Vector: A plasmid designed to carry the gene of interest, typically containing essential features like restriction enzyme sites, antibiotic resistance genes, and a reporter gene.

• Restriction Enzymes: Enzymes that cut DNA at specific sequences, allowing for the insertion of the gene of interest into the plasmid.

• DNA Ligase: An enzyme that joins DNA fragments by forming phosphodiester bonds, crucial for creating a stable recombinant plasmid.

Steps to Create a Recombinant Plasmid

Step-by-Step Process

• Step 1: Identify the Gene of Interest: Select the gene that encodes the desired protein, such as insulin, which is crucial for diabetes treatment.

• Step 2: Choose a Plasmid Vector: Select a plasmid that has the necessary features: restriction sites, antibiotic resistance, a reporter gene, and an origin of replication (ORI).

• Step 3: Use Restriction Enzymes: Cut both the plasmid and the gene of interest with the same restriction enzyme to create complementary sticky ends for easier ligation.

• Step 4: Ligate the DNA: Add DNA ligase to join the gene of interest to the plasmid vector, forming a recombinant plasmid.

• Step 5: Transformation: Introduce the recombinant plasmid into bacterial cells through methods like heat shock or electroporation.

Visual Representation of Steps

1. Gene of Interest + Plasmid Vector
2. Cut with Restriction Enzymes
3. Ligate with DNA Ligase
4. Transformation into Bacteria

Bacterial Transformation Process

Mechanisms of Transformation

• Natural Transformation: Some bacteria can naturally uptake free-floating DNA from their environment, a process exploited in genetic engineering.

• Heat Shock Method: Involves exposing bacteria to a sudden increase in temperature, making their membranes more permeable to plasmids.

• Electroporation: Uses an electric field to increase the permeability of the bacterial cell membrane, facilitating plasmid uptake.

Selection of Transformed Bacteria

• After transformation, bacteria are cultured on two types of agar plates: one with nutrients only and another with nutrients plus an antibiotic.

• Only bacteria that have successfully taken up the recombinant plasmid (which contains an antibiotic resistance gene) will survive on the antibiotic-containing plate.

• This selection process allows researchers to identify and isolate transformed bacteria for further study or protein production.

Applications of Recombinant Plasmids

Production of Human Insulin

• Recombinant plasmids are used to produce human insulin by inserting the human insulin gene into bacterial plasmids, allowing bacteria to synthesize insulin.

• This method provides a reliable and cost-effective source of insulin for diabetes treatment, compared to previous methods of extraction from animal sources.

• Other proteins produced using recombinant plasmids include erythropoietin for anemia treatment, growth hormones for growth disorders, and chymosin for cheese production.

Broader Implications in Biotechnology

• The ability to manipulate plasmids has revolutionized genetic engineering, enabling advancements in medicine, agriculture, and research.

• Recombinant DNA technology has led to the development of genetically modified organisms (GMOs), which can enhance food production and resistance to pests.

• Ethical considerations and regulations surrounding the use of recombinant DNA technology are critical in ensuring safe and responsible applications.

1. Bacterial Transformation and Identification

1.1 Overview of Bacterial Transformation

• Transformation is the process of incorporating foreign DNA into a cell, which can occur in both prokaryotic and eukaryotic cells.

• Bacteria are commonly transformed using plasmids as vectors to transport desired genes, such as the human insulin gene.

• The transformed bacteria can be identified by the presence of a reporter gene, such as GFP, which causes them to fluoresce under UV light.

1.2 Identification of Transformed Bacteria

• Transformed bacteria can be confirmed by culturing them and checking for fluorescence under UV light, indicating the presence of the gfp gene.

• This method ensures that only bacteria containing the plasmid with the desired gene are selected for further study.

• The use of antibiotic resistance genes in plasmids allows for the selection of successfully transformed bacteria, as only those that have taken up the plasmid will survive in the presence of the antibiotic.

2. Making a Recombinant Plasmid

2.1 Creation of Recombinant Plasmids

• Genetic engineers utilize restriction enzymes to cut plasmids, allowing foreign DNA to be inserted.

• DNA ligase is then used to seal the plasmid, creating a recombinant plasmid that can be reintroduced into bacteria or other host organisms.

• The process of creating recombinant plasmids is crucial for gene cloning and the production of proteins such as insulin.

2.2 Components of a Recombinant Plasmid

• A typical recombinant plasmid includes an enzyme recognition site, sticky ends for DNA ligation, a promoter region, and an antibiotic resistance gene.

• The inclusion of a beta-galactosidase gene allows for easy identification of successful transformations, as it can be used as a marker.

• The diagram below illustrates the structure of a recombinant plasmid:

| Component | Description |
|------------------------|-----------------------------------------------|
| Enzyme Recognition Site | Site where restriction enzymes cut the DNA |
| Sticky Ends | Overhanging ends that facilitate ligation |
| Promoter Region | Initiates transcription of the inserted gene |
| Antibiotic Resistance | Allows for selection of transformed bacteria |

3. Insulin Production Using Recombinant DNA Technology

3.1 Understanding Insulin

• Insulin is a peptide hormone essential for glucose metabolism, promoting sugar uptake and storage in tissues.

• Type I diabetes results from the autoimmune destruction of insulin-producing cells in the pancreas, leading to insufficient insulin production.

• The structure of insulin consists of two polypeptide chains (A and B) linked by disulfide bridges, totaling 51 amino acids.

3.2 Steps in Insulin Production

• Step 1: Isolate the genes coding for the A and B chains of insulin using restriction enzymes.

• Step 2: Insert the insulin genes into plasmid vectors alongside a beta-galactosidase gene and a promoter region.

• Step 3: Introduce the recombinant plasmids into suitable bacterial hosts, such as E. coli, for expression.

• Step 4: Select transformed cells using antibiotic resistance or reporter genes to ensure only successful transformations are cultured.

• Step 5: Induce expression of the insulin genes by adding lactose, which promotes transcription and translation of the fusion proteins.

4. Purification of Insulin

4.1 Purification Process

• The fusion protein produced consists of beta-galactosidase and the insulin chains, which must be separated for functional insulin.

• Chemical methods are used to remove beta-galactosidase, allowing the A and B chains of insulin to be joined by disulfide bonds, forming active insulin.

• The final product, Humulin®, is a commercially available form of human insulin used for diabetes treatment.